SECTION 4 Immunological mechanisms

4.1 The innate immune system 307

4.1 The innate immune system 307

ESSENTIALS The innate immune system comprises evolutionarily ancient mech- anisms that mediate first-​line responses against microbial patho- gens, and are also important in priming and execution of adaptive immune responses, and in defence against tumours. These re- sponses, which recognize microbial non-​self, damaged self, and absent self, are characterized by rapidity of action and involve various different cell types, cell-​associated receptors, and soluble factors. Previously believed to lack plasticity or memory, certain in- nate immune responses have recently been shown to be capable of ‘learning’ or ‘training’. Cellular components of the innate immune system—​these are mainly derived from myeloid precursors in the bone marrow and include monocytes, dendritic cells, and granulocytes (neutrophils, eosinophils, and basophils). It has recently been discovered that, at least in mice, most tissue macrophages are derived from a sep- arate progenitor population before birth. Two small populations of lymphoid cells—​natural killer and natural killer T cells—​are also in- cluded because they lack the clonally rearranged receptors of B and T lymphocytes. Receptors—​the innate immune system uses a relatively small rep- ertoire of germline-​encoded largely non-​rearranging receptors. (1)  Pattern recognition receptors include Toll-​like receptors and mannose receptors and recognize invariant molecular signatures, usually microbial in origin, known as pathogen-​associated molecular patterns. (2) Natural killer family of receptors—​these largely have spe- cificity for self or altered self molecules (e.g. recognizing conserved features of human leucocyte antigen class 1 molecules; can be either stimulatory or inhibitory). Soluble mediators—​these include (1) complement; (2) defensins—​ typically small microbicidal proteins, with other actions to stimulate both innate and adaptive immune responses; and (3) cytokines—​ frequently act over relatively short distances by binding to cell-​ surface receptors and initiating signalling via intracellular second messengers; play major roles in stimulating immune cell differenti- ation and proliferation. Clinical features of dysregulation of the innate immune system—​ (1) hypofunction—​can result in uncontrolled infections (e.g. in chronic granulomatous disease); (2)  excess activity—​can result in autoinflammatory disease (e.g. periodic fever syndromes); (3) dysfunction—​may contribute to common conditions of multi- factorial aetiology (e.g. Crohn’s disease). Introduction The innate immune system is an evolutionary ancient defence system, with elements present in invertebrates, that mediates defence against microbial pathogens. It is also important in both the priming and the execution of adaptive immune responses. Although it is increasingly appreciated that the innate and adaptive responses are tightly inter- woven, innate immune responses are characterized by rapidity of action and with limited capacity for ‘learning’ or memory. The innate immune system frequently relies upon recognition of conserved molecular fea- tures of microbial pathogens—​pathogen-​associated molecular pat- terns (PAMPs). The innate immune system also recognizes damaged self and absent self, and hence plays an important role in rooting out malignant cells. It is useful to think of the components of the innate system separately. Most cells of the innate immune system are derived from myeloid precursors in the bone marrow. These include mono- cytes and their derivatives—​macrophages and dendritic cells, blood granulocytes (neutrophils, basophils, and eosinophils), and tissue mast cells. Natural killer (NK) and natural killer T (NKT) cells, which are derived from the lymphoid cell lineage, are also included within the in- nate immune system as they lack the clonotypic receptors of lymphoid T and B cells characteristic of the adaptive immune system. An increasing number of receptor recognition systems for non-​ self, damaged self, and missing self are being identified. The para- digm family of innate immune receptors are the Toll-​like receptors (TLRs). These are present in Drosophila melanogaster (fruit flies), where for example the absence of Toll predisposes to overwhelming fungal infection. Important soluble factors include innate immune system cytokines, defensins, and pentraxins. Dysregulation of innate immune responses is increasingly rec- ognized as causing inflammatory human disease. Hypofunction of the innate immune system can result in uncontrolled infections, as seen in chronic granulomatous disease. Excess innate immune ac- tivity can result in autoinflammatory disease; examples of this type of disease include the periodic fever syndromes. Innate immune 4.1 The innate immune system Paul Bowness

308 SECTION 4  Immunological mechanisms dysfunction also contributes to common conditions of multifac- torial aetiology such as Crohn’s disease. Cells of the innate immune system Cells of the innate system are predominantly of the myeloid lineage, and arise from either fetal progenitors or in the bone marrow (see Table 4.1.1 and Fig. 4.1.1). Myeloid cell lineage Mononuclear phagocytes: Macrophages monocytes
and dendritic cells Macrophages and monocytes are related cell types, the former found in tissues and the latter in the blood. Macrophages (‘big eaters’) are able to phagocytose (engulf) large particles including whole bacteria and apoptotic or necrotic dying cells. Phagocytosis is initiated following either recognition of antibody/​complement coating (opsonization), or recognition of PAMPs by PRR such as the mannose receptors. Phagocytosed microbes are usually killed by mechanisms including production of reactive oxygen species. Organisms that are able to survive within macrophages, such as the mycobacterial species responsible for tuberculosis and lep- rosy, can cause major disease. Different types of macrophages are found in different anatomical locations. Recent studies in mice have shown that most tissue macrophages are embryonic- ally seeded into tissues, from yolk sac or fetal liver, where they are capable of self-​renewal. The same is probably true for hu- mans, since for example skin macrophages (Langerhans cells) are found in normal numbers in patients with GATA2 mutations who lack blood monocytes, and are likely therefore to be of distinct (embryonic) origin. Table 4.1.1  Cell types of the innate immune system Myeloid lineage Origin Site-​circulating Site-​tissue resident Monocyte/​macrophage family Largely derived from fetal erythromyeloid progenitors Tissue macrophages including Kuppfer cells (liver), alveolar macrophages (lung) Microglia (CNS) Langerhans cells (skin epidermis) Common myeloid progenitors in the bone marrow Monocytes (blood) Dendritic cells Osteoclasts (bone) Intestinal macrophages Myelocytic family Common myeloid progenitors in the bone marrow Neutrophils Eosinophils Basophils Mast cells Lymphoid lineage Common lymphoid progenitors in bone marrow Natural killer (NK) cells Natural killer T cells (NKT) Dendritic cell Monocyte Lymphocytic lineage (circulating and tissue- resident) Myelocytic lineage Circulating Tissue-resident NK cell NKT cell CD56+, absent CD3 CD56+, CD3+ Basophil neutrophil eosinophil macrophage Cytoplasmic granules a b c Fig. 4.1.1  Cell types of the innate immune system.

4.1  The innate immune system 309 Macrophages are also able to secrete and respond to cytokines (see following paragraphs), and secrete proteases and growth fac- tors important in tissue remodelling and repair. Uptake of modi- fied low-​density lipoprotein (LDL) by macrophages in arterial vessel walls gives rise to foam cell formation and is thought to be critical in the pathogenesis of atherosclerosis. Excessive systemic macrophage activation can result in the life-​threatening haemophagocytic syn- drome seen in children with viral infection and systemic juvenile idiopathic arthritis. The macrophage population within the spinal cord, known as microglial cells, have recently been demonstrated to play a key role in modulation of pain sensation in male but not female mice (where the adaptive immune system plays a major role), both implicating the innate immune system in nociception and pointing to important sex differences. Monocytes are derived from haematopoietic stem cell pre- cursors in the bone marrow and make up 3 to 9% of circulating blood leucocytes in adults. Although lacking cytoplasmic gran- ules, monocytes have lysosomes containing acid phosphatase and express the CD14 and CD68 markers. Monocytes them- selves usually leave the blood within 48 h to further mature and reside in tissues as monocyte-​derived tissue-​resident cells (MCs) with phenotypic and functional properties very similar to macro- phages or dendritic cells (although these monocyte-​derived cells are ontogenically different). For example, recent evidence sug- gests that most ‘macrophage-​like’ cells in the intestine are such monocyte-​derived cells. Recently monocytes have been shown to mediate the phenomena of both innate immune ‘priming’ and postsepsis immunoparalysis. The former denotes a form of innate immune memory whereby previous exposure to microbial products results in enhanced im- munity, and the latter reduced responsiveness. Immune priming can be induced with the fungal product β glucan. Immunoparalysis can follow exposure to bacterial lipopolysaccharide (LPS). This endotoxin-​induced tolerization is mediated by epigenetic mech- anisms and manifested by altered metabolic pathways. Thus, these novel examples show how programming of the innate immune system results in its ability to ‘learn’ from environmental challenges. It is likely that further examples of epigenetic modification of innate immune responses will be shown in future to result in innate im- mune memory. Dendritic cells are now recognized as vital players in the im- mune system. Although immature dendritic cells are largely tolerogenic, mature dendritic cells are the most potent known stimulators of immune responses. Dendritic cells are characterized by their ability to produce long cellular extensions known as den- drites (see Fig. 4.1.1). Dendrites are important both for sampling their environment for antigens and danger signals and for con- tacting other cell types. Immature dendritic cells patrol the tissues and transduce danger signals through recognition of PAMPs by PAMP receptors. These serve to drive both maturation of dendritic cells as well as their migration to adjacent lymphoid tissues, such as draining lymph nodes, where priming of adaptive immune re- sponses occurs. A programme of cellular and molecular changes occurs, outlined in Table 4.1.2, which facilitate this migration and immune stimulation. Thus, for example, expression of the CCR7 receptor facilitates homing to lymph nodes or spleen. Mature den- dritic cells show reduced antigen uptake but increased expression of HLA class II molecules, carrying antigen already taken up in the periphery for presentation to T cells. The HLA/​antigen com- plex provides ‘signal 1’ and the costimulatory molecules CD80 and CD86 (formerly known as B7.1 and B7.2) give ‘signal 2’ to initiate T-​cell adaptive immune responses. It has recently become clear that several different types of den- dritic cells exist, with differing functional properties, as shown in Table 4.1.2. The two principal types of dendritic cells are classical (formerly known as myeloid) and plasmacytoid dendritic cells. Classical dendritic cells are derived from common myeloid pro- genitors in the bone marrow and are now themselves recognized to include various subpopulations, their subsequent phenotype and behaviour exhibit plasticity depending on tissue environmental factors. Plasmacytoid dendritic cells (pDCs) are now thought to originate from pre-​pDC which derive from common dendritic cell precursors under the stimulus of FLT3 ligand. Plasmacytoid dendritic cells produce both α-​ and β-​type 1 interferons, which have potent antiviral properties. Their likely role in defence against viral infections is supported by their expression of the Toll-​like re- ceptor TLR9 (see following paragraphs). Excessive α-​interferon production by plasmacytoid dendritic cells has been implicated in the pathogenesis of skin psoriasis and juvenile systemic lupus erythematosus. Polymorphonuclear phagocytic cells or granulocytes Neutrophils, eosinophils, and basophils comprise the polymorpho- nuclear cells, all of which, together with monocytes, are capable of phagocytosis. Neutrophils are the most abundant white cells in the blood, and also the principle component of pus. They can rapidly leave the circulation to migrate to areas of inflammation. They are drawn down concentration gradients of cytokines such as interleukin (IL)-​8 and γ-​interferon (see next) in a process known as chemotaxis. Neutrophil granules contain abundant toxic defensins, cathepsins, and enzymes such as elastase. Deficiency of the NADPH oxidase en- zyme (EC 1.6.3.1) in individuals (usually young males) with chronic granulomatous disease results in inability of phagocytes to generate Table 4.1.2  Human dendritic cell subtypes Cell type Example Function Phenotypic markers Classical DC Immature Lung CD103+ DC Antigen surveillance and capture by endocytosis and phagocytosis CD11c, CD1a Mature Lymph node DC Stimulation of adaptive immune response Type 3 (lambda) interferon production CD11c, CCR7, HLA class 2, CD40, CD80/​86 Plasmacytoid pDC Type 1 interferon production CD123 hi, lack CD11c

310 SECTION 4  Immunological mechanisms superoxide and its bactericidal derivatives peroxynitrite, hydroxyl radicals, and hydrogen peroxide. As a result, bacterial and fungal infections are not cleared and large inflammatory granulomas may form. By contrast, familial Mediterranean fever (OMIM 240100), another genetic disorder (of the pyrin gene) primarily affecting neutrophils, results in excessive inflammatory activity causing peri- tonitis, arthritis, and amyloidosis. Eosinophils are short-​lived gran- ulocytes whose granules stain red on staining with eosin. IL-​5 is a key mediator of eosinophil activation. Eosinophils are important in combating parasitic infections but are also implicated in asthma and allergy. Basophils are the least common form of granulocyte. Basophils are capable of releasing histamine and cytokines including IL-​4 (see next). Mast cells Mast cells, also known as mastocytes, are tissue-​resident granulo- cytic cells that may originate from a distinct lineage. They are found in the skin, lungs, and gastrointestinal tract and express the high-​ affinity receptor for IgE (FcεRI). Mast cells play an important role in allergy, anaphylaxis, and immunity to parasites. Mast cells coated with antigen-​specific IgE release granules containing histamine, cytokines, and eicosanoids upon antigen binding. The ‘weal and flare’ reaction is an example of such a response. Lymphoid cells of the innate immune system NK cells Natural killer or NK cells are a small but important blood lympho- cyte population (c.2%), distinct from T cells and B cells. They do not express the T-​cell receptor for antigen (or CD3), nor the surface immunoglobulin B-​cell receptor, and in contrast to adaptive im- mune responses mediated by T cells, have the ability to kill target cells without prior sensitization. This is known as ‘natural’ killing. They therefore play a key early defence role against many infectious pathogens. NK cells are also currently being used in clinical trials as adoptive cellular immunotherapeutics against numerous cancers. NK cells can be activated by NK receptors (see next), by the binding of antibody–​antigen complexes to their Fc receptors, and by inter- ferons and cytokines. Human NK cells are classified into two populations according to the intensity of CD56 (neural cell adhesion marker, NCAM) sur- face expression, as well as possession of CD16, the FcγIII receptor. CD56dim CD16bright make up approximately 90% of circulating NK cells and CD56bright CD16negative/​dim comprises the remaining 10%. By contrast, CD56bright NK cells predominate in lymph nodes and sites of inflammation. CD56bright NK cells produce abundant cyto- kines (e.g. γ-​interferon) and have immunoregulatory function, while CD56dim play a key role in natural and antibody-​mediated cell cytotoxicity. They are capable of rapidly killing infected or ma- lignant cells, sharing with cytotoxic (CD8) T cells the ability to induce apoptosis, and the cytolytic granules containing perforin and granzymes. The cytotoxic activity of NK cells is controlled by a balance of stimulatory and inhibitory receptors. Stimulatory re- ceptors include the natural cytotoxicity receptors, some of which recognize microbial products, and some of the NK family of re- ceptors (NKRs) including NKG2D, described later. Almost all NK cells also express inhibitory receptors for self HLA (usually HLA class 1 with or without self peptide), which serve to limit killing of self cells under normal circumstances. Consequently, NK cells have the ability to recognize absence of self or ‘missing self’. NK cells have a major role in the early innate immune response to viruses, and can also kill antibody-​coated cells through their FcγR3 receptors. Innate Lymphoid Cells Innate lymphoid cells (ILC) are a recently described family of tissue resident innate lymphocytes. ILC are related to NK cells, lack classical T or B cell lineage markers, and play important roles in lymphoid or- ganogenesis, tissue homeostasis and local tissue immune responses. NKT cells NKT cells are a minor population of lymphocytes (0.2% of periph- eral blood lymphocytes) that coexpress both NK markers including CD56 and the T-​cell receptor for antigen (TCR). They recognize foreign or self glycolipids presented by the non​polymorphic major histocompatibility complex (MHC) class 1-​like molecule CD1. NKT cells can recognize relatively conserved glycolipids derived from bacteria and parasites, although the best-​character- ized ligand, α-​galactosylceramide, is derived from a sponge. These glycolipids are bound and ‘presented’ by CD1 to the NKT cell TCR. Two types of human NKT cells are currently distinguished. Type 1 or iNKT express an invariant T-​cell receptor (using the TCR α-​ chain AV24AJ18) and recognize α-​galactosylceramide presented by CD1d. Type 2 NKT express variable TCRs and are CD1-​re- stricted but do not respond to α-​galactosylceramide, presumably recognizing distinct glycolipids. Upon activation NKT cells pro- duce IL-​4, γ-​interferon and granulocyte colony stimulating factor (G-​CSF; see next). The function of NKT cells is currently under intense investigation, with recent evidence in a murine model for a role in causing asthma. They may also play a role in immunity to tumours, and it is possibly relevant that the glycolipids lysosomal glycosphingolipid iGb3 and ganglioside GD3 are overexpressed by melanoma cells. Receptors of the innate immune system Unlike the adaptive immune system, the innate immune system uses a relatively small repertoire of germline-​encoded largely non-​rearranging receptors. Charles Janeway first proposed that conserved molecular patterns in microbes would be recognized by pattern recognition receptors (PRRs). These PAMPs would be both essential for the pathogen and distinct to host molecules. Recognition of such PAMPs is increasingly seen as a major func- tion of the innate immune system. It is now recognized that innate immune receptor recognition systems can also have specificity for damaged self (e.g. necrotic cells) and missing self. The term DAMPS is used interchangeably to signify both damage-​ or danger-​ associated molecular patterns. Recognition of PAMPs or DAMPS by the innate immune system—​the immunological ‘danger’ signals proposed by Matzinger—​provides a key trigger in initiating both in- nate and adaptive immune responses. ‘Missing self’ is detected by loss of the inhibitory signals provided by receptors for self molecules including those for self HLA molecules. The TLRs principally recog- nize PAMPs; another very different grouping of receptors, the NKRs frequently recognize self and altered self.

4.1  The innate immune system 311 Toll-​like receptors TLRs are transmembrane receptors, largely expressed at the cell sur- face, that recognize conserved microbial and, to a lesser extent, self molecules. These include conserved nucleic acids, lipoproteins, and lipopolysaccharides. The principle TLRs and their ligands are shown in Fig. 4.1.2. One of the most important TLRs is TLR4, which was identified by Beutler and colleagues as a critical component of the receptor for bacterial lipopolysaccharide (LPS). LPS is a major com- ponent of the outer cell wall of Gram-​negative bacteria, (and hence known as an endotoxin), and is the principle cause of the fever associ- ated with Gram-​negative bacterial infection. Mice with a natural mu- tation in TLR4 exhibit both increased susceptibility to Gram-​negative bacterial infection and resistance to LPS-​induced fever. TLR4 is part of a cell-​surface receptor complex, which includes CD14, the secreted helper molecule MD2, CCR5, and the intracellular signalling adaptor protein MyD88. In addition to bacterial LPS and certain viral pro- teins, some self molecules including heparan sulphate, fibrinogen, and hyaluronan fragments can signal through TLR4. Other TLRs, illustrated in Fig. 4.1.2, include TLR3 and TLR10, which recog- nize viral double-​stranded RNAs. Another bacterial component, flagellin, is recognized by TLR5. TLR9 recognizes unmethylated CpG dinucleic acids, common in bacteria but very rare in mamma- lian DNA. Modulation of immune responses through therapeutic use of TLR ligands has huge potential for human therapy (e.g. the TLR7/​8 ligand imiquimod is used in the treatment of skin malignancy). Natural killer receptors (NKRs) The term NKR loosely describes several groups of receptors that are frequently but not uniquely expressed on NK cells. These receptors can be either stimulatory or inhibitory and can recognize either self or foreign antigens. Killer cell immunoglobulin-​like receptors (KIR) recognize groups of HLA class 1 molecules, as illustrated in Table 4.1.3. KIRs can have either inhibitory functions, mediated through immunoreceptor tyrosine-​based inhibitory motifs, or stimulatory functions mediated by adaptor proteins. Many KIRs have numerous different allelic variants. The KIR genes are located on chromosome 19q13.4 and are in linkage disequilibrium, thus a group of different variants are commonly inherited together, with two major haplotypes recently recognized. Allelic forms have re- cently been implicated in HIV progression to AIDS, and in suscep- tibility to autoimmune arthritis including psoriatic arthritis. In HIV progression and psoriatic arthritis, it is the inheritance of a specific combination of KIR allele with HLA allele that determines disease progression/​susceptibility. Leucocyte immunoglobulin-​like recep- tors (LILR), formally known as ILTs, are generally inhibitory, are expressed on a group of leucocytes and have broader specificity for most class 1 HLA molecules. NKG2D is important in cancer sur- veillance, at least in murine studies, and recognizes the MHC-​like invariant molecules MICA and B. The natural cytotoxicity recep- tors are activatory molecules with poorly defined ligands that are also implicated in recognition of malignant cells. The outcome of an interaction of an NK cell with a potential target is likely to depend on the net balance of positive and negative signals. Other cell-​associated receptors The discovery of new PRRs is occurring rapidly. Emerging cyto- plasmic receptors of importance are the NLRs (nucleotide-​binding domain, leucine-​rich repeat) and the viral RNA sensors, retinoic TLR agonists Leucine-rich repeats Plasma membrane Chromosomal location TIR domain TLR1 4 TLR2 4 TLR6 4 TLR3 4 TLR4 9 TLR5 1 TLR7 X TLR8 X TLR10 4 dsRNA ssRNA miR-21 TLR9 3 CpG DNA dsRNA MALP-2 (mycoplasma) LAM (mycobacteria) Lipoproteins (Gram- bacteria) Zymozan (yeast) LPS (Gram–bacteria) Protein F (RSV) Hsp 60 (host-derived) Fibronectin (host-derived) Hyluronan (host-derived) ssRNA Flagellin Spaetzle Toll Drosophila Fig. 4.1.2  Toll-​like receptors and their ligands.

312 SECTION 4  Immunological mechanisms acid-​inducible gene I (RIG-​I, and other RIG-​1-​like helicases) and melanoma differentiation-​associated gene 5 (MDA5) which are important in responses to pathogens. The NLR family is large and increasing and includes the NOD, NALP, NAIP, and CIITA subfam- ilies. NOD2 variants are associated with Crohn’s disease. Scavenger receptors (class A–​H), sialic-​acid-​binding Ig-​like lectins (Siglecs), and C-​type lectins (e.g. DC-​SIGN and mannose receptor) also have roles in recognition of pathogen determinants as well as some host molecules. Soluble factors Complement The complement proteins comprise a vital arm of the innate im- mune response described in detail in Chapter  4.2. The classical, lectin, and alternative pathways comprise cascades that ultimately activate the membrane attack complex resulting in lysis of targeted cells. Covalent attachment of activated C3 to microorganisms is a key signal to the innate immune system to take up and destroy for- eign material. The complement pathway is particularly important in immune responses to polysaccharide antigens. Defensins These are small microbicidal proteins of usually 29 to 40 amino acids. α-​Defensins are largely stored in the granules of neutrophils and, to a lesser degree, macrophages. Once released they exert direct antimicrobial (including anti-​HIV) activity, and can also induce mast cell degranulation and attract both naive T cells and imma- ture dendritic cells. β-​Defensins are chemotactic for immature den- dritic cells and memory T cells bearing CCR6. Other peptides with antiviral, antibacterial, or antifungal activity include cathelicidin, histatins, cathepsin G, azurocidin, chymase, eosinophil-​derived neurotoxin, and lactoferrin. Mannose-​binding lectin (MBL) MBL is a member of the collectin subfamily of C-​type lectins. MBL and the related surfactant proteins A and D have an antimicrobial role in pulmonary defence against bacterial infections. MBL initi- ates the lectin pathway of complement activation following binding to mannose, N-​acetylglucosamine, fucose, or glucose residues on microorganisms. Pentraxins The pentraxins are a family of proteins with a ring structure made up of five monomers. They include C-​reactive protein, a liver-​ derived acute-​phase protein, induced by inflammatory cytokines such as IL-​1 and IL-​6, which protects against endotoxin-​mediated mortality in animals. Cytokines Cytokines are a group of proteins important in host defence that are secreted by cells of the innate and adaptive immune systems. The first to be described was interferon, a compound produced by vir- ally infected tissue that ‘interfered’ with subsequent viral infection of uninfected tissue. Several overlapping terminologies are used to describe groups of cytokines, based upon their function. Thus, monokines are made by monocytes, and lymphokines by lympho- cytes; chemokines are cytokines with chemotactic activities; and interleukins are cytokines made by one leucocyte and acting on other leucocytes. Cytokines commonly have autocrine actions on the cells that secrete them, and paracrine actions on nearby cells. Cytokines are synthesized de novo in response to specific stimuli, frequently act over relatively short distances, and bring about their effects by binding to cell-​surface receptors and initiating signalling via intracel- lular second messengers. Cytokines frequently exhibit redundancy and pleiotropy (i.e. one cytokine can act on different cell types). Cytokine receptors fall into several categories: haematopoietin re- ceptors such as the IL-​2 receptor; tumour necrosis factor (TNF) family receptors; interferon and chemokine receptors (see next). Many cytokines play major roles in stimulating immune cell dif- ferentiation and proliferation. The functions of some of the major cytokines are briefly summarized in Table 4.1.4. Cytokines are in- creasingly being targeted in human disease therapy, as their roles are elucidated. Thus, tumour necrosis factor alpha (TNFα), also de- scribed as cachexin, is now known to play a central role in the joint pathology, malaise, and systemic features of rheumatoid arthritis as well as other inflammatory arthropathies including psoriatic arth- ritis, ankylosing spondylitis, and Crohn’s disease. Treatment with monoclonal anti-​TNF antibodies or recombinant TNF receptors is highly effective in most patients. IL-​1 has been shown to be im- portant in Muckle–​Wells syndrome and treatment with interleukin receptor antagonists is effective. Table 4.1.3  Natural killer (NK) and related innate immune receptors Receptor Type Cellular expression Ligand KIR Ig NK and T cells HLA class 1, specific alleles (e.g. KIR3DL1 recognizes
HLA-​B27 and related HLA-​B alleles) LILR Ig Monocytes, DC, B HLA class 1, general NKG2D Lectin activatory All NK/​some T MICA/​B NKG2A/​CD94 Lectin NK/​T HLA-​E with HLA-​derived peptide Natural cytotoxicity receptors (NCR) NKp30, 46 Ig NK Malignant cells NKp44 Ig Activated NK Malignant cells NKp80 Ig NK AICL on monocytes Ig denotes immunoglobulin family.

4.1  The innate immune system 313 Chemokines These are small (c.8–​10 kDa) glycoproteins, which are usually proinflammatory and result in cellular attraction along concentra- tion gradient. Four major groups are recognized, C, CC, CxC, and CxxxC, based on the relative separation of their two N-​terminal cysteines. CC and CxC are the major groups, C and CxxxC having only one member each, lymphotactin and fractalkine, respectively. Most CxC chemokines are chemoattractants for neutrophils me- diated by an ELR motif adjacent to the cysteines (e.g. IL-​8, which induces migration from the bloodstream into tissues). By contrast, CC chemokines attract lymphocytes, monocytes, basophils, and/​or eosinophils. Examples of CC chemokines are MCP-​1, RANTES, and MIP-​1α (CCL3). Monocyte chemoattractant protein MCP-​1 (CCL2) is a potent monocyte chemoattractant that induces monocyte migra- tion from the bloodstream into tissues and subsequent maturation into macrophages. Chemokine receptors have seven transmembrane helices and signal through intracellular G proteins. CC chemokines bind to CC chemokine receptors, and CxC chemokines bind to CxC chemokine receptors, of which at least seven are described. The chemokine receptors CCR5 and CxCR4 are also coreceptors for HIV infection of macrophages and CD4 T cells. Interferons The interferons are a group of cytokines with potent antimicrobial and antiproliferative effects that are produced in response to prod- ucts of bacterial or viral infection, such as double-​stranded RNAs, cytokines, or mitogens. They have multiple effects, characteristic- ally mediated through JAK-​Stat pathways, that include upregulation of HLA class 1 and 2 expression, and generally have potent anti- viral activity. Type 1 interferons include α-​interferons produced by lymphocytes (of which at least 13 are recognized), β-​interferons produced by fibroblasts, and others cell type. All type 1 interferons bind to a unique cell-​surface receptor, the interferon-​α receptor. Only a single type 2 interferon is recognized, γ-​interferon. This is Table 4.1.4  Production and action of selected cytokines Cytokine Cell source Target cell Action GM-​CSF Th cells Myeloid progenitor cells Growth and differentiation Monocytes Inflammation Il-​1α Monocytes T, B NK, other Stimulation IL-​1β Macrophages Fever, inflammation Dendritic cells B lymphocytes IL-​2 Th1 CD4 T T, B, NK Activation, proliferation IL-​4 Th2 CD4 T T, B, macrophages Activation, proliferation, class switching IL-​6 Monocytes B, plasma cells Differentiation, antibody secretion Macrophages, etc. Il-​8 Macrophages Neutrophils Chemotaxis Endothelial cells IL-​12 Macrophages NK cells Activation IL-17 T cells Many Inflammation Host defense Gut homeostasis IFN-​α Leucocytes Many Inhibition of viral replication Increased HLA class 1 expression IFN-​β Fibroblasts, pDCs Many Inhibition of viral replication Increased HLA class 1 expression IFN-​γ Leucocytes Many Inhibition of viral replication Increased HLA class 1 expression Macrophage activation T-​cell proliferation B-​cell class switching MIP-​1α (CCL3) Macrophages Monocytes, T cells Chemotaxis MIP-​1β (CCL4) Lymphocytes Monocytes, T cells Chemotaxis TNFα Macrophages Macrophages Cytokine production including IL-​1 NK, lymphocytes Tumour killing TGFβ Monocytes, T lymphocytes Monocytes, T cells Cell differentiation and proliferation GM-​CSF. granulocyte macrophage colony stimulating factor; IFN, interferon; IL, interleukin; TGF, tissue growth factor.

314 SECTION 4  Immunological mechanisms produced by lymphocytes and activated NK cells, and binds to the type 2 interferon receptor. γ-​interferon has a variety of activities including macrophage activation. Type 3 (λ) interferons have re- cently been recognized. Exogenously administered α-​interferons 2a and 2b, which can be PEGylated to prolong their action, are effective in the treatment of hepatitis C infections, acting to greatly reduce viral replication. Interferons are also used therapeutically in other viral infections, including hepatitis B. FURTHER READING Banchereau J, Steinman RM (1998). Dendritic cells and the control of immunity. Nature, 392, 255–​62. Bedoui S, Gebhardt T, Gasteiger G, Kastenmüller W (2016). Parallels and differences between innate and adaptive lymphocytes. Nat Immunol, 17(5), 490–4. Beutler B, et al. (2006). Genetic analysis of host resistance: toll-​like receptor signalling and immunity at large. Annu Rev Immunol, 24, 353–​89. Branzk N, Gronke K, Diefenbach A (2018). Innate lymphoid cells, mediators of tissue homeostasis, adaptation and disease tolerance. Immunol Rev, 286(1), 86–101. Farag SS, Caligiuri MA (2006). Human natural killer cell development and biology. Blood Rev, 20, 123–​37. Feldmann M, Maini RN (2003). TNF defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases. Nat Med, 9, 1245–​50. Ganz T (2003). Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol, 3, 710–​20. Gomez Perdiguero E (2015). Tissue-​resident macrophages originate from yolk-​sac-​derived erythro-​myeloid progenitors. Nature, 518, 547–​51. Guilliams M, et al. (2014). Dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny. Nat Rev Immunol, 14, 94–​108. Janeway C, et al. (2005). Immunobiology: the immune system in health and disease. Churchill Livingstone, Edinburgh. Marcus A, et al. (2014). Recognition of tumors by the innate immune system and natural killer cells. Adv Immunol, 122, 91–​128. Ng LG, Ostuni R, Hidalgo A (2019). Heterogeneity of neutrophils. Nat Rev Immunol, doi: 10.1038/s41577-019-0141-8. Plüddemann A, et al. (2006). The interaction of macrophage receptors with bacterial ligands. Exp Rev Mol Med, 8, 1–​25. Pure E, Allison JP, Schreiber RD (2005). Breaking down the barriers to cancer immunotherapy. Nat Immunol, 12, 1207–​10. Saeed S, et  al. (2014). Epigenetic programming of monocyte-​to-​ macrophage differentiation and trained innate immunity. Science, 345, 1251086. Sorge RE, et al. (2015). Different immune cells mediate mechanical pain hypersensitivity in male and female mice. Nat Neurosci, 18, 1081–​3. Steinman R, Cohn Z (1973). Identification of a novel cell type in per- ipheral organs of mice. J Exp Med, 137, 1142–​62. Takeda K, Kaisho T, Akira S (2003). Toll-​like receptors. Ann Rev Immunol, 21, 335–​76. Wagner AK, Alici E, Lowdell MW (2019). Characterization of human natural killer cells for therapeutic use. Cytotherapy. doi: 10.1016. Wantanabe Y (2004). Fifty years of interference. Nat Immunol, 5, 1193.

4.2 The complement system 315

4.2 The complement system 315

ESSENTIALS The complement system, consisting of soluble and membrane-​ bound proteins, is a major effector mechanism of host defence against infection and inflammatory responses. It has an important role in the removal of immune complexes and dying cells, and also modulates humoral and cell-​mediated immune responses. Complement activation and regulation Complement activation occurs through three pathways, each generating enzyme complexes, termed C3 convertases. These cleave native C3 to form C3b and C3a. C3b can covalently attach to sur- faces (e.g. pathogen surfaces) where it triggers biological responses following interaction with membrane-​bound receptors and can also trigger cleavage of native C5 to C5b and C5a. C5b triggers the for- mation of the membrane attack complex, which disrupts target cell membrane integrity and may result in cell lysis. The physiological role of complement requires that activation specifically occurs upon non​host or altered host surfaces, which is achieved by recognition proteins (e.g. C1q, mannose-​binding lectin) within the activation cascades (recognition proteins). Complement in disease Disease may arise from:  (1) impaired complement activation, re- sulting in immunodeficiency, autoimmune disease, and/​or develop- mental abnormalities; (2) defective complement regulation, resulting in abnormal complement activation on host surfaces, leading to con- ditions including paroxysmal nocturnal haemoglobinuria, atypical haemolytic uraemic syndrome, and C3 glomerulopathy. Hereditary angio-​oedema is associated with deficiency of C1 inhibitor. Measurement of complement in clinical practice The most useful and widely available complement assays are anti- genic measurements of serum C4 and C3: hypocomplementaemia is typical in systemic lupus erythematosus. Functional tests are im- portant when immunodeficiency is suspected (e.g. recurrent pyo- genic infections in childhood). Complement therapeutics Eculizumab is a monoclonal antibody that blocks C5 activation pre­ venting formation of both C5a and the membrane attack complex. It is licensed for the treatment of transfusion-​dependent haemolytic anaemia in paroxysmal nocturnal haemoglobinuria, generalised my- asthenia gravis and atypical haemolytic uraemic syndrome. Introduction Complement was discovered as a heat-​labile plasma component that augmented (‘complemented’) antibody-​mediated killing of bacteria. It consists of a large number of plasma and cell-​bound proteins that interact with each other in an enzymatic cascade (Fig. 4.2.1). We limit our review of complement biology to aspects relevant for the clinician to understand complement-​associated diseases. There are three activation pathways: the classical pathway, the al- ternative pathway, and the lectin pathway. The classical pathway is a predominantly antibody-​dependent pathway. It is activated by ag- gregated IgG or IgM in immune complexes. The lectin pathway is antibody-​independent and is initiated by the binding of pattern rec- ognition molecules to, for example, mannose-containing structures on bacteria. The pattern recognition molecules include mannose- binding lectin (MBL), ficolins (ficolin 1, -2 and -3), collectin kidney 1 (CL-K1, collectin 11) and collectin liver 1 (CL-L1, collectin 10). The alternative pathway is constitutively active. Complement activation results in the generation of enzymatically active complexes (termed convertases) that cleave C3 and C5. Activated C3 (termed C3b) can be rapidly amplified through a posi- tive feedback cycle, termed the C3b amplification loop. Amplification of C3b can occur irrespective of which pathway generated the C3 convertase. Activation of C5 triggers the formation of the membrane attack complex (MAC), A large array of regulatory proteins (com- plement regulators) prevent inappropriate complement activation and limit host surface damage. Complement biological roles A wide range of roles has been ascribed to the complement system, but they can be categorized into two main activities:  (1) the de- struction/​removal of anything that is recognized as foreign or is not 4.2 The complement system Marina Botto and Matthew C. Pickering

316 SECTION 4  Immunological mechanisms adequately protected by soluble or cell-​associated complement regu- lators; and (2) the modulation of humoral and cell-​mediated immune responses. There is evidence of unrelated or alternative roles of the complement system outside immunity, mainly in regenerative and developmental processes (see following paragraphs regarding defects in the lectin components). The most important function of complement is the host defence against infectious disease. Complement provides mechanisms for the killing and clearance of microorganisms. It does this by the co- valent binding to their surface of complement fragments (e.g. C3b) that are ligands for receptors on phagocytic cells that ingest and kill the microorganisms. The activation of complement also causes the generation of anaphylatoxins (e.g. C5a and C3a), which have chemo- tactic activity and recruit leucocytes to sites of infection and inflam- mation. These small complement fragments signal through specific receptors (C5aR and C3aR) and attract neutrophils and monocytes to the site of complement activation. A further role of complement in host defence against infections is the generation of the MAC, which may disrupt the cell membrane and kill the microorganism. As part of its clearance role the complement system promotes the non​inflammatory disposal of dying cells and immune complexes. It is in this role that complement may prevent the development of systemic lupus erythematosus (SLE). Activation of complement by immune complexes facilitates the clearance of antigen and thereby helps to prevent immune complexes from causing inflammatory damage to tissues, although, as outlined to follow, complement may also contribute to inflammatory tissue injury in circumstances when immune complexes persist. The other main activity of complement is bridging innate and adaptive immunity. Activation of complement augments antibody responses and thereby enhances host defence against pathogens. The binding of complement to antigens reduces the threshold for B-​cell activation and enhances antigen presentation and B-​cell memory. Similarly, complement can modulate the T-​cell responses control- ling T-​cell homeostasis and activation. Complement activation pathways and regulation Classical pathway The initial step in classical pathway activation (Fig. 4.2.2) is the binding of C1q, the first component of this pathway, to the Fc portion of antibodies complexed with antigens. The various IgG isotypes have different capacities to bind to and activate C1q. In humans, IgG3 is the most potent activator, followed by IgG1 and then IgG2. IgG4 does not bind C1q and cannot activate the classical pathway. C1q also binds to the CH3 domain of IgM that has adopted a staple configuration fol- lowing binding of antigen. The general order of complement-​fixing potential to human antibodies is thus IgM > IgG3 > IgG1 > IgG2 >> IgG4. IgA can activate the alternative pathway, whereas IgE is not an effective complement-​activating isotype. The classical pathway can also be triggered in an antibody-​ independent manner by the binding of C1q directly to the surface of certain pathogens or host proteins such C-​reactive protein bound to its ligand or amyloid fibrils. C1q is part of a complex, the C1 complex, which includes two serine proteases (C1s and C1r). Once activated, ALTERNATIVE LECTIN CLASSICAL PATHWAY TRIGGERS C3 CONVERTASE C3b C3a C5 CONVERTASE C5a C5b TERMINAL MAC C3b AMPLIFICATION Fig. 4.2.1  Schematic depiction of the complement system. There are three activation pathways: the classical, lectin, and alternative pathways. The classical and lectin pathways are activated by specific triggers; the alternative pathway is constitutively active. On activation the pathways result in the formation of an enzyme complex termed C3 convertase. The C3 convertase proteolytically cleaves C3 to form the anaphylatoxin C3a and the opsonin C3b. Further generation of C3b occurs through a positive feedback loop termed the C3b amplification pathway. The addition of another molecule of C3b to a C3 convertase results in a complex (C5 convertase) that can proteolytically cleave C5. The C5 convertase generates the anaphylatoxin C5a and C5b. The C5b molecule triggers formation of the terminal pathway which, through the sequential addition of C6, C7, C8, and multiple C9 molecules, results in the membrane attack complex (MAC, also denoted C5b-​9).

4.2  The complement system 317 the C1 complex acts on the next two components of the classical pathway, cleaving C4 and then C2, to generate the classical pathway C3 convertase (C4b2a). This surface-​bound convertase cleaves C3 resulting in the generation of C3b, which coats the activating sur- face, a phenomenon termed opsonization. The activation of C3 is followed by the formation of a multiprotein complex that cleaves C5. Alternative pathway The main feature of the alternative pathway (Fig. 4.2.2) is that is in a constant state of activation or ‘tick-​over’ that results in the generation of low levels of activated C3 fragment (C3b) in the fluid phase. Most of this fluid-​phase C3b is rapidly inactivated by hydrolysis, but a small amount can bind to surfaces in the immediate vicinity of the C3 acti- vation and initiate the amplification of the alternative pathway. The fate of surface-​bound C3b is controlled by two mechanisms. The first mechanism is the presence of membrane-​bound comple- ment regulatory molecules, which serve to protect host cells by inhibiting further C3 activation. The second mechanism depends on the affinity of the surface-​bound C3b for factor H. Factor H is the major fluid-​phase regulator of C3 activation but also is an important regulator of surface-​bound C3b, particularly along the renal endo- thelium. Factor H binds preferentially to C3b bound to vertebrate cells as it has a high affinity for the sialic acid residues present on these cells. In contrast, pathogen surfaces lack sialic acid residues, rendering the bound C3b resistant to inactivation by factor H and allowing amplification to proceed. The alternative pathway hence works through an amplification loop (Fig. 4.2.3) that can proceed efficiently on the surface of a pathogen, but not on a host cell. The same amplification loop enables the alternative pathway to amplify complement activation initially triggered through the classical or the lectin pathway (C3b amplification loop, Fig. 4.2.1). Lectin pathway The lectin pathway is initiated by the binding of MBL, ficolins 1–​3, or CL-L1 and CL-K1 Fig. 4.2.2) to targets on the surface of pathogens or damaged tissue. All these proteins belong to a family of collagenous lectins, named collectins, which are capable of rec- ognizing carbohydrate domains. After recognition, initiation pro- ceeds through the activation of serine proteases, known as MASPs (mannose-​binding lectin-​associated serine proteases). The activa- tion of the MASP system, which contains three different enzymes—​ MASP-​1, MASP-​2, and MASP-​3, also a protein with no proteolytic activity named MAP19 or sMAP—​results in cleavage of C4, C2, and then C3 in a similar way to the classical pathway. The relationship between the lectin pattern recognition molecules and the MASPs is complex and incompletely understood. MASP-3 contributes to alternative pathway activation since it is required for activation of factor D (i.e. its conversion from pro-Factor D to active Factor D). ALTERNATIVE LECTIN CLASSICAL C1q C1r, C1s C4 C2 Mannose-binding lectin (MBL) Ficolin-1, Ficolin-2, and Ficolin-3 CL-K1 and CL-L1 MBL-associated serine protease (MASP)-1, -2, -3 MAp19, MAp44 C4 C2 Hydrolysed C3 Factor B Factor D Properdin (P) Immune-complex (antibody-antigen complex) Carbohydrate or acetyl patterns Spontaneous Main pathway triggers: Pathway components: Recognition molecules: Other components: C4b2a C3bC4b2a C4b2a C3bC4b2a C3bBb C3bC3bBbP Final enzyme complexes: C3 convertase: converts C3 to C3b and C3a C5 convertase: converts C5 to C5b and C5a Fig. 4.2.2  Complement activation pathways. Examples of the triggers of the three activation pathways are shown. These triggers are recognized by specific proteins within the activation pathways. Examples include the interaction of the classical pathway recognition protein, C1q, with immune complexes; and the interaction of the lectin pathway recognition protein, mannose-​binding lectin (MBL), with carbohydrates on bacteria. There are multiple recognition proteins in the lectin pathway and the biological role of many of these is incompletely understood. Further activation is achieved through the actions of enzymes within the classical (C1r, C1s, C2), lectin (MASP-​1, MASP-​2, MASP-​3, C2), and alternative (factor B, factor D) pathways. The final enzyme complexes are able to proteolytically cleave C3 to C3b and C3a. These complexes are termed C3 convertases. The addition of a further C3b molecule to the C3 convertases enables the resultant complex to cleave C5. These complexes are termed C5 convertases.

318 SECTION 4  Immunological mechanisms Terminal pathway The final phase of complement activation is the assembly and for- mation of the membrane attack complex (Fig. 4.2.4). The end result is a pore in the lipid bilayer membrane, firstly identified in electron micrographs as membrane ‘pores’ and ‘hollow cylinders’ that des- troy the membrane integrity. Structural studies have shown that the MAC has a ‘split-washer’ configuration which partially penetrates the lipid bilayer, resulting in an irregular β-barrel pore. The first step of the terminal pathway is the enzymatic cleavage of C5 to release C5a and a larger fragment, C5b, that binds sequentially and non​enzymatically to the plasma proteins C6, C7, C8, and C9. The polymerization of C9 produces a hydrophobic complex, com- monly denoted as C5b-​9n, where n represents the number of poly- merized C9 molecules that forms ‘pores’ in lipid bilayers resulting in target lysis. The lytic effect of MAC is particularly evident in condi- tions in which red cells are targeted by complement activation and in host defence against Neisseria. Regulation of complement activation Given the destructive effects of complement and the way in which its activation is rapidly amplified through a triggered-​enzyme cascade, it is not surprising that its activation is tightly regulated both in the fluid phase and on cell surfaces, serving not only to prevent tissue damage from autologous complement activation but also to prevent the depletion of complement proteins. The molecular mechanisms utilized to block complement activa- tion include: (i) inhibiting activating proteases such as C1s (see later paragraph on C1 inhibitor deficiency); (ii) acting as competitor/​ decay factor for enzyme complexes; (iii) performing cofactor activ- ities in proteolytic cleavage; and (iv) working as a substrate-​specific protease. The evolution of the complement system has therefore been accompanied by the development of a sophisticated regulatory system consisting of fluid-​phase and membrane-​bound regulatory proteins that act at many steps of the pathway (Fig. 4.2.5). Complement deficiency and disease Disease may arise when there is (1) impaired complement activation or (2) defective complement regulation (Table 4.2.1). Impaired complement activation and disease Increased susceptibility to infections Increased susceptibility to childhood pyogenic bacterial infections is frequently seen in many of the homozygous deficiencies, while heterozygous deficiency is usually asymptomatic. Homozygous defi- ciencies are rare, with the exception of C2, MBL, and (in the Japanese population, where about 1/​1000 are affected) C9. C2 deficiency occurs in 1:20 000 white individuals and is usu- ally asymptomatic. MBL deficiency occurs in 5–​10% of individuals, but increased infection risk is only seen if there are immunosup- pressive comorbidities. Examples include cancer patients receiving chemotherapeutic treatment and organ-​transplant recipients on immunosuppressive medication. Impaired complement activation should be suspected in an individual with recurrent pyogenic child- hood infections, a family history of infection and consanguinity. Patients with hereditary C3 deficiency or with mutations in mol- ecules leading to C3 consumption show increased susceptibility to recurrent and severe bacterial infections, particularly those C3b AMPLIFICATION Factor B Factor D C3 Properdin C3b Pathway trigger: Pathway components: Recognition molecules: Other components: C3bBb Pathway enzymes: C3 convertase: converts C3 to C3b and C3a Fig. 4.2.3  C3b amplification pathway. C3b on a surface can be rapidly amplified through a feedback cycle. This pathway utilizes the same components as the alternative pathway, hence it is sometimes referred to as the amplification loop of the alternative pathway. Surface C3b can interact with factor B to form a pro-​C3 convertase (C3bB). Factor B in complex with C3b can be cleaved by the enzyme factor D. This converts factor B to two fragments termed Bb (an enzymatically active fragment which remains attached to C3b) and Ba, which is released from the complex. This results in the conversion of the pro-​convertase (C3bB) to a convertase (C3bBb). The C3 convertase now cleaves C3 generating further C3b. Note that C3b can be amplified via this pathway irrespective of how it was formed. TERMINAL C6 C7 C8 C9 C5b Pathway trigger: Pathway components: Recognition molecules: Other components: C5b-9 Final complex: Membrane attack complex: Fig. 4.2.4  Terminal pathway. This is triggered by the generation of C5b and the sequential addition of a single molecule of C6, C7, C8, and multiple (n) C9 molecules. This results in the formation of a macromolecular complex (C5b, C6, C7, C8, C9n, typically denoted C5b-​9) termed the membrane attack complex (MAC). The MAC damages cell membranes by creating membrane pores. In cells that are unable to repair the damaged membrane, cell death or lysis may occur. Erythrocytes are particularly susceptible to MAC-​induced lysis since, unlike many nucleated cells, they are unable to actively remove MAC
that has been incorporated into their membranes.

4.2  The complement system 319 caused by encapsulated organisms (e.g. Streptococcus pneumoniae and Staphylococci). Similar infections are seen among individuals lacking antibodies or normal phagocytic function. This indicates that the normal pathway for eradication of these bacteria requires antibody, complement, and phagocytes. In patients with C3 defi- ciency infections become less frequent during adulthood when the protective antibody repertoire has expanded following repeated in- fectious challenges. Eradication of Neisseria requires alternative pathway C3 activa- tion on the surface of the pathogen and subsequent formation of the MAC through the terminal pathway. Homozygous deficiencies of the alternative and terminal pathway components are associ- ated with increased susceptibility to Neisserial infections. Patients treated with the anticomplement C5 antibody (eculizumab) are also at risk of Neisserial infections because eculizumab prevents terminal pathway activation. Susceptibility to meningitis is also influenced by genetic poly- morphism across the complement factor H gene family. This is because some Neisserial strains bind the alternative pathway negative regulator (factor H) and evade complement-​mediated eradication. Autoimmunity There is a strong association between classical pathway deficiency and SLE. Over 95% of homozygous C1q deficient individuals de- velop SLE. The reasons for this association are complex and include abnormal immune complex processing and impaired tolerance to autoantigens through defective clearance of apoptotic cells (see Chapter 19.11.2). The association between C2 deficiency and SLE is much less strong and suspected to be approximately 10%. Developmental abnormalities Mutations in the lectin pathway components, MASP1 and CL-​K1, are associated with a developmental syndrome, termed 3MC (Mingarelli, Malpuech, Michels, Carnevale) syndrome. This condition includes multiple developmental defects including severe growth retardation, facial dysmorphism, and skeletal abnormalities. The contribution of the lectin pathway components to its pathogenesis was unexpected and remains incompletely understood. Defective complement regulation and disease C1 inhibitor deficiency (Also see Chapter 4.5.) Aetiology and pathogenesis The disease hereditary angio-​oedema (OMIM 106100) is caused by deficiency of C1 inhibitor. This is inherited as an autosomal dom- inant disorder with partial penetrance. The disease is dominantly inherited because the production of C1 inhibitor from a single, normal allele is insufficient to maintain normal homeostasis of the complement and kinin pathways. The mutations may have two ef- fects on protein production. In type I  hereditary angio-​oedema, which accounts for approximately 85% of cases of the disease, the mutant prevents any expression of protein from the mutant allele and hence there are reduced levels of C1 inhibitor. Type II heredi- tary angio-​oedema is caused by a series of point mutations in the C1 inhibitor gene that alter one of the amino acids at the active centre of the protein and abolish its activity as a serine proteinase inhibitor. These mutations allow expression of normal amount of protein, ALTERNATIVE LECTIN CLASSICAL PATHWAY TRIGGERS C3 CONVERTASE C3b C3a C5 CONVERTASE C5a C5b TERMINAL MAC C3b AMPLIFICATION CD59 Factor H CD46 (MCP) Factor I Factor H CD55 (DAF) C1INH Fig. 4.2.5  Complement regulation. There is a complex network of proteins in plasma and within cell membranes that negatively regulate complement activation. These proteins act at different steps in the pathways. Key examples are shown. These include the membrane-​bound protein CD59 that prevents the assembly of the MAC. Factor H and CD55 (also termed decay-​accelerating factor, DAF) enhance the disassembly of the C3 convertases. C3b can be proteolytically cleaved by the enzyme factor I to form a product (termed iC3b) that can no longer bind to factor B. The conversion of C3b to iC3b therefore prevents further C3b amplification. Factor I requires cofactors to mediate the cleavage of C3b to iC3b. These cofactors are factor H and CD46 (also termed membrane cofactor protein, MCP), a membrane-​bound cofactor. Factor I and factor H are the key inhibitors of the alternative pathway (see text). C1 inhibitor (C1INH) negatively regulates both the classical and lectin pathways (see text).

320 SECTION 4  Immunological mechanisms which is non​functional, or even abnormally high C1 inhibitor levels, because the mutant protein is not consumed by normal interaction with activated serine proteinases. It is easy to miss the diagnosis of this variant of hereditary angio-​oedema if it is not appreciated that levels of C1 inhibitor can be normal or high in patients with the dis- ease: functional C1 inhibitor assays are required. In normal circumstances, C1 inhibitor binds to and inactivates enzymatically active C1r and C1s. It also inhibits plasmin, kallikrein, and activated coagulation factors XIIa and XIa. Deficiency results in uncontrolled fluid-​phase classical pathway activation and conse- quently reduced levels of both C4 and C2. Acute angio-​oedema attacks are characterized by increased vas- cular permeability at the affected sites. The swellings are believed to be caused by the action of small peptides, called kinins, in par- ticular bradykinin, that induce increased vascular permeability by their actions on vascular endothelium and smooth muscle. These kinins are produced by the action of serine proteinases that are ineffectively regulated in the presence of reduced activity of C1 in- hibitor. Plasmin activation may be important in the precipitation of attacks by consuming the reduced amounts of available C1 in- hibitor in individuals with only half normal functional expression of the protein. Individuals exhibiting clinical features of hereditary angio-​ oedema who have normal C1 inhibitor concentration and function have also been described. This type of hereditary angio-​oedema (OMIM 610 618) has been termed hereditary angio-​oedema type III or, more comprehensively, oestrogen-​related hereditary angio-​ oedema or oestrogen-​sensitive hereditary angio-​oedema. In contrast to hereditary angio-​oedema types I and II, hereditary angio-​oedema type III has been observed exclusively in women, where it appears to be correlated with conditions of high oestrogen levels (e.g. preg- nancy or the use of oral contraceptives). The aetiology of type III hereditary angio-​oedema appears to be heterogeneous, with some patients having gain-​of-​function mutations in F12, the gene Table 4.2.1  Complement deficiency and disease Complement deficiency Phenotype Comments ACTIVATION PROTEINS Classical pathway deficiency: C1q, C1r, C1s, C2, C4 SLE Recurrent encapsulated bacterial infections All extremely rare except C2 deficiency where estimated prevalence is 1:20 000 Association with SLE weakest for C2 deficiency Alternative pathway deficiency: Factor B, factor D Recurrent meningococcal infections Recurrent encapsulated bacterial infections All extremely rare Lectin pathway deficiency: MBL, ficolins 1–​3, MASP-​1, MASP-​2, and MASP-​3, CL-​K1 Increased infection among immunocompromised individuals (MBL deficiency) Ficolin-​3 deficiency associated with necrotizing enterocolitis Mutations in the genes encoding CL-​K1 (COLEC11),
CL-L1 (COLEC10) and MASP-​3 and MASP-​1 (MASP1) associated with an autosomal recessive developmental syndrome termed ‘3MC syndrome’a All extremely rare except MBL deficiency where estimated prevalence is 5% in white populations Terminal pathway C5, C6, C7, C8, and C9 Recurrent meningococcal infections All rare except C9 deficiency in Japanese where estimated prevalence is 1:1000 C3 Recurrent encapsulated bacterial infections Membranoproliferative glomerulonephritis (rare) SLE-​like illness (rare) Extremely rare REGULATORY PROTEINS C1INH Negative regulator of: classical and lectin pathways contact system coagulation system fibrinolytic system Hereditary angioedema Estimated prevalence 1:50 000 Angioedema results from uncontrolled production of bradykinin due to dysregulation of the contact system and does not arise from uncontrolled complement activation. Associated with low C4 due to uncontrolled classical pathway activation Factor H, Factor I, and CD46 Negative regulators of the alternative pathway and C3b amplification loop Atypical haemolytic uraemic syndrome (aHUS) C3 glomerulopathy All rare aHUS manifests in heterozygous deficiency states Complete factor H deficiency associated with C3 glomerulopathy (e.g. dense deposit disease) Factor H-​related protein 5 Putative regulator of C3 processing within the kidney—​biological function incompletely understood C3 glomerulopathy (‘CFHR5 nephropathy’) Rare Predominantly individuals with Cypriot ancestry CD59 Negative regulator of terminal pathway activation Paroxysmal nocturnal haemoglobinuria Rare Acquired somatic mutation Renders CD59-​deficient erythrocytes susceptible to complement-​mediated intravascular haemolysis Properdin Positive regulator of C3 activation Recurrent meningococcal infections Rare X-​linked deficiency MBL, mannose-​associated lectin; MASP-​2, MBL-​associated serine protease; C1INH, C1 inhibitor; CD46, also known as membrane cofactor protein; CL-​K1, also known as collectin-​11. a 3MC syndrome is a term used to describe clinically identical syndromes that were independently described: the Mingarelli, Malpuech, Michels, and Carnevale syndromes.

4.2  The complement system 321 encoding human coagulation factor XII (FXII, or Hageman factor), as a possible cause. Angio-​oedema may be acquired due to the development of auto- antibodies to C1 inhibitor. This may occur in association with lymphoproliferative disease, particularly B-​cell lymphoma. The clin- ical features are similar to hereditary angio-​oedema but with later age of onset. Typically, C4 and C1 inhibitor levels are low. Complement C1q level is reduced in patients with acquired angio-​oedema, but normal in patients with hereditary angio-​oedema. Clinical features and diagnosis Allergy is much more common than hereditary angio-​oedema as a cause of angio-​oedema. In hereditary angio-​oedema, the swelling is not itchy and is not accompanied by other features of allergy such as asthma and urticaria. Oedema can affect any part of the integu- ment but is most common in the extremities. Classically, the oedema and swelling develop gradually over several hours and then subside over 2 to 3 days. Involvement of the upper airways (including the tongue, pharynx, and larynx) may result in life-​threatening airway obstruction. Swelling of the bowel mucosa may produce severe ab- dominal pain, mimicking common surgical emergencies. In general, women have a more severe course of the disease than men. Patients with early onset of clinical symptoms are affected more severely than those with late onset. Diagnosis of hereditary angio-​oedema is made on the basis of the clinical findings described earlier, the presence of family his- tory, and blood tests. A family history of angio-​oedema makes diag- nosis much easier but is not always present because some cases are due to new mutations in the C1 inhibitor gene. In other families, other members with C1 inhibitor deficiency may have no clinical symptoms. All patients who are suspected of having hereditary angio-​oedema should have serum C4 levels measured, which is a good screening test for hereditary angio-​oedema as it is invariably low in untreated patients with the condition. This is because the reduced C1 inhibitor activity allows C1s to cleave C4 and C2 in an unregulated fashion. In patients with type I hereditary angio-​oedema C1 inhibitor pro- tein levels are typically low (usually <30% of normal levels), but they may be normal or high in the 15% of patients with type II disease, and functional assays of C1 inhibitor are necessary to make the diag- nosis. Genetic tests are not indicated routinely and are usually not necessary to confirm the diagnosis of hereditary angio-​oedema. Management Acute attacks of angio-​oedema may be stopped by infusion of puri- fied C1 inhibitor concentrate. Three preparations are available for clinical use: two are derived from plasma (Berinert P and Cinryze) and the third is a recombinant molecule (Rhucin). All are adminis- tered intravenously. An alternative is fresh frozen plasma, but this is less satisfactory because plasma not only contains C1 inhibitor but also kallikrein, C1r, and C1s, which may generate further kinin production. Acute attacks of hereditary angio-​oedema do not respond to adrenaline (epinephrine), though if there is any cause to suspect al- lergic rather than hereditary angio-​oedema, then administration of epinephrine is unlikely to cause any harm and may be life-​saving. C1 inhibitor is rapidly effective in acute attacks of oedema. In one study of laryngeal oedema, the most feared complication, administration of the inhibitor reduced the median duration of the attack from 100+/​–​26 h to 15+/​–​9 h. C1 inhibitor should be given rapidly at the first sign of an attack and prophylaxis considered if attacks are frequent or patients are undergoing procedures that may trigger an attack (e.g. dental procedures). C1 inhibitor levels originating from the single normal allele in- crease in response to treatment with attenuated androgens, such as danazol, stanozolol, and oxandrolone. These are moderately ef- fective treatments, although these compounds retain some virilizing activity. An alternative is the proteinase inhibitor tranexamic acid, which may reduce the consumption of C1 inhibitor by blocking the activity of the serine proteinases that interact with C1 inhibitor. With the knowledge that the pathogenesis of the angioedema results from dysregulated bradykinin production, two other thera- peutic approaches have been utilized:  bradykinin B2 receptor blockade (icatibant, a 10 amino acid peptide) and inhibition of kallikrein (ecallantide, a 60 amino acid protein). Both are effective in acute attacks, can be administered subcutaneously and—​unlike C1 inhibitor preparations—​are effective in type III angioedema. Advice on use of contraceptives and hormone replacement therapy should emphasize avoidance of oestrogen. Angiotensin-​ converting enzyme (ACE) inhibitors need to be avoided because of their effects on the kallikrein–​bradykinin pathway. Angiotensin-​II receptor antagonists may be used with caution in patients with her- editary angio-​oedema. Atypical haemolytic uraemic syndrome (aHUS) (See also Chapter 21.10.6.) aHUS is characterized by renal failure due to thrombotic microangiopathy (TMA). This condition, which is distinct from HUS associated with Shiga toxin-​producing strains of E.  coli, is linked with defective regulation of the alternative pathway along the renal endothelium. The defective regulation in aHUS can be inherited or acquired. Inherited causes include (1) loss-​of-​function mutations in the nega- tive regulators of the alternative pathway—​factor H, factor I, and CD46; and (2) gain-​of-​function mutations in the activation proteins of the alternative pathway—​C3 and factor B. Acquired causes in- clude autoantibodies to factor H. Antifactor H autoantibodies, like aHUS-​associated factor H mutations, impair the ability of factor H to regulate C3 activation along the renal endothelium. Alternative pathway activation triggers C5 activation, which is now known to be critical for the development of the renal TMA. Eculizumab (an anticomplement C5 antibody) is highly effective in treating patients with complement-​associated aHUS and is now licensed for this indication. C3 glomerulopathy (C3G) (See Chapter 21.8.6.) C3 glomerulopathy is characterized by predominant or isolated accumulation of C3 within glomeruli. This condition is associated with impaired regulation of the alternative pathway within plasma or along the glomerular basement membrane (GBM). The path- ology is thought to develop as a consequence of either accumulation of serum-​derived C3 metabolites along the GBM or activation of C3 directly on the GBM. The abnormal alternative pathway activation may be genetic or acquired. Loss of function of regulators and gain-​ of-​function of activation proteins within the alternative pathway can

322 SECTION 4  Immunological mechanisms result in familial C3G. Acquired factors include autoantibodies that stabilize the alternative pathway C3 convertase, termed C3 nephritic factors (C3NeF). C3NeF is frequently associated with low plasma C3 levels. However, since C3NeF does not commonly affect the clas- sical or lectin pathways, plasma C4 levels are usually normal. C3 nephritic factor is also associated with partial lipodystrophy (loss of fat from the face and upper part of the body). C3NeF potentiates alternative pathway activation on, or in the vicinity of, adipocytes resulting in complement-​mediated damage. Adipocytes are suscep- tible since they produce alternative pathway components: C3 and factor D (also termed adipsin). Loss-​of-​function mutations in factor H are associated with sus- ceptibility to both aHUS and C3G. The association with two distinct renal phenotypes (renal TMA versus glomerulonephritis) can be ex- plained at the molecular level. aHUS-​associated factor H mutations target domains of the protein that are required for its interaction with surface ligands (e.g. polyanions) along the renal endothelium. These mutations do not impair the ability of factor H to regulate C3 activation in plasma. Consequently, plasma C3 levels are typic- ally normal in affected patients. The result is defective regulation of C3 activation specifically along the renal endothelium. In contrast, C3G-​associated factor H mutations result in impaired C3 regulation in plasma and low plasma C3 levels. Pigs and mice with complete factor H deficiency develop impaired plasma C3 regulation (with severe falls in C3 levels) and accumulation of C3 along the GBM. Distinct entities within the C3G classification include dense deposit disease (DDD, formerly termed membranoproliferative glomerulo- nephritis type II) and complement factor H-​related 5 (CFHR5) neph- ropathy. DDD is associated with ocular drusen, similar to that seen in age-​related macular degeneration (AMD). The pathogenesis of DDD-​ associated ocular drusen is unknown, but it is interesting that gen- etic susceptibility to AMD is strongly influenced by polymorphisms within complement genes. These include genes encoding factor H, factor I, C3, and factor B. These findings suggest a role for alternative pathway activation in the pathogenesis of ocular drusen. CFHR5 nephropathy is a C3G that is endemic in Cyprus and as- sociated with heterozygous mutations in the gene encoding CFHR5. The role of the factor H-​related proteins is unclear. CFHR5 might act as a competitive antagonist with factor H. CFHR5, unlike factor H, does not have C3 regulatory functions. When it interacts with activated C3 (C3b) it prevents factor H binding and enables further complement activation. The mutant CFHR5 protein in CFHR5 neph- ropathy is thought to potentiate C3 activation along the GBM even in the presence of normal factor H. Unlike complement-​associated aHUS, the contribution of C5 activation to kidney damage is less clear. Eculizumab has not been licensed for C3G, but there are case reports of its effectiveness in this condition, particularly where there is crescentic glomerulonephritis. Paroxysmal nocturnal haemoglobinuria (See also Chapter 22.5.3.) Paroxysmal nocturnal haemoglobinuria (PNH; OMIM 311770) il- lustrates the critical role of membrane-​bound complement regula- tory proteins in protection against complement-​mediated lysis. In PNH a somatic mutation results in a clone of erythrocytes that lack glycosylphosphatidylinositol-​linked proteins. These include the membrane-​bound complement regulators CD59 and CD55 (also termed decay-​accelerating factor, DAF). Affected cells (PNH erythrocytes) are susceptible to complement-​mediated lysis within the circulation. Activated C3 (C3b) accumulates on PNH erythrocytes through alternative pathway activation. Subsequent terminal pathway acti- vation results in cell lysis by the MAC. CD59 is an inhibitor of the terminal pathway and its deficiency appears to be responsible for PNH since isolated CD59 deficiency, but not CD55 deficiency, is as- sociated with a PNH-​like phenotype. Prevention of terminal pathway activation with eculizumab (a monoclonal antibody against complement C5) prevents complement-​mediated intravascular lysis of PNH erythrocytes. Eculizumab is licensed for the treatment of transfusion-​dependent PNH. During treatment C3b may accumulate on PNH erythrocytes since, unlike terminal pathway activation, the alternative pathway is unaffected by eculizumab. PNH erythrocytes coated with C3b may be prematurely removed from the circulation by phagocytosis in the liver and spleen (extravascular haemolysis). Complement investigations Standard and specialized complement investigations are listed in Table 4.2.2. The most frequently available complement assays are antigenic measurements of serum C4 and C3, but the results of such assays need to be interpreted cautiously. The normal ranges are wide because there is substantial genetic variation in the levels of these proteins. Furthermore, proteins levels are a product of both synthetic and catabolic rates, and both of these may vary in health and disease. Both C3 and C4 are acute phase reactants and concen- trations of these proteins may rise, in the case of C3 by as much as 0.5 g/​litre, in response to acute phase stimuli. Several approaches have been devised to assess the presence of complement activation in vivo. Many assays have been developed which identify the product of activation of the complement system (e.g. C3a, C3d). Although these assays are attractive in principle, the products of complement activation are only present in plasma very transiently and, in routine clinical practice, measurement of total C4 and C3 levels have not been supplanted as the best ‘rough and ready’ estimates of complement activation. A fall in serum C4 with or without a fall in C3 usually indicates classical pathway activation (Table 4.2.2). Formation of immune complexes, either in the circulation or in tissues, may result in clas- sical pathway activation sufficient to cause circulating C4 levels to fall. This is most likely to indicate SLE, and in many patients with this condition low complement levels (fall in C4 with or without a fall in C3) are a useful marker of active disease. A low C4 level may be due to the presence of a C4 null allele. C4 null alleles are more common in SLE patients. In this case the low C4 level may remain low during disease quiescence. Other important causes of classical pathway activation in- clude C1 inhibitor deficiency, chronic infections (e.g. subacute bacterial endocarditis, mixed essential cryoglobulinaemia, and hypocomplementaemic urticarial vasculitis syndrome). Cryoglobulins should be tested in patients presenting with unex- plained renal disease or peripheral neuropathy and low C4. A low C3 level in the setting of a normal C4 level indicates alterna- tive pathway activation. This is rare and is associated with the acute phase of postinfectious glomerulonephritis and C3 glomerulopathy.

4.2  The complement system 323 Other standard assays include functional tests of the classical and alternative pathways. These are useful screening tests for assessing complement activation defects, but the most common cause of ab- normal functional activity is delays in ex vivo sample processing. However, if a persistent abnormality is demonstrable, then it is ap- propriate for specialist laboratories to proceed to measure individual components to identify the abnormal or missing component. C1 in- hibitor testing is routinely available and straightforward. Specialized assays include anti-​C1q antibodies, C3NeF testing, anti-factor H autoantibodies, and genetic screening for alternative pathway mutations associated with aHUS and C3G. These assays are useful in defined settings (see Table 4.2.2), but their clinical in- terpretation requires experience and care. Anti-​C1q antibodies are associated with glomerulonephritis in patients with SLE and are part of the defining criteria in hypocomplementaemic urticarial vasculitis syndrome (HUVS). These IgG autoantibodies are dir- ected against an epitope in the collagenous region of C1q, which becomes exposed when C1q is dissociated from the other proteins of the C1 complex, C1r and C1s. This type of epitope is known as neoepitope. Up to one-​third of patients with SLE develop anti-​C1q autoantibodies. These are associated with activation of the classical pathway, causing very low C4 levels and, to a lesser extent, reduced C3 levels. They are thought to amplify complement activation by immune complexes in tissues, by binding to C1q fixed to immune complexes enlarging the complexes and promoting further com- plement activation. Increases in anti-​C1q antibody titres have been shown to precede renal involvement in SLE and, in contrast to rises in anti-​DNA antibody titres, appear to increase specifically prior to renal relapse. In HUVS very high titres of anti-​C1q antibodies are typically found together with marked reduction in C4, C3, and C1q levels. HUVS is characterized by urticarial vasculitis (histologically is usually a leukocytoclastic vasculitis), polyarthritis/​polyarthralgia, membranoproliferative glomerulonephritis, angio-​oedema, neur- opathy, and obstructive pulmonary disease. Complement therapeutics Patients with chronic hypocomplementaemia are at particular risk of developing serious infection with encapsulated organisms Table 4.2.2  Complement investigations Standard assays Comments Serum C4 Reduced levels usually indicate classical pathway activation or presence of C4 null alleles. If C4 is low with normal C3 this indicates predominant classical pathway activation and typical causes include: • Active SLE • Hypocomplementaemic urticarial vasculitis (HUVS) • C1 inhibitor deficiency • Mixed essential cryoglobulinaemia • Rheumatoid vasculitis • Chronic infections associated with immune complex formation (e.g. subacute bacterial endocarditis) Serum C3 Reduced levels indicate complement activation. Commonly reduced in active SLE, usually in combination with reduced C4 level, indicating classical pathway activation Low C3 with normal C4 indicates predominant alternative pathway activation. This is rare, and causes include: • Poststreptococcal glomerulonephritis • C3 glomerulopathy (e.g. dense deposit disease) CH100 Functional test in which classical pathway activation is triggered in vitro and terminal pathway activation assessed by measurement of the membrane attack complex either directly (using anti-​C5b-​9 antibodies) or indirectly (using red cell lysis). Test calculates % of test sera required to cause 100% terminal pathway activation Normal CH100 requires an intact classical pathway, functional C3, and intact terminal pathway Important to screen for complement deficiency AP100 Functional test in which alternative pathway activation is triggered in vitro and terminal pathway activation assessed by measurement of the membrane attack complex either directly (using anti-​C5b-​9 antibodies) or indirectly (using red cell lysis) Normal AP100 requires intact alternative pathway, functional C3, and intact terminal pathway. Important test to screen for complement deficiency C1 inhibitor (C1INH) assays Standard assays test both antigenic and functional activity of C1INH Essential test to detect type I and type II hereditary angioedema Specialized assays Comments Anti-​C1q antibodies Associated with SLE nephritis but not routinely measured in clinical practice Indicated in suspected hypocomplementaemic urticarial vasculitis syndrome (HUVS) C3 nephritic factor Represents an autoantibody that enhances the C3 convertase of the alternative pathway Associated with enhanced alternative pathway activation Indicated in atypical haemolytic uraemic syndrome and C3 glomerulopathy Anti-Factor H autoantibodies Associated with enhanced alternative pathway activation Indicated in atypical haemolytic uraemic syndrome and C3 glomerulopathy Mutation screening for structural and sequence variation in complement genes encoding alternative pathway proteins and regulators Indicated in patients with atypical haemolytic uraemic syndrome prior to renal transplantation

324 SECTION 4  Immunological mechanisms such as Streptococcus pneumoniae and Neisseria meningitidis. The hypocomplementaemia, in addition to causing defective opsoniza- tion, results in reduced splenic clearance of these organisms. These patients can be thought of as functionally asplenic and we recom- mend prophylactic penicillin therapy together with pneumococcal and meningococcal vaccination. There is currently one complement inhibitor, eculizumab, that is in use in routine clinical practice. Eculizumab is a monoclonal antibody that blocks C5 activation, preventing the formation of the anaphylatoxin C5a and the generation of the MAC. It is licensed for the treatment of transfusion-​dependent haemolytic anaemia in PNH, generalised myasthenia gravis, and aHUS. It is admin- istered intravenously and its use is associated with increased sus- ceptibility to Neisserial infection. This is a predictable side-​effect since terminal pathway deficiencies are associated with increased susceptibility to this infection and eculizumab induces an acquired C5 (and therefore terminal pathway) deficiency state. Patients re- ceiving this agent must therefore be immunized against Neisserial strains and treated with prophylactic antibiotic therapy. C1 inhibitor deficiency can be treated by the intravenous admin- istration of C1 inhibitor preparations. Two are derived from plasma, Berinert P and Cinryze. A third is a recombinant C1 inhibitor mol- ecule, Rhucin. In some of the rare cases of complement activation protein deficiency, fresh frozen plasma has been used as a source of the missing component. For example, in complete C1q deficiency associated with a SLE-​like illness, short or long-​term plasma in- fusions have been beneficial. In severe cases, bone marrow trans- plantation has been used to achieve permanent restoration of C1q levels since C1q is synthesized by bone marrow-​derived cells. FURTHER READING Bordron A, et al. (2019). Complement system: a neglected pathway in immunotherapy. Clin Rev Allergy Immunol, doi: 10.1007/ s12016-019-08741-0. Conigliaro P, et al. (2019). Complement, infection, and autoimmunity. Curr Opin Rheumatol. doi: 10.1097/BOR.0000000000000633. Degn SE, Jensenius JC, Thiel S (2011). Disease-​causing muta- tions in genes of the complement system. Am J Hum Genet, 88, 689–​705. Lambris JD, Ricklin D, Geisbrecht BV (2008). Complement evasion by human pathogens. Nat Rev Microbiol, 6, 132–​42. Merle NS, et al. (2015). Complement system part I—​molecular mech- anisms of activation and regulation. Front Immunol, 6, 262. Merle NS, et al. (2015). Complement system part II—​role in immunity. Front Immunol, 6, 257. Thurman JM, Yapa R (2019). Complement therapeutics in autoimmune disease. Front Immunol, 10, 672. doi: 10.3389/fimmu.2019.00672. eCollection 2019.

4.3 Adaptive immunity 325

4.3 Adaptive immunity 325

ESSENTIALS Following the innate immune response, which acts very rapidly, the adaptive immune response plays a critical role in host defence against infectious disease. Both types of immune response work together in order to achieve immunity (protection from disease). Unlike the in- nate response, which is triggered by pattern recognition of patho- gens (i.e. features that are common to many bacteria or viruses, the adaptive response is triggered by structural features—​known as antigens or epitopes—​that are typically unique to a single organism). Cells involved in the adaptive immune response—​these are B lympho- cytes and T lymphocytes, the latter divided into CD4+ (helper) and CD8+ (cytotoxic) populations. All of these lymphocyte subsets can potentially respond to a huge variety of antigens through the gener- ation of great diversity in their antigen receptors (T-​cell receptors and B-​cell receptors), some of which is genetically encoded, but much is created by recombination between gene segments as the receptors are expressed. Recognition of antigens—​(1) B cells—​a membrane-​bound form of the soluble antibody molecules that the cell is destined to secrete acts as the B-​cell receptor, which can bind to a range of antigens, including non​protein antigens such as carbohydrates. (2) T cells—​ these can only survey antigens that have been cleaved to short pep- tides and presented on surface of cells bound in the groove of the hugely diverse major histocompatibility complex class I and class II molecules. Dendritic cells have a central role since they can not only present the peptides efficiently, but also provide critical extra signalling in the form of specialized ‘costimulatory’ surface molecules and soluble cytokines. Response to antigens—​once T cell and B cells have been triggered by antigen, they proliferate rapidly and display a range of effector functions. (1) B cells—​secrete antibodies, initially in the form of im- munoglobulin M (IgM), but subsequently ‘class switching’ to IgG, IgA, or IgE. (2) T cells—​(a) CD8+ T cells—​response includes migration to sites of infection, killing of infected cells, and secretion of soluble me- diators; (b) CD4+ T cells—​play a key role in providing ‘help’ for B cells (e.g. in class switching), ‘help’ for proliferation of CD8+ T cells, and also conditioning of dendritic cells. CD4+ T cells which secrete a panel of cytokines promoting cell mediated immunity (such as interferon-​γ (IFNγ)) are described as Th1 (T helper 1), while others which secrete cytokines involved in responses to extracellular parasites such as hel- minths (such as interleukin 4 (IL-​4)) are Th2, and a third group which secrete IL-​17 are termed Th17. A further recently delineated set of follicular helper T cells (Tfh) are able to provide help for B cells in lymphoid organs. T cells which counterregulate these responses can also be induced and are described as regulatory T cells (Treg). Immunological memory—​once an infection is contained, the B-​ and T-​cell populations contract and enter a phase of immunological ‘memory’ keeping the antigen selected receptors and some of the cell changes acquired during the response. These memory popu- lations are found largely in lymph nodes, although some ‘effector memory’ T cells may be found in non​lymphoid organs (e.g. liver). They are retained long term at much higher cell frequencies than are found in an unexposed person, and can respond to re-​encounter with antigen with very rapid proliferation and effector function. A set of ‘tissue-​resident’ memory cells (Trm) have been described which do not recirculate and provide very early memory responses in tissues. Regulation of immune responses—​the functions of the adaptive immune system are tightly regulated to limit immune-​mediated pathology. T cells develop initially within the thymus, where those which recognize host (‘self’) antigens are eliminated (‘central toler- ance’). Self-​reactive T cells may be further controlled in the periphery through a variety of mechanisms, including Tregs and expression of inhibitory molecules. B cells develop mainly in the bone marrow where self-​reactive cells can be eliminated or undergo receptor editing that change cell specificity rescuing them from elimination. However, this multilayered control sometimes breaks down, thereby allowing pathological responses to harmless antigens (hypersensi- tivity) or self-​antigens (autoreactivity). Pathogens such as HIV, and various cancer types may exploit downregulatory mechanisms to allow their long-​term persistence in the body. Clinical impact of understanding the adaptive immune system—​ this may be harnessed to generate novel diagnostics, therapies
(e.g. monoclonal antibodies) and vaccines, but many challenges
remain in translating our increasing knowledge about molecular control of adaptive immunity into protection against complex per- sistent infections. 4.3 Adaptive immunity Paul Klenerman and Constantino López-Macias

326 SECTION 4  Immunological mechanisms Introduction The adaptive immune response is distinguished from the innate im- mune response by two main features: its capacity to respond flexibly to new, previously unencountered antigens (antigenic specificity), and its enhanced capacity to respond to previously encountered antigens (immunological memory). These two features have pro- vided the focus for much research attention, from the time of Jenner, through Pasteur onwards. In recent years, the molecular basis for these phenomena has become much better understood. Antigen recognition by the adaptive immune system is performed through the T-​cell receptor (TCR) and B-​cell receptor (BCR) expressed on the surface of T or B cells, whereas molecular recognition by innate immune system is performed by ‘pathogen recognition receptors’ (PRRs). PRRs do not undergo changes during immune responses (innate response), leading to a repertoire of around 102, whereas TCRs and BCRs are formed by a process of gene rearrangement that can also undergo additional changes after antigen recognition (adaptive responses), leading to a repertoire of orders of magnitude larger. T and B cells activated by the specific antigen proliferate and differentiate, and some of these will be retained long term and repre- sent the pool of memory cells. Historically, innate and adaptive immune responses have often been treated as separate, with the latter being considered more ‘ad- vanced’ because of its flexibility. It is now clear this not the case. Innate immune responses provide the essential early controls and conditioning required for an adaptive immune response to function. This arises because of the differential speed of the two responses. Innate responses occur within minutes or hours of infection, whereas initiation of effective adaptive immunity may take days. Not only do mediators such as type I interferons have direct antiviral effects, but they also activate antigen presentation pathways and thus have a critical role in priming the adaptive immune response. Thus, the adaptive immune response to a given antigen may be vig- orous or absent depending on the quality of innate signalling that accompanies it. To integrate this further, T and B cells themselves can express innate receptors that contribute to their development and function. Several lymphocyte types with a ‘bridging’ innate and adaptive role have been discovered, including ‘innate lymphoid cells’ which share many phenotypic features of T cells, but lack TCR and respond to innate signals. The immune response evolved to deal with pathogens, of which viruses are good examples. This is the focus of this chapter, but the same responses against self-​, allo-​ or environmental antigens lead to autoimmunity (Chapter 4.6), transplant rejection (Chapter 4.7) and hypersensitivity/​allergy (Chapter 4.5). Antigen specificity of adaptive immune responses Antigen is a word with a long history, originally associated with anti- body binding, but currently used broadly to mean anything to which BCR or TCR can bind. An alternative description is ‘immunogen’ meaning anything can trigger B-​ or T-​cell responses. Largely these are protein structures, and in the case of T cells short peptides and in some cases lipopeptides, but for B cells the targets may be much more diverse such as lipoproteins, lipids, carbohydrates, and nucleic acids. If a large molecule, such as influenza matrix protein, is defined as the antigen, the small regions within it which are recognized by the cells of the adaptive immune response are termed ‘epitopes’. Antigen recognition by T cells T cells are divided simply into two lineages according to the type of TCR: αβ and γδ. αβ T cells are in general divided into CD4+ T cells (or T helper, Th cells) and CD8+ T cells (or cytotoxic T cells). There are also unconventional αβ T cells comprising invariant nat- ural killer T (iNKT) cells and mucosal associated invariant T cells (MAIT), which can be CD8+, CD4+, or double-​negative. CD4+ and CD8+ T cells have distinct, if overlapping functions, but crucially they recognize antigen delivered through distinct path- ways. Both sets of T cells can recognize antigenic peptides only if they are presented by specific host major histocompatibility com- plex (MHC) molecules at the cell surface. These molecules vary substantially between individuals, which is the basis of MHC restric- tion of the capacity of T cells to recognize a given antigen when pre- sented by a specific MHC molecule. In contrast, unconventional T cells and γδ T cells recognize non​peptide antigens and do not bind classical MHC molecules. Antigen presentation to CD8+ T cells CD8+ T cells recognize antigen presented largely from intracellular compartments. In the case of a virus infection, this means newly synthesized viral proteins can be presented on the surface of an in- fected cell (Fig. 4.3.1). The proteasome Proteins destined for the antigen presentation pathway are tagged with ubiquitin and delivered to the cellular proteasome, a multicomponent proteolytic complex present constitutively in all cells (although modified under inflammatory conditions to an immunoproteasome). The outputs from these proteasomes are sets of short peptides derived by specific cleavage of the larger input pro- tein. Typically, these are 9 to 11 amino acids in length. Further pep- tide ‘trimming’ may occur at later stages. Peptide transport The next stage of antigen presentation is transport through an ATP-​dependent peptide transporter (TAP, transporters associ- ated with antigen processing) into the endoplasmic reticulum. Patients with genetically deficient TAP transporters have been de- scribed, which fail to present peptides at their cell surfaces bound to MHC class I molecules (see paragraphs to follow). Their clinical presentation is with bacterial and vasculitic disease and they show overactivated natural killer (NK) cells. Binding to MHC class I The next step for antigenic peptides is loading on to an MHC class I molecule. These comprise a heavy chain with three major extracellular domains (α1–​3), which dimerizes with an invariant light chain β2-​microglobulin (β2m). The α3 domain acts as a membrane-​ proximal stalk, which provides the binding site for β2m, and stability for the complex. The α1 and α2 domains form a groove with closed ends lying above the stalk. Peptides lie stretched out lengthways in the groove, with

4.3  Adaptive immunity 327 two to four specific amino acid residues bound into ‘pockets’ in the floor. These act as ‘anchors’ and provide stability for the peptide–​ MHC interaction. Only specific amino acids can form anchor res- idues in any given MHC class I molecule. As a result, many peptides cleaved from a given protein will not be presented by host MHC molecules and the cellular immune system is essentially ‘blind’ to these. The other non​anchor amino acids within the peptide are dis- played above the lips of the groove and are available for binding by the T-​cell receptor. Recognition by the T-​cell receptor The T-​cell receptor is the molecule responsible for sensitive and spe- cific recognition of peptide–​MHC complexes by T cells. TCRs are made up of pairs of chains—​either α and β chains or γ and δ chains. TCRs comprising α and β chains (αβ T cells) are able to recognize MHC class I molecules through an interaction of low avidity but high specificity. Cocrystallization studies of a limited number of molecules have revealed that the tips of the TCR αβ complex interact in a diagonal fashion with the top surface of the MHC class I pep- tide complex. Thus, the strength of the interaction comes not only from binding of the TCR to the available peptide residues, but also from binding to the MHC class I molecule. This encapsulates the fundamental principle of the cellular immune response. The MHC molecule provides the central focus (restriction), but the peptide provides the essential specificity. Even subtle changes in the pep- tide sequence such as conservative amino acid exchanges (e.g. ly- sine to arginine) can substantially change the strength of the TCR interaction and radically affect the recognition by the T cell, a feature that has been fully exploited by variable viruses such as the human immunodeficiency virus (HIV; see Chapter 8.5.23). Antigen presentation to CD4+ T cells Like CD8+ T cells, CD4+ T cells also survey antigens as peptides presented on the cell surface through MHC molecules. Unlike CD8+ T cells, these peptides are not principally derived from cyto- solic antigens (Fig. 4.3.1). Two key points must be noted. First, while MHC class I molecules are present on virtually all cells, class II molecules are normally present on only a limited number of cell types. Secondly, the pathway requires specific machinery for soluble or particulate antigen outside the cell to be taken into the cellular endosome, which is notably greater in phagocytic cells. The pathway of autophagy may also provide a route into the class II pathway for intracellular proteins. MHC class II molecules Proteins within the endosome, after fusion with lysosomes, are de- graded to peptides, which subsequently bind MHC class II mol- ecules. These are polymorphic dimeric molecules, like class I, but differ in that both α and β chains are equal partners in peptide binding and presentation. An important difference from MHC class I is that the ends of the groove are open, allowing longer pep- tides to be bound (12–​15 residues). MHC class II molecules initially bind an ‘invariant chain’ (CLIP) in the endoplasmic reticulum which protects the binding groove. In CD4+Tcell CD8+ Tcell B cell Virus protein proteasome TAP peptide E.R MHC Class I β2-microglobulin Anchor residues Phagosome/ lysosome Virus peptide TCR contact residues T-cell receptor (TCR) B-cell receptor (BCR) CLIP MHC Class II exchange α-chain β-chain Virus protein replication Viral antigen Target cell Target cell Antigen-presenting cell Fig. 4.3.1  Antigen presentation to T and B cells. A viral antigen is used as an example. Peptides are generated in infected (target) cells and in the case of class I transported to the endoplasmic reticulum (ER) via the TAP (transporters associated with antigen processing) complex. CD4+ T cells are triggered by professional antigen-​presenting cells, while B cells engage antigen directly.

328 SECTION 4  Immunological mechanisms the lysosome/​endosome fusion compartment, the invariant chain is exchanged for peptides that can specifically bind into the groove if they possess the key anchor residues. Class II–​peptide complexes are then presented at the surface of the cell, and can be recognized by specific CD4+ T cells. Antigen recognition by ‘bridging’ T-​cell subsets Some subsets of human αβ T cells are described as possessing in- variant or ‘semi-​invariant’ TCRs and show a distinct biology. MAIT cells make up around 10% of human CD8+ T cells in blood and are highly enriched in the liver and lung. These cells recognize small metabolic intermediates derived from the bacterial riboflavin syn- thesis pathway, presented in the groove of the conserved surface molecule MR1. The much rarer invariant NK-​T cells or iNKTs rec- ognize bacterial glycolipids presented by CD1d. Both of these sets of T cells, and related cells recognizing other members of the CD1 family, play a bridging role between innate and adaptive immunity, and likely a primary role in host defence in epithelia. T cells in which the TCR comprises γδ chain (γδ T cells) make up around 5% of the normal human T-​cell pool in blood, although they may be concentrated in tissues. The molecular targets of such cells are not fully established, but they are able to recognize small phosphorylated antigens (e.g. derived from bacteria), with a role for Butyrophilin 3A1 as a presenting molecule. Functionally, γδ
T cells are thought to play a role more aligned with innate immune responses. Antigen recognition by B cells Unlike T cells, which can mainly survey peptides (or other lipid related molecules) antigens displayed on cells bound to MHC molecules, the range of antigens which can be bound by B cells and antibodies is much more diverse and includes non​protein antigens, such as carbohydrates, lipids, and nucleic acids. Recognition by B cells occurs through the B-​cell receptor, a membrane-​bound form of the soluble antibody molecules that the cell is destined to secrete (Fig. 4.3.1). The basic structure of an antibody (immunoglobulin G, IgG, in this case) is illustrated in Fig. 4.3.2. Essentially each antibody unit comprises one heavy (H) and one light (L) chain. Despite the functional differences from T cells, the basic structure of the TCR and the BCR/​Ig is quite similar. The TCR resembles the key antigen-​ recognizing subunit of the Ig known as a Fab fragment. Because B cells can react to intact antigen, no specific antigen-​ presenting pathway is required (Fig. 4.3.1). However, there are simi- larities with T-​cell recognition of antigen, such as the size of the epitope recognized. Detailed mapping studies, using antigens such as influenza haemagglutinin, have revealed that the sites of B-​cell recognition are discrete, each comprising less than 10 amino acids. Similar studies of carbohydrate antigens reveal a footprint of around seven sugars. Small synthetic molecules, typically described as haptens (haptenes), can also act as B-​cell targets but priming occurs only if they are linked to a conventional protein antigen. One important difference from the peptide antigens recognized by T cells is that B-​cell epitopes may be conformational. This means that the amino acids which interact with the B-​cell receptor need not be in a continuous sequence, but may come together as the pro- tein folds. Other B-​cell responses may be directed against so-​called ‘linear’ epitopes, in which case they can be mimicked by a shorter peptide. Perhaps more important functionally is the definition of epitopes that, when bound, lead to neutralization of a virus (i.e. loss of the capacity of the virion to enter a cell). Typically these epitopes CD4+ T cell CD8+ T cell B cell C V C V α-chain β-chain C V C V α-chain β-chain C C C C C C V C C Antigen recognition region Disulphide bonds C C V TCR IgG Fab papain Cytoplasmic tail V V V V Fig. 4.3.2  Antigen binding by T and B cells. The T-​cell receptor (TCR) is composed of two chains, bound by disulphide bonds, with antigen recognition occurring in a variable region. This is analogous to the variable region of an immunoglobulin molecule (IgG is illustrated here); the TCR-​equivalent region (Fab fragment) may be cleaved from the full molecule using papain.

4.3  Adaptive immunity 329 occur on viral glycoproteins required for binding cellular entry re- ceptors. The development of such neutralizing antibodies is the basis for many vaccines and for sterilizing immunity. Generation of diversity within the immune system The previous discussion gives some indication of the nature of antigens and the common features of their presentation to T and B cells. However, the key feature of the system is its huge adapt- ability to diverse antigens. How is this diversity of antigen receptors generated? Generation of diverse T-​cell receptors T-​cell receptor diversity arises initially through genetically encoded variation, and is hugely expanded through combinatorial processes (Fig. 4.3.3). First, consider the β chain destined to become part of a TCR αβ complex. The genetic organization of this chain includes a variable (V), a diversity (D), and a joining (J) segment, which are spliced and recombined with a constant (C) chain to form the final sequence. The organization is similar for the α chain, but with the absence of the small D segment. The diversity arises first from the fact that the V, D, and J regions exist in multiple copies, each distinct. It is expanded by the process of recombination whereby, in the pro- cess of generating a new T cell from a precursor, any combination of V, D, and J can be used to generate the new β chain. During this process, the action of the terminal deoxynucleotide transferase en- zyme creates further diversity at junctions. Since α and β chains re- combine independently, further diversity is generated as these form heterodimers. It has been estimated that this process could generate 1014 distinct T-​cell receptors. Diversity of B-​cell receptors and antibody The generation of diversity is similar to that of the TCR α and β chains described here (Fig. 4.3.3). V, D, and J regions exist in mul- tiple copies which, as the B cell develops, are recombined randomly (D regions are only present in the H chain), incorporating add- itional diversity as this occurs. Further variability is introduced as two loci for the generation of light chains exist, producing either κ or λ chains. However, in any given cell, only one heavy and one light chain is used, a process known as allelic exclusion. The aforementioned process allows for a huge variability in the key regions which act as binding sites, within the ‘variable’ domain at the N-​terminus. However, there is an additional biological variation between antibodies, not dependent on their specificity, which is pro- vided by further recombination with a constant region (Table 4.3.1). In the heavy chain locus, nine potential C chains can be used to create a palette of diverse antibody types. For the heavy chains, the initial constant (C) chain used is µ, leading to the creation of an early IgM antibody in typical immune responses. Subsequent class switching, which is a one-​way process through further recombination events, may lead to use of any of the other chains. This switching event is largely but not exclusively determined by T-​cell help and cytokines. CD8+ T cell CD4+ T cell B cell α- chain β- chain V J C V D J C N and P nucleotides TCR genes α-chain β-chain T-cell receptor (αβ TCR) κ/λ- L chain H chain BCR/Ig genes Recombination β2-m A B C DRA DRB1 DRB3-5 DQa/b DPa/b B-cell receptor (sIg) MHC complex Class I Class II V D J C Somatic hypermutation Polymorphism Fig. 4.3.3  Generation of diversity in the immune system. Both TCRs and BCRs are generated through recombination. For Ig H chains there are 65, 27, and 8, V, D, and J genes encoded in the germ line, respectively. Somatic hypermutation occurs only in B cells. The MHC complex is highly polymorphic—​the class II genes actually lie upstream of the class I genes on chromosome 6. DRA encodes the α chain which is conserved and pairs with polymorphic β chains from the DRB1 locus.

330 SECTION 4  Immunological mechanisms The constant regions determine critical factors in the distribution of the antibodies. Notably, only IgG can cross the placenta and protect the fetus, but IgA is secreted across epithelial membranes, including into breast milk for protection of the newborn. Finally, an important process which creates further diversity, and one which distinguishes B cells from T cells, is the emergence of somatic hypermutation. Once B cells have developed during an initial immune response, the action of the mutagenic enzyme AID within the hypervariable regions leads to the creation of somatic mutants. The B-​cell receptors of some of these mutant clones may have increased avidity for their original antigen, and are further selected. This molecular process is observed functionally by affinity maturation of the antibody response over time. Diversity among MHC molecules MHC molecules are glycoproteins expressed on the cell surface that are highly polymorphic and therefore a molecular ‘self-​marker’ of the organism. These molecules are fundamental for the activation and regulation of the immune system in health and disease, and in the context of transplantation. Class I molecules These molecules provide the key platforms for antigen presentation, but only a small number of peptides can bind any given class I mol- ecule, potentially limiting the T-​cell response. This limitation has been solved for human MHC molecules (human leucocyte antigens, HLA) first by reduplication of these genes, such that for class I, there are three loci—​A, B, and C (Fig. 4.3.2). However, more important, at each locus there exists a huge range of alleles or HLA types, which are rep- resented at varying frequencies in different populations. In Western populations the commonest A allele is HLA A*0201, which occurs in up to 50% of individuals, but there are over 3000 alleles, all of which are much less common. The B locus is even more diverse, with nearly 4000 alleles described. HLA-​C molecules are of slightly more limited diversity, and also play a major role in signalling to NK cells. Class II molecules The principles are similar for HLA class II, although there are four loci, the most diverse of which is DRB1, encoding the β chain of a range of DR molecules (the α chain is invariant). The next locus en- codes only three alleles, DRB3, 4 and 5 (previously DR51, 52, and 53). DR molecules are highly expressed on antigen-​presenting cells and are restricting elements for important CD4+ T-​cell epitopes. DQ and DP are also polymorphic loci, although the latter is less so, and is also expressed in lower amounts. In total there are over 3000 HLA Class II alleles described. Thus an antigen-​presenting cell will present potentially six dif- ferent class I molecules and eight different HLA class II molecules, each binding distinct peptides from a given antigen. However, these molecules are not typically inherited independently, since they are often in strong linkage disequilibrium. Adaptive immune responses and the basis of immunological memory The previous discussion has outlined the molecular basis for antigen recognition, but how is this process coordinated in order to establish and maintain immune responses? The naive state Lymphocytes are generated from common lymphoid progenitors within the bone marrow and undergo a series of maturation steps to create naive B and T cells (Fig. 4.3.4). Naive in this context means ready to respond functionally to an as yet unencountered antigen. Reaching this stage requires education within the thymus (for T cells) and bone marrow (for B cells). T-​cell development T-​cell education occurs in the thymus, through interaction of thymocytes with specialized thymic cells (cortical epithelial cells and bone marrow derived cells). These present a range of self-​antigens, including non​thymic tissue-​specific antigens (e.g. from pancreas), the expression of which is liberated by the AIRE gene. The process of thymic development is initially similar for CD4+ and CD8+ thymocytes, which pass through a CD4+ CD8+ phase, before downregulating either receptor. Those that interact strongly with host MHC and self peptides receive signals which Table 4.3.1  Diversity of immunoglobulin types. The different immunoglobulins have different biological properties, some of which are illustrated here. There are further subtypes of the IgG classes Isotype H+L chains Mr (kDa) Serum conc. (mg/​ml) t1/​2 (days) C1q bound Placental transport Mast-​cell binding IgG1 2+2 146 9 21 + + ± IgG2 2+2 146 3 20 + + –​ IgG3 2+2 165 1 7 + + ± IgG4 2+2 146 0.5 21 –​ + ± IgM 10+10 970 1.2 5 + –​ –​ IgA1 2+2 160 2 6 –​ –​ –​ IgA2 2+2 160 0.5 –​ –​ –​ –​ sIgA 4+4 405 0.5 –​ –​ –​ –​ IgD 2+2 170 0.06 3 –​ –​ –​ IgE 2+2 190 0.0002 3 –​ –​ ++ sIgA, secretory IgA; this also contains a J chain linking the multimeric structure.

4.3  Adaptive immunity 331 lead to elimination through a process termed negative selection. Thymocytes which fail to interact with host MHC molecules do not receive sufficient signals to survive and thus many thymocytes are eliminated at this stage. Positively selected CD8+ or CD4+ T cells are therefore those that interact weakly with MHC–​self-​peptide com- plexes (class I or II, respectively), providing a broad but non-​self-​ reactive naive repertoire. B-​cell development Early B-​cell development within the bone marrow occurs through pro-​ and pre-​B-​cell stages, during which time immunoglobulin genes are rearranged. Immature B cells possess rearranged surface IgM. Self-​reactive B cells may be eliminated at this stage through clonal deletion, induction of an anergic state, or by secondary re- arrangements of the BCR genes (receptor editing). Further B-​cell maturation towards functional antibody-​secreting cells occurs within lymphoid follicles. As for T cells, a ‘bridging’ subset of B cells with innate-​like characteristics also develops, known as mar- ginal zone (MZ) B cells and B1 B cells. These B-​cell populations are the main contributors of T-​cell-​independent antibody responses and are important players in the immunity to pathogens. Marginal zone B cells (MZB) cells are key components of the first line of de- fence against blood-​borne pathogens in the spleen. B1 B cells are capable of self-​renewal, are enriched in peripheral sites such as the peritoneal and pleural cavity, and are responsible for the secretion of ‘natural’ (pre-​existing) antibodies and contribute substantially to mucosal immunity. Lymphocyte localization Naive lymphocytes are found in blood and in lymphoid organs (spleen, lymph nodes, and the gut-​associated lymphoid tissue or GALT). Within lymph nodes, the B and T cells segregate into the primary follicles and paracortex, respectively (Fig. 4.3.4). The re- circulation and organization of these cells is important in under- standing the restrictions on priming of immune responses. Naive T cells do not home to tissues, even inflamed tissues. This is because their homing receptors and chemokine receptors (most import- antly CD62L, or L-​selectin, and CCR7) allow for entry through high endothelial venules into lymphoid organs. From there they can re- circulate back into the blood via the efferent lymphatics. Since they cannot meet antigen at a peripheral site, it is therefore essential that antigen is delivered appropriately to the lymph node, and this is achieved by the dendritic cell (DC). The scaffolding of the lymph node in the form of stromal cells and follicular dendritic cells also plays an important role in guiding this process. Priming of an immune response DCs are the most important antigen-​presenting cells as they pos- sess not only the appropriate class  I  and class  II molecules, but three further important biological features. First, within tissue, they are very efficient at taking up antigen and delivering this to the class II pathway. Some antigen may enter the cytosol and thus the class I pathway through a process known as cross-​presentation. Secondly, they are mobile, and once antigen is taken up, they are able to migrate through chemokine signalling through afferent lymph- atics to local lymph nodes. Thirdly, they possess an array of cell surface molecules and soluble mediators which allow for primary activation (priming) of naive T cells. All three of these functions are strongly influenced by local innate immune signalling including sol- uble factors such as interferon (IFN)-​α, or direct signalling through Toll-​like receptors (TLRs) that promote ‘maturation’ of the DC, improving its priming ability. Thymus Bone marrow Lymphoid progenitor Early CD4-CD8- thymocyte CD4+CD8+ T cell aβTCR CD4+ T cell CD8+ T cell γδT cell T-cell precursor Pro B cell Pre B cell Immature B cell sIgM H+L chains H chain Lymph node Naive T cells Naive B cell DC Antigen Fig. 4.3.4  Generation of a naive B-​ and T-​cell repertoire. T-​ and B-​cell precursors are generated in the bone marrow, but T-​cell development occurs in the thymus. This includes negative selection and more than 90% of thymocytes die through apoptosis. Deletion, anergy, and receptor editing of autoreactive immature B cells occur in the bone marrow.

332 SECTION 4  Immunological mechanisms T-​cell priming requires TCR interaction with cognate MHC–​ peptide. However, the TCR triggering requires support through signals from other cell surface molecules. A vast array of these is present, but some of the most important are shown in Fig. 4.3.5, including the interaction between CD28 on the T cell, with CD80/​86 on the DC. Additionally, soluble cytokines (e.g. interleukin (IL)-​12) are secreted by the DC, which signal through cytokine receptors on the T cell to promote maturation to a full effector cell. Triggering within the T cell requires the integration of all of these signals for full activation. Important signalling pathways within the cell include a cascade of tyrosine kinases starting at the TCR CD3 complex. Critical motifs on the cytoplasmic tails of signalling mol- ecules (immunoreceptor tyrosine-​based activatory motifs, ITAMs) initiate these cascades. Downstream, some cellular pathways are in- volved leading to a calcium flux and induction of key transcription factors such as NFAT, NF-​κB, and AP-​1. Similar intracellular path- ways are involved in B-​cell triggering. T-​cell effector functions Proliferation The most important consequence of T-​cell priming is cellular prolif- eration. This is crucial because while there is huge diversity among the naive repertoire, the precursor frequency is extremely low, less than 1 in 1 000 000. Rapid clonal proliferation of responding CD4+ and CD8+ T cell may be observed, but particularly the latter. CD8+ T-​cell responses to specific epitopes may reach 20 to 50% of the CD8+ T-​cell pool within a few days of encounter with viruses such as Epstein–​Barr virus (EBV). Homing A second key feature of T-​cell activation is altered homing poten- tial. Instead of homing to lymphoid tissue, these cells refocus their attention on peripheral organs (Fig. 4.3.6). They lose expression of CD62L and CCR7 and gain expression of chemokine recep- tors such as CCR5, which allow for distribution to inflamed sites (CCR5 is also the coreceptor for HIV entry). A diaspora of such cells therefore occurs to many organs, where innate mediators such as macrophages may secrete appropriate chemokines—​a family of small molecules which play a crucial role in regulating cellular mi- gration. Several other cell surface receptors change in primed cells; one useful marker is CD45, which switches isoform from RA to RO. CD8+ T-​cell functions These functions are broadly divided into killing and secretory. Killing of target cells occurs by two major means. (1) Lytic function is mediated through secretion of lytic granules, specialized secretory lysosomes which contain perforin, and a set of granzymes. When CD8+ T cells encounter a target cell, the molecules at the contact point reorganize to form an immunological synapse. Lytic granules are released across the synapse and lead to disruption of the target cell membrane and apoptosis. (2) Killing may also occur through interaction of Fas Ligand (FasL) on the CD8+ T cell with Fas on the appropriate target, leading to apoptosis. CD8+ T cells also secrete a range of cytokines, the most important of which is IFN-​γ, which has proinflammatory and antiviral effects. They may also secrete tumour necrosis factor (TNFα), interleukins, and chemokines. Some of the latter serve to attract further lympho- cytes; they may also have important inhibitory effects on HIV entry, as they compete for binding to key entry receptors for the virus. Overall these functions are critical in the control of intracellular pathogens such as viruses. Mice where CD8+ T cells are deficient (e.g. CD8 or perforin knockout) are susceptible to infection with lymphocytic choriomeningitis virus. Similar inferences are made for human persistent virus infections. MHC class I genes such as HLA B27 and B57 are associated with protection against both HIV and hepatitis C (HCV), probably through promoting efficient antiviral CD8+ T-​cell responses. CD4+ T-​cell functions CD4+ T cells provide essential help for other cell types such as CD8+ T cells, B cells, DCs, and macrophages. This is largely through cytokine se- cretion, but one important interaction, through CD40L/​CD40, serves as a further important maturation signal for DCs. The type of cytokines secreted by the priming DC, in turn influenced by the original innate CD8+ T cell CD4+ T cell B cell CD40L CD40 IL-2 IL-4 Antigen Innate signals TLR CD28 IL-12 Priming DC MHC class I CD80 CD86 MHC class II Fig. 4.3.5  Priming of naive B and T cells. Priming of T cells occurs through antigen presentation by a mature DC. Signals for maturation include innate signalling and also CD40L signals from primed T cells (not shown for the DC, but also occurring on B cells). A large number of other costimulatory molecules, and regulatory molecules are also expressed. Expression of these strongly modifies the quality of the T-​cell response and thus the B-​cell response.

4.3  Adaptive immunity 333 signalling, has an important influence on the quality of the CD4+ T cell. Broadly, CD4+ T cells were first described to mature in the dir- ection of Th1 cells (which secrete IFN-​γ, driven by the transcription factor Tbet), or Th2 cells, which secrete IL-​4, IL-​5, IL-​10, and IL-​13, driven by GATA3. The former are critical in responses against intra- cellular pathogens such as viruses and mycobacteria, while the latter are involved in extracellular defence, including IgE production and eo- sinophilia. A third effector subset includes cells secreting IL-​17 (Th17 cells). Their evolution is driven by IL-​6 and IL-​23 and the transcription factor RORγT, and this pathway has been linked to autoimmunity and antibacterial/​antifungal defence. ‘Regulatory’ CD4+ T cells (Treg) can also emerge, under control of the master regulator FoxP3 (see next). More recently a set of follicular helper cells (Tfh) have been identified, controlled by BCl-​6 and function in the germinal centre reaction. Overall, CD4+ T cells play a central role in host defence, and, in their absence, there is failure of CD8+ T-​cell-​mediated immunity, generation of new antibody responses, and macrophage-​mediated immunity, creating susceptibility to viruses, mycobacteria, and tu- mours. This is most evident in the case of HIV, where CD4+ T-​cell populations are depleted. Genetically determined variation in CD4+ T-​cell responses plays a major role in host defence and also auto- immunity (e.g. the association of specific HLA class II alleles with protection and susceptibility to viral hepatitis). Priming and functions of B cells As with T cells, full activation of B cells requires encounter with antigen binding the B-​cell receptor on the lymphocyte surface, but also further signals. These can be provided through innate signalling via TLRs, but additionally signals from CD4+ T cells, both cell:cell (CD40L/​CD40) and soluble (IL-​4). Mutations in the gene for CD40L result in a failure of class switching and presents clinically as the hyper-​IgM syndrome. B cells also undergo clonal proliferation upon appropriate signalling, and move from the state of naive B cell, through lympho- blast and plasmablast to plasma cell (Fig. 4.3.6). Unlike T cells, a diaspora through the body is not seen, but reorganization within the lymph node occurs. Accumulation of B cells during an immune response leads to generation of a secondary follicle, containing add- itionally CD4+ T cells. Ultimately immunoglobulin secreting plasma cells migrate to cords within the medulla (and also bone marrow). As well as secreting antibody, B cells have roles as antigen-​ presenting cells. They express MHC class II and can take up antigen, most efficiently cognate antigen via their BCR, for presentation to CD4+ T cells. They also secrete cytokines, including IL-​10, which has an immunoregulatory role. Generation and maintenance of memory Once an immune response has been initiated, the first acute phase may last days to weeks, depending on the type of challenge, but typ- ically the antigen is controlled through the effector mechanisms just outlined. Thus, the expanded populations seen in the acute phase col- lapse down to smaller levels, although still much greater than seen previously. This elevated precursor frequency of cells with the selected antigen-​specific receptor is the hallmark of immunological memory. Lymph node Naive T cells Naive B cell Germinal centre Plasma cells Activated B cells Th1 Th2 Th17 Treg Effector CD4+ T cells TBet GATA3 RORgT FOXp3 Effector CD8+ T cells Nonlymphoid organs Memory CD8+ and CD4+ T cells Effector memory T cells Central memory T cells Medulla CD62L+ CCR7+ CD62L- CCR7- High endothelial venule Afferent lymph DC Antigen Tfh Bcl-6 Tissue-resident memory cells Fig. 4.3.6  Induction and maintenance of memory responses. Effector T-​cell populations migrate from the lymph node and may enter non​lymphoid tissue. Subsequently they may revert to a central memory pool, in the absence of further antigenic encounter, or retain some effector functions and continue recirculating through non​lymphoid organs (effector memory). Some may remain long-​term in tissues as tissue-​resident memory cells.

334 SECTION 4  Immunological mechanisms T-​cell central and resident memory For antigens that are not re-​encountered or do not persist, memory T-​cell pools over time lose their capacity for immediate effector functions (e.g. secretion of perforin), and their tendency to home to organs. They may regain expression of CD62L and CCR7 and home to lymph nodes (Fig. 4.3.6). These populations are termed central memory. They retain the capacity to respond very rapidly to antigen (within hours), proliferate, and regenerate effector populations. Specialized memory T cell populations are retained at rest in tissues (tissue resident memory), with local protective functions. T-​cell effector memory For antigens which persist or are re-​encountered, ‘memory’ popula- tions exist which retain some features of effector cells (e.g. expression of perforin), and are found distributed throughout organs. These are termed effector memory cells and are thought to provide more im- mediate protective function. Within these pools, there is still further variation between cells which are considered more or less ‘mature’ as judged by a range of surface and intracellular markers which may be lost or gained over time. The net result of this is that such cells re- ceive less costimulation (e.g. via CD28) and more inhibitory signals (via NK-​type receptors). The proportion of such cells varies in dif- ferent infections, with CMV-​specific memory CD8+ T cells showing the most mature phenotype, as well as the largest populations (often 1–​10% of CD8+ T cells specific for a single epitope; Fig. 4.3.7). B-​cell memory Immunological memory due to the B cells is composed of two main populations, memory B cells and long-​lived plasma cells (PCs). Memory B cells express the BCR that has been selected during the first infection or exposure to the antigen. These cells are in a resting state, but have unique properties such as longevity, robust respon- siveness, and a capacity for rediversification. In contrast, long-​lived PCs are terminally differentiated cells that continuously secrete the antibodies induced during the immune response. These cells are lo- cated mainly in the bone marrow and are responsible for maintaining circulating antibody levels over long periods of time. Downregulation of immune responses The focus so far has been on the initiation of responses against pathogens, but these responses must also be controlled to limit immune-​mediated pathology. Responses against self-​antigens must also be minimized. The limitation of responses against self is termed tolerance, but many of the same mechanisms also limit responses against pathogens. These issues are briefly outlined next, but are also discussed further in Chapter 4.7 on transplantation and Chapter 4.6 on autoimmunity. Mechanisms of T-​cell tolerance As discussed earlier, negative selection within the thymus serves to delete many autoreactive T cells. This process of central tolerance is, however, leaky and further peripheral tolerance mechanisms are required. First, since naive T-​cell priming occurs predominantly within lymphoid tissue, failure of antigen to reach this tissue provides an important checkpoint. This has been described as ‘ignorance’ and may be relevant so-​called immune privileged sites, or rare antigens. Secondly, anergy may occur through triggering of a T cell via its T-​cell receptor, without costimulation (via cell surface signals and cytokines). Such anergic cells subsequently fail to respond to antigen. This may occur if the antigen is encountered on a non-​ professional antigen-​presenting cell that lacks costimulatory cap- acity. Another important contributor to anergy may be encounter of T cells with DCs which have not been fully activated. Antigens encountered without appropriate inflammatory or ‘danger’ signals (e.g. through TLRs) may lead to self-​tolerance. The corollary of this is that self-​antigens encountered under conditions of ‘danger’ may prime autoreactive responses. Thirdly, CD4+ T-​cell subsets with a regulatory role (Tregs) may emerge, driven by expression of the transcription factor FOXP3. Such regulatory subsets may be generated in the thymus against self-​ antigens (natural Tregs), or after antigen/​cytokine stimulation (adap- tive Tregs). Their modes of action include secretion of transforming growth factor β (TGFβ) and IL-​10, and upregulation of CD39, an ecto-​enzyme which leads to breakdown of pro-​inflammatory ATP (derived from damaged cells) to adenosine, which is inhibitory. Finally, downregulation may also be achieved through upregu­ lation of inhibitory molecules on activated T cells. These include spe- cific inhibitory molecules such as CTLA-​4 and PD-​1 (programmed death 1), which bind specific ligands on target cells or DCs, causing a downregulation of T-​cell triggering. Many such inhibitory mol- ecules act through so-​called immunoreceptor tyrosine-​based in- hibitory motifs (ITIMs) which recruit phosphatases and compete with ITAMs. Blockade of inhibitory molecules by biologic agents (‘checkpoint inhibitors’) can very effectively augment responses to and tumours, although potentially risk immunopathology. Mechanisms of B-​cell tolerance Since B cells do not receive education regarding self in the thymus, a potential self-​reactive antibody repertoire is being generated Tetramer 104 103 102 101 100 100 101 102 103 104 CD8 CD8+ T cells specific for a single epitope from CMV Fig. 4.3.7  Direct ex vivo evaluation of human antigen-​specific T cells using MHC class I peptide tetramers. The example is a healthy donor with a memory response to CMV. The peptide is derived from pp. 65 and the HLA restriction is A2. Approximately 1% of CD8+ T cells are visible after staining, using a flow cytometer. Courtesy of Alison Turner.

4.3  Adaptive immunity 335 continuously. One important mechanism for containing this is through the requirement for T-​cell help for full maturation of anti- body responses—​in other words a crucial mechanism for B-​cell tol- erance is induction and maintenance of CD4+ T-​cell tolerance. The use of transgenic mouse models, where both a model antigen and antigen-​specific antibody are expressed (classically hen egg lysozyme, HEL) has shed important light on other mech- anisms of B-​cell tolerance. As they mature, B cells encountering self-​antigens may be controlled through deletion and anergy, as for T cells, or later suffer exclusion from germinal centres. Unlike T cells, autoreactive B cells get a second chance to rearrange their immunoglobulin genes through a process of receptor editing. This may rescue the B cell and allow further normal maturation. Additionally, B cells can contribute to the control of immune re- sponses generated by other cell types. A cell population termed regulatory B cell (Breg) have been described as preventing immunopathology by inhibiting inflammatory lymphocytes through the production of IL-​10 and TGF​β. Regulatory mechanisms and immune responses to pathogens Induction of T-​cell tolerance at high levels of viral replication has been observed in murine models and is probably occurring to some extent in chronic hepatitis B (HBV), hepatitis C (HCV), and HIV infection. Functional failure or anergy of T-​cell responses under such conditions is termed T-​cell exhaustion, and ultimately there may be deletion of such cells. It may be that such mechanisms have evolved to avoid potentially lethal immunopathology (e.g. in brain or liver), especially in non​cytopathic virus infections. Induction of PD-​1 and associated inhibitory molecules play a crucial role in such regulation. Induction of Tregs may play an important role in persistent in- fection and such populations have been implicated in tubercu- losis, leishmania, HCV, and HIV. To what extent they are a cause or consequence of persistent infection remains to be established. Upregulation of PD-​1 and IL-​10 have been similarly implicated. Failure of regulation of responses The mechanisms of tolerance outlined here may limit self-​reactivity, and also responses against pathogens. However, in all responses to pathogens, some immune-​mediated pathology may occur as in acute hepatitis B or C. In most circumstances the benefit of the pro- tective response outweighs the short-​term cost of the tissue damage. However, immune-​mediated pathology can occur against harmless environmental antigens, where there is no net benefit and here it is termed hypersensitivity, or allergy (in the case of IgE-​mediated responses; see Chapter 4.5). The underlying immunological mech- anisms for this (and also for autoimmunity) are identical to those against pathogens. Classically they are divided into four forms, using the criteria of Coombs. • Type I hypersensitivity describes immediate responses mediated by IgE and mast cells and is clinically the most critical (including anaphylactic responses to insect venom and asthma). Underlying this is a T-​cell response predominated by Th2 cells. • Type II responses are based on antibody binding antigen on the cell surface and fixation of complement, for example in red-​cell or platelet sensitizing syndromes induced by drugs (e.g. penicillin). • Type III responses involve antibody binding soluble antigen (e.g. drugs or therapeutic anserum) to form complexes. These may again fix complement in tissues leading to an Arthus reaction or lead to a more generalized syndrome described as serum sickness. Both type II and III responses are mediated by IgG antibodies. • Type IV responses are cell mediated, and thus delayed, requiring activation and migration of memory T cells. The best example of this is in the tuberculin test. Harnessing adaptive immune responses Diagnostics B cells The use of antibody induction to track exposure to specific patho- gens relies on the specificity of responses. Early responses induce IgM, which is ultimately switched to IgG, except in the case of carbo- hydrate antigens. Direct detection of antibody-​secreting or memory B cells may be performed using specific B-​cell ELISpot analysis, but these populations are very rare in blood, boosted transiently by vac- cination. Affinity maturation of B-​cell responses leads to increased antibody avidity over time, which may be used to date the onset of IgG responses. Antibody detection in patient sera using immuno- assays such ELISA and Western blot forms the basis of many clinical tests. In addition, antibodies specific for multiple molecules have been developed in animals as diagnostic reagents, expanding diag- nostics for many infectious and non​infectious diseases using tech- nologies such as flow cytometry. T cells In the past, detection of antigen-​specific T cells usually required cul- ture in vitro and was cumbersome and poorly quantitative. However, techniques such as ex vivo ELISpot, intracellular cytokine staining, and MHC–​peptide tetramer analysis have revolutionized the ability to measure T-​cell responses in human disease. ELISpot and intracellular cytokine staining rely on detection of cytokine (typically IFN-​γ) after exposure to antigen, and subsequent capture of these single cell events on plates, or using a flow cytometer. Alternatively total IFNg can be captured using an ELISA. Direct ex vivo analysis of such populations has been used clinically to evaluate the T-​cell response against tuberculosis, in a manner similar to the tuberculin skin test (interferon-​γ-release assay or IGRA). Tetramer analysis relies on in vitro synthesis of fluorescently la- belled MHC–​peptide complexes. These bind specifically to the T-​ cell populations of interest, which may be identified using a flow cytometer (Fig. 4.3.7). Such analyses may be of value in tracking the immune responses to infection or vaccines, or during immuno- suppression. Novel technologies based on cytometer coupled with mass-​spectrometry (CyTOF) or coupled to a microscope can define multiple functional and phenotypic parameters per cell. Prophylaxis Clearly the most successful clinical harnessing of the adaptive im- mune response is in the form of immunization, which relies on the antigen specificity and memory induction just described. This is dealt with in detail in Chapter 8.3. Overall, vaccines that induce neutralizing antibodies and provide sterilizing immunity have been

336 SECTION 4  Immunological mechanisms the most successful. CD4+ T-​cell-​based vaccines already exist for tuberculosis in the form of bacille Calmette–​Guérin (BCG) and may be relevant in other settings. The ability to generate specific CD8+ T responses may be needed for complex infections such as HIV and HCV, where antibody responses are insufficient or are confounded by strain variation. Experimental vaccines, especially based on re- combinant virus technology such as adenoviruses to deliver viral antigens, are in trial in such settings. Therapy B cells Transfusion of serum enriched for particular antibodies has been used for many years (e.g. in postexposure prophylaxis of herpes zoster, ra- bies, and hepatitis B). Such antibodies are polyclonal (i.e. derived from a range of B cells). Fusion of antibody-​secreting B cells with myeloma partners in vitro, followed by selection of specific hybridomas al- lows the generation of highly potent and specific monoclonal anti- bodies. This technology was first developed by Milstein and Kohler in 1975 and has in recent years led to the generation of a large range of ‘biologics’ antibodies with therapeutic potential (e.g. targeting cytokines/​receptors, adhesion molecules, and tumour receptors). ‘Humanization’ of murine monoclonal antibodies by subsequent mo- lecular modifications may be required for optimization. T cells In contrast with B-​cell based interventions, transfusion of specific T cells has been limited by the inability to grow such cells in vitro, and problems of MHC restriction and rejection. However, the ability to detect and isolate specific T cells has allowed some intervention in specific cases, such as cytomegalovirus disease after bone marrow transplantation. In vivo expansion of donor-​derived transfused T cells may be observed after bone marrow transplantation and clinical effects even against established EBV-​driven lymphomas have been reported. Augmentation of pre-​existing T-​cell responses through use of ‘checkpoint inhibitors’ can liberate a marked antitumour ef- fect in cancers such as melanoma. In addition, in vitro expanded tumour-​specific T lymphocytes can form the basis of the adoptive T-​cell therapies such as prostate cancer while chimeric antigen re- ceptor (CAR) T cells with modified and targeted receptors have been successfully used to treat several haematological malignancies. Possible future developments The molecular dissection of the adaptive immune response has re- cently allowed a clearer view of the basis for antigenic diversity and the mechanisms involved in induction and maintenance of func- tional responses. This has allowed improved diagnostics and tar- geted therapies to enhance or suppress specific responses. Further developments in this area leading to rationally designed immuno- suppressant or adjuvant approaches are to be expected. The use of monoclonal antibodies or small molecules to interrupt or target spe- cific pathways has been greatly expanded recently, although there is caution since severe reactions can occur, as in the case of a trial anti- body to CD28. Further complexity of the cellular components of im- mune responses is likely to be revealed, including defining further the role for novel ‘bridging subsets’ (see Fig. 4.3.8) and integration of innate and adaptive responses. Finally, pathogens are by far the best immunologists. Learning from them could lead us to novel and successful immunotherapy strategies. While their ability to manipu- late host responses will remain a challenge to vaccine development, their ingenuity may be fruitfully harnessed further to provide novel immunization strategies. FURTHER READING Beck A, et al. (2010). Strategies and challenges for the next generation of therapeutic antibodies. Nat Rev Immunol, 10, 345–​52. Blum J, et al. (2013). Pathways of antigen processing. Ann Rev Immunol, 31, 443–​73. Davis SJ, van der Merwe PA (2006). The kinetic-​segregation model: TCR triggering and beyond. Nat Immunol, 7, 803–​9. Guedan S, Ruella M, June CH (2018). Emerging cellular therapies for cancer. Annu Rev Immunol, doi: 10.1146/annurev-immunol-
042718-041407. Horton R, et al. (2004). Gene map of the extended human MHC. Nat Rev Genet, 5, 889–​99. Kurosaki T, et al. (2015). Memory B cells. Nat Rev Immunol, 15, 140–​50. Mueller SN, Mackay LK (2016). Tissue-resident memory T cells: local specialists in immune defence. Nat Rev Immunol, 16, 79–89. Natoli G, Ostuni R (2019). Adaptation and memory in immune re- sponses. Nat Immunol, 20, 983–92. Qi H, et al. (2015). Spatiotemporal basis of innate and adaptive immunity in secondary lymphoid tissue. Annu Rev Cell Dev Biol, 30, 141–​67. Rammensee H, et al. (1999). SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics, 50, 213–​9. Rosser E, Mauri C (2015). Regulatory B cells: origin, phenotype and function. Immunity, 42, 607–​12. Sallusto F, et al. (2010). From vaccines to memory and back. Immunity, 33, 451–​63. Sharma P, Allison JP (2015). The future of immune checkpoint therapy. Science, 348, 56–​61. Virgin HW, Wherry EJ, Ahmed R (2009). Redefining chronic viral in- fection. Cell, 138, 30–​50. Xing Y, Hogquist KA (2012). T-​cell tolerance: central and peripheral. Cold Spring Harb Perspect Biol, 4, a006957. Zinkernagel RM (1996). Immunology taught by viruses. Science, 271, 173–​8. Innate Adaptive B cell CD4+ T cell CD8+ T cell DCs Granulocytes MAIT cell NKT cell γδ T cell ILCs NK cells MZ B cell Fig. 4.3.8  Innate, adaptive, and ‘bridging’ immune cell subsets. From left to right are depicted components of the immune response with a transition from ‘innate’ to adaptive. The innate lymphoid cells, and NK cells show broadly innate behaviour, although share many phenotypic and functional features with T and B cells, while MAIT, NKT, and γδ T cells are T-​cell subsets which show significant innate responsiveness, as do marginal zone and B1 B cells.

4.4 Immunodeficiency 337

4.4 Immunodeficiency 337

ESSENTIALS Immunodeficiency is caused by failure of a component of the im- mune system and results in increased susceptibility to infections. The possibility of an underlying immunodeficiency should be considered if a patient has: (1) serious, persistent, unusual, or recurrent infections; (2) failure to thrive in infancy; (3) known family history of immuno- deficiency; (4) unexplained lymphopenia in infancy; (5) combination of clinical features characteristic of a particular immunodeficiency syndrome. The nature of the microbial infection in a particular pa- tient provides a clue to the likely cause of immunodeficiency. Primary immunodeficiency diseases are potentially heritable dis- orders that result in defects in an intrinsic component of the immune system. Increasingly, the alternative term ‘inborn error of immunity’ is preferred to embrace other manifestations of impaired immune func- tion such as autoimmunity, lymphoproliferation and autoinflammation. Secondary immunodeficiencies are caused by conditions that impair the normal function of the immune system and include viral infections, myelomatosis, non-​Hodgkin’s lymphoma, severe renal or liver failure, and use of therapeutic agents which impair immunity. Defects in anatomical and physiological barriers to infection These are some the commonest predisposing causes of infection (e.g. obstruction of the biliary tract, urinary tract, or bronchi; presence of foreign bodies or avascular areas). Recurrent infections within the same anatomical locations are a characteristic feature, with typical organisms including pyogenic bacteria such as staphylococci, commensal organ- isms from the skin or intestinal tract, and fungi, especially candida. Combined immunodeficiency (T-​cell immunodeficiency) T-​cell hypofunction is accompanied by variable degrees of humoral immunodeficiency. In the most severe types a complete block in T-​cell development leads to catastrophic failure of the adaptive im- mune system. Clinical features—​these include (1) susceptibility to all types of in- fection (especially intracellular pathogens); (2) immune dysregulation; (3) increased risk of neoplasia, especially lymphomatous and/​or vir- ally associated; (4)  variable primary effects on other components of the immune system +/​–​ extrahaematological manifestations, depending on the molecular defect. Causes—​these may be inherited (rare) or acquired. Commonest causes of acquired T-​cell deficiency include HIV infection or immunosuppressive therapy. Inherited causes include (1)  severe combined immunodeficiency—​caused by a variety of molecular de- fects; (2) other conditions including MHC class II deficiency, Wiskott–​ Aldrich syndrome, LRBA deficiency, hyper IgM syndrome, X-​linked lymphoproliferative syndrome, thymic defects, DNA repair defects associated with immunodeficiency, autosomal dominant hyper IgE syndrome. Management—​(1) severe inherited T-​cell disorders—​invariably fatal unless treated with haematopoietic stem cell transplantation or (in a very few instances) with gene therapy; (2) secondary T-​cell deficiency—​requires supportive therapy with antiviral and antibac- terial chemotherapy agents. Primary antibody deficiencies Antibody deficiency diseases are characterized by a decrease in the levels of serum immunoglobulins below the fifth centile for age. The reduction may be in all classes of immunoglobulins or a single isotype. Clinical features—​typical presentation is with recurrent infec- tions by encapsulated bacteria (e.g. Streptococcus pneumoniae, Haemophilus influenzae type B); most patients suffer from re- peated sinopulmonary infections, eventually resulting in struc- tural lung damage; arthritis occurs in a few patients; diarrhoea and malabsorption may occur in a few patients; due to chronic infec- tion with intestinal pathogens or bacterial overgrowth in the small intestine. Causes—​major forms of antibody deficiency include (1) common variable immune deficiency—​the commonest primary immuno- deficiency disease; underlying molecular defect usually unknown; clinically defined by susceptibility to infection accompanied by low serum IgG and evidence of impaired specific antibody pro- duction in response to natural microbial exposure or vaccination. (2) X-​linked agammaglobulinaemia—​caused by a defect in a cyto- plasmic tyrosine kinase that results in the arrest of B-​cell maturation; affected boys usually develop recurrent infections typical of antibody deficiency from around 6  months of age. (3)  Other conditions—​ including (a)  autosomal recessive antibody deficiencies with B lymphopenia; (b) physiological antibody deficiencies; (c) transient hypogammaglobulinaemia of infancy; (d)  selective antibody defi- ciency with normal immunoglobulins; (e) antibody deficiency asso- ciated with thymoma; (f) IgA deficiency; (g) IgG subclass deficiency. 4.4 Immunodeficiency Sophie Hambleton, Sara Marshall, and Dinakantha S. Kumararatne

338 SECTION 4  Immunological mechanisms Management—​immunoglobulin replacement therapy through the intravenous (IVIG) or subcutaneous (SCIG) routes is the mainstay of therapy. Diseases of immune dysregulation Complex regulatory mechanisms ensure that innate and adaptive im- mune responses are held in check within the healthy immune system. Haemophagocytic lymphohistiocytosis is a life-​threatening systemic illness in which there is excessive but ineffective immune activation. An increasing number of monogenic disorders are being recognized as causing autoimmunity, often in association with lymphoproliferation. Phagocyte deficiencies Clinical features—​these typically include repeated visceral abscesses caused by Staphylococcus aureus or some species of Gram-​negative bacteria, and invasive fungal infections are a particular risk. Causes—​these include (1) neutropenia—​the commonest phago- cyte deficiency seen in clinical practice; a neutrophil count less than 0.5 × 109/​litre is associated with a high risk of life-​threatening bac- terial sepsis; (2) defects in bacterial killing—​the best-​characterized condition is chronic granulomatous disease (CGD), which is due to faulty postphagocytic activation of the NADPH oxidase complex; (3) defects in leucocyte adhesion and migration. Management—​this requires prophylactic antibacterial and antifungal agents, with the aggressive use of antibiotic chemotherapy of infections when they occur. Haemopoietic Stem Cell Transplantation (HSCT) is required for patients with defective leucocyte migration. HSCT is in- creasingly used for CGD, and gene therapy is in development. Introduction The primary function of the immune system is to resist infection; a role in restraining neoplasia is also increasingly recognized. In the well-​functioning immune system, these tasks are achieved without inappropriate host-​damaging responses. Immunodeficiency dis- orders are typically characterized by an increased susceptibility to infection with or without autoimmunity and/​or neoplasia (es- pecially of the haemopoietic system); in certain disorders there is also excessive autoinflammation and/​or allergic sensitization. While classically described immunodeficiencies predispose to a broad range of infections, we now recognize more subtle lesions of pathogen-​specific immunity that may not be fully penetrant. On a philosophical level, every clinically evident infection results from a pathogen overcoming the immune defences of the body. However, most patients who suffer an infection do not have an underlying im- munodeficiency, and the infectious episode is due to a shifting of the dynamic balance between the resistance of the host and the viru- lence of the pathogen. The possibility of immunodeficiency should be considered under the following circumstances: • Severe, potentially life-​threatening infections—​immunodeficient patients may present for the first time with this type of infection • Persistent infection—​despite adequate and appropriate therapy • Recurrent infection—​assessment of this criterion depends on age and clinical circumstances. For example, six to eight upper respiratory tract infections a year may not be unusual in young children, especially if they have recently joined a playgroup or started school, but such a pattern in adults would need investiga- tion to exclude immunodeficiency • Unusual infection—​infections caused by pathogens of low-​grade virulence are pathognomonic of immunodeficiency. Examples are Pneumocystis jirovecii pneumonitis, atypical mycobacterial disease, or persistent oral candidiasis in an adult without a predisposing factor • Failure to thrive in infancy—​often reflecting an infective enter- opathy; possible associated skin rash, organomegaly, and lymph- adenopathy. Immunodeficiency needs to be considered in the differential diagnosis, ideally as early as possible, since treatment of primary immunodeficiency is most successful if instituted be- fore the onset of significant infections • Known family history of immunodeficiency—​especially if pre- senting with repeated or persistent infections • Unexplained lymphopenia in infancy—​lymphocyte counts in infancy are significantly higher than in adults (around 6 × 109/​ litre in infancy, compared with 1.0–​3.0 × 109/​litre in adults). Retrospective review of children with severe combined immuno- deficiency (SCID) showed that many had absolute lymphocyte counts below the age-​specific normal range at presentation • Combination of clinical features characteristic of a specific im- munodeficiency syndrome—​for example, recurrent respiratory infections, eczema, and thrombocytopenia associated with small-​ sized platelets in a boy raises the possibility of Wiskott–​Aldrich syndrome • Susceptibility to infection with otherwise unexplained lymph­ oproliferation such as lymphadenopathy, hepatosplenomegaly +/​–​ lymphoid hyperplasia of gut or respiratory tract mucosae—​ sometimes associated with systemic features such as fever and weight loss; progression to lymphoma may occur in some cases • Recurrent sterile fever with other evidence of end-​organ inflam- mation such as skin and joint involvement or uveitis—​may indicate autoinflammatory disorders such as periodic fever syndromes. • Defects in the immune system can impair immuno-​regulatory mechanisms which normally prevent autoimmunity, and hence autoimmunity may be a presenting feature of immunodeficiency—​ a combination of autoimmunity and susceptibility to infection is particularly suggestive of an underlying immune deficiency The type of microbial pathogen causing infection in a particular pa- tient may be a clue to the likelihood of immunodeficiency and will often indicate the category of immunodeficiency. Classification of immunodeficiency disease Primary immunodeficiency diseases are heritable disorders which result from defects within the immune system. Most primary im- munodeficiency disorders are caused by single-​gene defects. Others may represent the end result of an interaction between the geno- type and environmental influences, including infections. Primary immunodeficiencies are rare, although it is difficult to give precise estimates due to the paucity of data, as well as variations between different ethnic groups. On the basis of data from national regis- tries, these diseases are estimated to occur in 1 in 2000 to 1 in 10 000 live births.

4.4  Immunodeficiency 339 The International Union of Immunological Societies (IUIS) con- venes a committee which meets biannually to review the classifica- tion of primary immunodeficiency diseases. The main categories are: 1. Immunodeficiencies affecting cellular and humoral immunity, where T-​cell function is defective (humoral immunity is often impaired to a variable degree as a consequence of T-​cell dysfunction); 2. Combined immunodeficiencies with associated or syndromic features; 3. Predominantly antibody deficiencies, where cell-​mediated im- munity is substantially intact; 4. Diseases of immune dysregulation; 5. Congenital defects in phagocyte number, function, or both; 6. Defects of intrinsic or innate immunity; 7. Autoinflammatory disorders; 8. Complement deficiencies; and 9. Phenocopies of primary immunodeficiency are also recognized (e.g. due to anticytokine autoantibodies). Each of these categories of immunodeficiency is characterized by a pattern of infection and associated features, summarized in Table 4.4.1. The main primary immunodeficiency diseases cur- rently identified are summarized in Table 4.4.2. Secondary immunodeficiencies (Table 4.4.3), also known as ac- quired immunodeficiencies, are much more common than primary immune deficiencies. They occur when a previously functioning im- mune system is compromised by external factors. Common causes Table 4.4.1  Immunodeficiency: usual patterns of associated infection and local pattern of associated infections Physiological mechanism Abnormality Organismsa Site: types of infectionb Integumental barrier Burns, eczema, skull fracture, sinus tract Pyogenic and enteric bacteria occasionally fungi, especially candida Recurrent in same location Outflow Obstruction of eustachian tube, urinary tract, or bronchi Pyogenic and enteric bacteria Vascular perfusion Oedema, angiopathy, infarction Microbiological flora Alteration by antibiotic therapy Opportunistic infection, especially candida Phagocyte function Chemotaxis Defects of neutrophil migration, e.g. leucocyte adhesin deficiency Staphylococci, enteric bacteria Skin, any site/​localized and systemic Opsonin deficiency (See ‘Humoral systems’) Skin and respiratory tract Phagocytosis Neutropenia Staphylococci, enteric bacteria Pseudomonas species Any site/​localized and bacteraemic, stomatitis, perianal excoriation Asplenia Pneumococcus, Haemophilus influenzae type b, (malaria, babesia)c Septicaemia, meningitis, severe wound infection with Capnocytophaga canimorsus following animal bites Killing Intrinsic cellular defects, e.g. chronic granulomatous disease Staphylococci, enteric bacteria Aspergillus, Candida, BCG Skin, lymph node, and viscerald abscesses Humoral systems Circulating antibody Hypogammaglobulinaemia Pyogenic bacteria, less commonly enteric bacteria, enteroviruses Upper/​lower respiratory tract; gastrointestinal, any site/​localized and bacteraemic Complement Congenital deficiency C3, Factor I Pyogenic bacteria, especially pneumococci Bacteraemia, meningitis pyoderma Congenital deficiency C5, C6, C7, C8 Neisseria meningitidis or N. gonorrhoeae Meningitis, pyogenic arthritis C1 inhibitor No specific infection susceptibility Develop angio-​oedema C2, C4 No infections or occasionally pneumococcal sepsis Cell-​mediated immunity Primary T-​lymphocyte defects Viruses, fungi, protozoa, intracellular bacteria; plus infections typical of antibody deficiency Any site/​localized and systemic; mucocutaneous candida infections Th-​1 cytokine/​cytokine receptor defects, e.g. IFNγ receptor, IL-​12, IL-​12 receptor (see section on innate immunity for comprehensive list) Poorly pathogenic mycobacteria e.g. M. avium, BCG; salmonella Lymph node; bone; disseminated Defects in innate immunity Defects in pattern recognition receptors or relevant downstream signalling pathway Depending on pathway may present with predominant susceptibility to pyogenic, viral, fungal or mycobacterial infection Any site/​localized and systemic a Common infecting organisms are emphasized. ‘Pyogenic bacteria’ refers to pneumococci, Streptococcus pyogenes, Haemophilus influenzae, meningococci, and staphylococci. ‘Enteric bacteria’ refers to enterococci and the Gram-​negative bacilli common to the intestinal tract, especially Escherichia coli, Pseudomonas, Klebsiella–​enterobacter, and proteus species. b Skin infections include furunculosis, subcutaneous abscesses, and cellulitis; respiratory tract infections include recurrent pneumonia, otitis media, and sinusitis. c Potentially fatal infections caused by blood-​borne parasites if exposed by travel/​residence in endemic area. d Liver, lungs, lymph nodes, and spleen. Source data from Johnston RB Jr. (1984). Recurrent bacterial infections in children. N Engl J Med, 310, 1237–​43, with permission.

340 SECTION 4  Immunological mechanisms Table 4.4.2  Classification of immunodeficiencies based on the International Union of Immunology Societies (IUIS) classification. This table only includes key immunological disorders in each category and does not aim to comprehensive. Readers should refer to latest version of the IUIS classification and online databases (e.g. OMIM) for a comprehensive list Antibody deficiencies Predominantly antibody deficiency diseases Mutated gene/​pathogenesis Associated features X-​linked agammaglobulinaemia BTK Antibody deficiency and B lymphopenia Autosomal recessive agammaglobulinaemia Mutations in genes for µ heavy chain (IGHM), IgA (CD79A), λ5 surrogate light chain (IGLL1), or BLNK, PIK3R1 Antibody deficiency and B lymphopenia Thymoma with antibody deficiency Unknown Antibody deficiency and B lymphopenia Hyper IgM syndrome (autosomal recessive) UNG or AICDA which encodes for AID or PMS2, resulting in defective mismatch repair Low IgG and IgA, raised IgM Common variable immunodeficiency Unknown in most; TNFRSF13B which encodes for TACI in c.10%, rarely ICOS, CD19, or TNFRSF13C which encodes for BAFFR, CD20, CD81, CD21, TWEAK, NFKB1, NFKB2 Antibody deficiency; may have autoimmunity, lymphoproliferation, systemic granulomata Selective IgA deficiency Most unknown; few due to mutations in TNFRSF13B, which encodes for TACI Most remain healthy; increase in autoimmunity, atopy, coeliac disease IgG subclass deficiency Unknown If associated with selective antibody deficiency may have recurrent sinopulmonary infections Specific antibody deficiency with normal serum immunoglobulins Unknown Deficient antibody responses to some antigens. Antipolysaccharide antibody deficiency may be associated with recurrent sinopulmonary infections IgG2 low plus poor responses to pneumococcal polysaccharide and haemophilus B (activated PI3K-​d syndrome) PIK3CD encoding for P110 subunit of PI3K Bronchiectasis, autoimmunity, nodular lymphoproliferation, increased susceptibility to Herpes family viruses (HZV, CMV, EBV), increased incidence of B-​cell lymphoma Congenital B lymphocytosis CARD11 gain-​of-​function mutations Lymphadenopathy, splenomegaly, bacterial and viral infections, chronic EBV infection, autoimmune cytopenias Transient antibody deficiency of infancy Unknown Reduced IgA and IgG; recovery by 3 years of age Cellular and humoral deficiencies Combined T (cellular) and
B-​cell (humoral) deficiency Example mutated gene Associated features Severe combined immunodeficiency (SCID) Lymphopenia, low serum Igs, failure to thrive, severe recurrent infections by viruses, bacteria, and parasites; fatal without corrective therapy such as BMT SCID due to failure of cytokine receptor signalling IL2RG (common g-​chain), IL7RA, JAK3 T Lymphopenia; B-​cell number normal (T-​B+ SCID) SCID due to defective VDJ gene recombination RAG 1, RAG2, DCLRE1C (Artemis) T-​B-​SCID SCID due to defective DNA repair PRKDC (PKcs), NHEJ1 (Cernunnos/​XLF), LIG4 Radiosensitivity, microcephaly, and developmental delay SCID due to defective nucleotide salvage ADA T-​, NK-​, and B-​cell lymphopenia (T-​B-​NK-​SCID) SCID due to defective T-​cell receptor function CD3D, CD3E, CD3Z, PTPRC (CD45) Normal B cell and NK numbers SCID due to lack of T-​cell egress from thymus CORO1A Causes a T-​B+NK+ SCID Reticular dysgenesis AK2 Profound neutropenia; sensorineural deafness; early presentation often with overwhelming sepsis in newborn period MHC class II deficiency CIITA, RFXANK, RFX5, RFXAP Lack of MHC class II expression resulting in CD4 lymphopenia and severe failure of T-​cell and B-​cell function Omenn’s syndrome Hypomorphic mutation of RAG1, RAG2, DCLRE1C (Artemis) or other genes Variant of SCID. Some T cells develop but are oligoclonal. Features include erythroderma, lymphadenopathy, hepatosplenomegaly, eosinophilia. Outcome poor without BMT

4.4  Immunodeficiency 341 Cellular and humoral deficiencies Combined T (cellular) and
B-cell (humoral) deficiency Example mutated gene Associated features MHC class I deficiency TAP1, TAP2, or TAPBP (TAP binding protein, tapasin) Lack of MHC class I expression on cells; CD8 lymphopenia; present with bronchiectasis or vasculitis X-​linked hyper IgM syndrome CD40LG Lack of CD40-​ligand on activated T cells. Failure of Ig class-​switching and affinity maturation; low IgG/​IgA, raised or normal IgM; may develop neutropenia, autoimmune cytopenias, opportunistic infections, and gastrointestinal and liver pathologies CD40 deficiency (a type of autosomal recessive hyper IgM syndrome) CD40 Lack of CD40 expression on B cells. Other features similar to CD40L deficiency DOCK 8 deficiency DOCK8 Recurrent sinopulmonary infections and cutaneous viral infections (Molluscum contagiosum and HPV); low serum IgM and variable IgG responses MHC class I deficiency TAP1, TAP2, or TAPBP (which encodes for the TAP binding protein tapasin) Lack of MHC class I expression on cells; CD8 lymphopenia; present with bronchiectasis or vasculitis IKBKB deficiency Defects in IKBKB, encoding IkB 2 kinase 2, a component of the NF-​kB pathway Normal total T cells; absent regulatory and γδ T cells; impaired TCR activation; normal B cell numbers; impaired B-​cell receptor (BCR) activation; hypogammaglobulinaemia; recurrent bacterial, viral, and fungal infections; clinical phenotype of SCID LRBA deficiency Mutations in LRBA (lipopolysaccharide responsive beige-​like anchor protein); AR Normal or decreased CD4 numbers; T-​cell dysregulation; low or normal numbers of B cells; reduced I IgG and IgA in most; recurrent infections, inflammatory bowel disease; autoimmunity; EBV infections CD27 deficiency Mutations in CD27 (TNFRSF7) required for generation and long-​term maintenance of T-​cell immunity; AR No memory B cells; low iNKT cells; clinical and immunologic features triggered by EBV infection; HLH; aplastic anaemia; lymphoma; hypogammaglobulinemia CID with syndromes Combined immunodeficiencies with associated or syndromic features Mutated gene/​pathogenesis Associated features

  1. Congenital thrombocytopenia Wiskott–​Aldrich syndrome (WAS) Mutations in WASP; cytoskeletal and immunologic synapse defect affecting haematopoietic stem cell derivatives. XL Progressive decrease, abnormal lymphocyte responses to
    anti-​CD3, normal numbers of B cells, thrombocytopenia with small platelets; eczema; lymphoma; autoimmune; decreased IgM; antibody to polysaccharides particularly decreased; often increased IgA and IgE, disease; IgA nephropathy; bacterial and viral infections. XL thrombocytopenia is a mild form of WAS, and XL neutropenia is caused by missense mutations in the GTPase binding domain of WASP WIP deficiency Mutations in WIPF1; cytoskeletal and immunologic synapse defect affecting haematopoietic stem cell derivatives. AR T cells reduced, defective lymphocyte responses to anti-​CD3; recurrent infections; eczema; thrombocytopenia. WAS-​like phenotype
  2. DNA repair defects (other than those in Table 4.4.1) Ataxia telangiectasia Mutations in ATM; disorder of cell-​cycle checkpoint and DNA double-​strand break repair; AR T cells progressive decrease, abnormal proliferation to mitogens; often decreased IgA, IgE, and IgG subclasses; increased IgM monomers; antibodies variably decreased; ataxia; telangiectasia; pulmonary infections; lymphoreticular and other malignancies; increased α-fetoprotein and increased radiosensitivity; chromosomal instability Nijmegen breakage syndrome (nibrin); disorder of cell-​cycle checkpoint and DNA double-​ strand break repair Hypomorphic mutations in NBN; AR T cells progressive decrease; often decreased IgA, IgE, and IgG subclasses; increased IgM; antibodies variably decreased; microcephaly; bird-​like face; lymphomas; solid tumours; increased radiosensitivity; chromosomal instability
  3. Thymic defects with additional congenital anomalies DiGeorge syndromea Contiguous gene deletion in chromosome 22q11.2 or mutation of a gene within this deletion region, TBX1, encoding a transcription factor critical for development of thymus and adjacent embryonic structures; De novo haploinsufficiency (majority) or AD; phenocopies may have other as yet undefined genetic lesions; Decreased or normal; 5% have <1500 CD3 T cells/​ul in neonatal period; hypoparathyroidism, conotruncal cardiac malformation, velopalatal insufficiency, abnormal facies, intellectual disability, and other abnormalities Table 4.4.2  Continued (continued)

342 SECTION 4  Immunological mechanisms CID with syndromes Combined immunodeficiencies with associated or syndromic features Mutated gene/pathogenesis Associated features
CHARGE syndrome due to CHD7 or SEMA3E defects Variable defects of the thymus and associated T-​cell abnormalities, often due to deletions or mutations in transcription regulator CHD7 semaphorin SEMA3E; de novo haploinsufficiency (majority) or AD T cells decreased or normal; response to PHA may be decreased; coloboma, heart anomaly, choanal atresia, mental retardation, genital, and ear anomalies; some are SCID-​like and have low TRECs Winged helix deficiency (nude) Defects in forkhead box N1 transcription factor encoded by FOXN1;AR T cells markedly decreased; alopecia; nail dystrophy; severe infections; abnormal thymic epithelium; impaired T-​cell maturation 4. Immune-​osseous dysplasias Mutations in RMRP (RNase MRP RNA) Involved in processing of mitochondrial RNA and cell-​ cycle control; AR T cells varies from severely decreased (SCID) to normal; impaired lymphocyte proliferation; immunoglobulins variably decreased; short-​limbed dwarfism with metaphyseal dysostosis; sparse hair; bone marrow failure; autoimmunity; susceptibility to lymphoma and other cancers; impaired spermatogenesis; neuronal dysplasia of the intestine 5. Hyper IgE syndromes (HIES); AD-​HIES (Job or Buckley syndrome) Dominant-​negative heterozygous mutations in signal transducer and activator of transcription STAT3; AD T cells normal overall Th-​17 and T-​follicular helper cells decreased; B cells normal; reduced switched and non​switched memory B cells; anti-​ B cell activation factor (BAFF) expression increased; normal; reduced switched and non​switched memory B cells; BAFF expression increased 6. Anhidrotic ectodermal dysplasia with immunodeficiency (EDA-​ID); (EDA-​ID. NEMO/​IKBKG deficiency); Mutations of NEMO (IKBKG), a modulator of NF-​κB activation; XL T cells normal or decreased; poor cell receptor (CR) activation function; low B memory B cells; decreased; poor specific antibody responses, absent antibody to polysaccharide antigens; anhidrotic ectodermal dysplasia + specific antibody deficiency (lack of Ab response to polysaccharides) + various infections (mycobacteria and pyrogens) EDA-​ID IKBA gain-​of-​function mutation Gain-​of-​function mutation in NFKBIA, encoding IκBα, a component of the NF-​κB pathway; AD Normal total T cells; impaired TCR activation; normal B cell numbers; impaired BCR activation; decreased; poor specific antibody responses, absent antibody to polysaccharide antigens; various infections (bacteria, mycobacteria, viruses, and fungi); colitis, EDA (not in all patients); variable defects of skin, hair, and teeth; T-​cell and monocyte dysfunction 7. Calcium channel defects ORAI-​I deficiency Mutation in ORAI1, a Ca++ release-​activated channel (CRAC) modulatory component; AR T cells normal; defective TCR mediated activation; autoimmunity, anhidrotic ectodermic dysplasia, non​progressive myopathy STIM1 deficiency Mutations in STIM1, a stromal interaction molecule 1; AR T cells normal; defective TCR mediated activation; autoimmunity, anhidrotic ectodermal dysplasia, non​progressive myopathy 8. Other defects Immunodeficiency with multiple intestinal atresias Mutation in TTC7A (tetratricopeptide repeat (TPR) domain 7A) protein, of unknown function; AR T cells variable, but sometimes absent; multiple intestinal atresias, often with intrauterine polyhydramnios and early demise; some with SCID phenotype Purine nucleoside phosphorylase (PNP) deficiency Mutation of PNP leading to absent PNP, T-​cell and neurologic defects from elevated toxic metabolites, especially dGTP; AR T cells progressive decrease; immunoglobulins normal or decreased; autoimmune haemolytic anaemia, neurological impairment Idiopathic CD4 cell lymphopenia CD4 lymphopenia of unknown cause Infections typical of T-​cell deficiency Defects in immunoregulation Defects in Immune regulation Mutated gene/​pathogenesis Associated features

  1. Familial hemophagocytic lymphohistiocytosis (FHL) syndromes 1.1. FHL syndromes without hypopigmentation Perforin deficiency (FHL2)
  2. Mutations in PRF1; perforin is a major cytolytic protein; AR Decreased to absent NK and cytotoxic T lymphocytes (CTL) activities cytotoxicity; fever, hepatosplenomegaly (HSMG); hemophagocytic lymphohistiocytosis (HLH), cytopenias UNC13D/​Munc13-​4 deficiency (FHL3) Mutations in UNC13D; required to prime vesicles for fusion; AR Decreased to absent NK and CTL activities cytotoxicity; fever, hepatosplenomegaly (HSMG); hemophagocytic lymphohistiocytosis (HLH); cytopenias Syntaxin 11 deficiency, (FHL4) Mutations in STX11, required for secretory vesicle fusion with the cell membrane; AR Decreased to absent NK and CTL activities cytotoxicity; fever; hepatosplenomegaly (HSMG); hemophagocytic lymphohistiocytosis (HLH); cytopenias STXBP2/​Munc18-​2 deficiency (FHL5) Mutations in STXBP2, required for secretory vesicle fusion with the cell membrane; AR or AD Decreased to absent NK and CTL activities cytotoxicity; fever, hepatosplenomegaly (HSMG); hemophagocytic lymphohistiocytosis (HLH), cytopenias Table 4.4.2  Continued

4.4  Immunodeficiency 343 Defects in immunoregulation Defects in Immune regulation Mutated gene/pathogenesis Associated features
SH2D1A deficiency (XLP1) Mutations in SH2D1A encoding an adaptor protein regulating intracellular signalling; XL Reduced memory B cells; partially defective NK cell and CTL cytotoxic activity; absent iNKT cells; clinical and immunologic features triggered by EBV infection: HLH, lymphoproliferation, hypogammaglobulinaemia; aplastic anaemia, lymphoma XIAP deficiency (XLP2) Mutations in XIAP/​BIRC4 encoding an inhibitor of apoptosis; XL Low iNKT cells; increased T-​cell susceptibility to apoptosis to CD95 and enhanced activation-​induced cell death (AICD); EBV infection, splenomegaly, lymphoproliferation HLH, colitis, inflammatory bowel disease (IBD), hepatitis 1.2. FHL syndromes with hypopigmentation Chediak–​Higashi syndrome Mutations in LYST, impaired lysosomal trafficking; AR Decreased NK and CTL activities (cytotoxicity and/​or degranulation); partial albinism, recurrent infections, fever, HSMG, HLH; giant lysosomes, neutropenia, cytopenias, bleeding tendency, progressive neurological dysfunction Griscelli syndrome, type 2 Mutations in RAB27A encoding a GTPase that promotes docking of secretory vesicles to the cell membrane; AR Decreased NK and CTL activities (cytotoxicity and/​or degranulation); partial albinism, fever, HSMG, HLH, cytopenias 2. T regulatory cells genetic defects IPEX, immune dysregulation, polyendocrinopathy, enteropathy X-​linked Mutations in FOXP3, encoding a T-​cell transcription factor; XL Lack of (and/​or impaired function of) CD4+ CD25+ FOXP3+ regulatory T cells (Tregs); autoimmune enteropathy, early onset diabetes, thyroiditis haemolytic anaemia, thrombocytopenia, eczema; elevated IgE, IgA CD25 deficiency Mutations in IL2RA, encoding IL-​2Rα chain; AR No CD4 + C25+ cells with impaired function of Tregs cells; lymphoproliferation, autoimmunity. Impaired T-​cell proliferation CTLA4 deficiency (ALPSV) Mutations in CTLA4, encoding Cytotoxic
T-​lymphocyte antigen 4, a protein that negatively regulate T-​cell receptor signalling and T-​cell activation; AD Impaired function of Treg cells; autoimmune cytopenias; enteropathy; interstitial lung disease; extralymphoid lymphocytic infiltration recurrent infections STAT3 GOF mutations Mutations in STAT3, encoding signal transducer and activator 3; AD Autoimmunity (especially cytopenias, enteropathy, pneumonitis), susceptibility to infection, variable lymphocyte numbers, low IgE; short stature in many 3. Autoimmunity with or without lymphoproliferation APECED (APS-​1), autoimmune polyendocrinopathy with candidiasis and ectodermal dystrophy Mutations in AIRE, encoding a transcription regulator needed to establish thymic self-​ tolerance; AIRE1 serves as checkpoint in the thymus for negative selection of autoreactive T cells and for generation of Tregs Autoimmunity: hypoparathyroidism hypothyroidism, adrenal insufficiency, diabetes, gonadal dysfunction, and other endocrine abnormalities, chronic mucocutaneous candidiasis, dental enamel hypoplasia, alopecia areata; enteropathy, pernicious anaemia, urticaria, pneumonitis Autoimmune lymphoproliferative syndrome (ALPS) ALPS-​FAS Germinal mutations in TNFRSF6, encoding CD95/​Fas cell surface apoptosis receptor; AD; AR: somatic Increased CD4−CD8−TCRαβ double-​negative (DN) T cells; apoptosis defect FAS mediated; splenomegaly, adenopathies, autoimmune cytopenias, increased lymphoma risk; IgG and A normal or increased; elevated FasL and IL-​10, vitamin B12 ALPS-​FASLG Mutations in TNFSF6, Fas ligand for CD95 apoptosis; AR Increased DN T cells; apoptosis defect FAS mediated; splenomegaly, adenopathies, autoimmune cytopenias, SLE; soluble FasL is not elevated ALPS-​caspase 10 Mutations in CASP10, intracellular apoptosis pathway; AD Lymphadenopathy and splenomegally; autoimmune diseases ALPS-​caspase 8 Mutations in CASP8, intracellular apoptosis, and activation pathways; AR Slightly increased DN T cells; defective lymphocyte apoptosis and activation; adenopathies, splenomegaly, bacterial and viral infections; hypogammaglobulinemia 4. Immune dysregulation with colitis IL-​10 deficiency Mutations in IL10, encoding IL-​10; AR No functional IL-​10 secretion; IBD; folliculitis, recurrent respiratory diseases; arthritis IL-​10Rα deficiency Mutations in IL10RA, encoding IL-​10R1; AR IBD; folliculitis; recurrent respiratory diseases; arthritis; lymphoma IL-​10Rβ deficiency Mutations in IL10RB, encoding IL-​10R2; AR Leukocytes no response to IL-​10, IL-​22, IL-​26, IL-​28A, IL-​28B, and IL-​ 29; IBD, folliculitis; recurrent respiratory diseases; arthritis; lymphoma 5. Type 1 interferonopathies Table 4.4.2  Continued (continued)

344 SECTION 4  Immunological mechanisms Phagocyte functional defects Congenital defects of phagocyte number, function, or both Mutated gene/​pathogenesis Associated features

  1. Congenital neutropenias See Chapter 22.3.1 on neutropenia See Chapter 22.3.1 on neutropenia
  2. Defects of motility Leukocyte adhesion deficiency Type 1 (LAD1) mutation in ITGB2: B chain for adhesion proteins CD18/​CD11; AR Neutrophilia, monocytosis; lymphocytosis. Impaired chemotaxis. Delayed cor separation, omphalitis, periodontitis, poor wound healing, pyoderma-​like skin ulcers, pyogenic sepsis Leukocyte adhesion deficiency type 2 (LAD2) Mutation in SLC35C1: GDP-​fucose transporter; AR Mild LAD type 1 features, plus hh-​blood group plus mental and growth retardation Leukocyte adhesion deficiency type 3 (LAD3) Mutation in KINDLIN3: AR LAD type 1 plus bleeding tendency Rac 2 deficiency Mutation in RAC2: regulation of actin cytoskeleton; AD impaired chemotaxis and Superoxide production; poor wound healing, leucocytosis Papillon–​Lefèvre syndrome Mutation in CTSC: cathepsin C activation of serine proteases; AR Periodontitis, palmoplantar hyperkeratosis in some patients Shwachman–​Diamond syndrome Mutation in SBDS: defective ribosome synthesis; AR Pancytopenia, exocrine pancreatic insufficiency, chondrodysplasia
  3. Defects of respiratory burst X-​linked chronic granulomatous disease (CGD) Mutation in CYBB: electron transport protein (gp91phox); XL Killing (faulty O2 production); McLeod phenotype in patients with deletions extending into the contiguous Kell locus; staphylococcal, Gram-​negative, and fungal infections Autosomal recessive CGD Mutation in CYBA: electron transport protein (p22phox); AR Killing (faulty O2 production); infections as in X-​linked chronic granulomatous disease (XLCGD), autoinflammatory phenotype Autosomal recessive CGD Mutation in NCF1: adapter protein (p47phox);
    AR Killing (faulty O2 production); infections as in XLCGD, autoinflammatory phenotype Autosomal recessive CGD Mutation in NCF2: activating protein (p67phox); AR Killing (faulty O2 production); infections as in XLCGD, autoinflammatory phenotype Autosomal recessive CGD Mutation in NCF4: activating protein (p40 phox); AR Killing (faulty O2 production); infections as in XLCGD, autoinflammatory phenotype
  4. Other defects GATA2 deficiency (mono MAC syndrome) Mutations in GATA2: master transcription factor in haematopoiesis; AD Low monocytes + peripheral dendritic cells; low NK cells; susceptibility to mycobacteria; papilloma viruses, EBV, histoplasmosis; alveolar proteinosis, MDS/​AML/​CMML Pulmonary alveolar proteinosisa Mutation in CSF2RA; biallelic mutations in pseudoautosomal gene defective GM-​CSF signalling; alveolar proteinosis Defects in innate immunity Defects in intrinsic and innate immunity Mutated gene/​pathogenesis Associated features
  5. Mendelian Susceptibility to Mycobacterial Disease (MSMD) IL-​12 and IL-​23 receptor β1 chain deficiency Mutation in IL12RB1: IL-​12 and IL-​23 receptor β1 chain; defective IFN-​γ secretion; AR Susceptibility to mycobacteria and salmonella IL-​12p40 deficiency Mutation in IL12B: subunit p40 of IL12/​IL23; defective IFN-​γ secretion; AR Susceptibility to mycobacteria and salmonella IFN-​γ receptor 1 deficiency Mutation in IFNGR1:IFN-​γR ligand binding chain; defective IFN-​γ binding and signalling; AR Susceptibility to mycobacteria, salmonella and viruses IFN-​γ receptor 1 deficiency Heterozygous mutation in IFNGR1:IFN-​γR ligand binding chain; AD Susceptibility to mycobacteria and salmonella IFN-​γ receptor 2 deficiency Mutation in IFNGR2: IFN-​γR accessory chain; defective IFN-​γ binding and signalling; AR Susceptibility to mycobacteria and salmonella STAT1 deficiency (AD form) Heterozygous mutation in STAT1 (loss of function); defective IFN-​γ signalling; AD Susceptibility to mycobacteria and salmonella Table 4.4.2  Continued

4.4  Immunodeficiency 345 Defects in innate immunity Defects in intrinsic and innate immunity Mutated gene/pathogenesis Associated features Macrophage gp91 phox Mutation in CYBB: electron transport protein (gp 91 phox), in macrophages only; intramacrophage killing faulty; XL Isolated susceptibility to mycobacteria IRF8-​deficiency (AD form) Mutation in IRF8: IL12 production by CD1c+ myeloid dendritic cells (MDC); AD Defective differentiation of CD1c + MDC subgroup; susceptibility to mycobacteria; leucocytosis Tyk2 deficiency Mutation in TYK2; multiple cytokine signalling defect; AR Susceptibility to intracellular bacteria (mycobacteria, salmonella), fungi, and viruses ISG15 deficiency Mutation in ISG15; AR; defective IFNγ production Susceptibility to mycobacteria (BCG); brain calcification RORC deficiency Mutation in RORC; lack of functional RORγT protein; complete absence of IL-​17A/​F-​producing T cells; defective IFNγ production; AR Mycobacteriosis and candidiasis 2. Epidermodysplasia verruciformis EVER1 deficiency Mutations of TMC6; EVER proteins may be involved in the regulation of cellular zinc homeostasis in lymphocytes; AR HPV (group B1) infections and cancer of the skin (typical EV) EVER2 deficiency Mutations of TMC8; EVER proteins may be involved in the regulation of cellular zinc homeostasis in lymphocytes; AR HPV (group B1) infections and cancer of the skin (typical EV) 3. WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome Gain-​of-​function mutations of CXCR4, the receptor for CXCL12; AD Warts/​human papilloma virus (HPV) infection; neutropenia; reduced B cell number; hypogammaglobulinemia 4. Predisposition to severe viral infection STAT1 deficiency Mutations of STAT1; defective STAT1-​dependent IFN-​α, and -​β response; AR Severe viral infections, mycobacterial infections STAT2 deficiency Mutations of STAT2, defective STAT2-​dependent IFN-​α, and -​β response; AR Severe viral infections (disseminated vaccine-​strain measles) IRF7 deficiency Mutation in IRF7, defective IFN-​α, and -​β and -​λ production; AR Severe influenza disease CD16 deficiency Mutation in CD16; deficient spontaneous NK cell cytotoxicity; AR Susceptibility to severe viral infections, inc. HSV, EBV, HPV 5. Herpes simplex encephalitis (incomplete clinical penetrance for all aetiologies listed here) TLR3 deficiency Mutations of TLR3; defective TLR3-​dependent IFN-​α, -​β, and -​λ induction in central nervous system (CNS) resident cells and fibroblasts; AD Herpes simplex virus 1 encephalitis (incomplete clinical penetrance for all aetiologies listed here) UNC93B1 deficiency Mutations of UNC93B1; defective UNC-​93B-​ dependent IFN-​α, -​β, and -​λ induction in CNS resident cells and fibroblasts; AR Herpes simplex virus 1 encephalitis TRAF3 deficiency Mutations of TRAF3; defective TRAF3-​dependent IFN-​α, -​β, and -​λ induction in CNS resident cells and fibroblasts; AD Herpes simplex virus 1 encephalitis TRIF deficiency Mutations of TRIF, also called TICAM1; defective TRIF-​dependent IFN-​α, -​β, and -​λ induction in CNS resident cells and fibroblasts; AD Herpes simplex virus 1 encephalitis TBK1 deficiency Mutations of TBK1; defective TBK1-​dependent IFN-​α, -​β, and -​λ induction in CNS resident cells and fibroblasts; AD Herpes simplex virus 1 encephalitis 6. Predisposition to invasive fungal diseases CARD9 deficiency Mutations of CARD9; defective CARD9 signalling pathway; AR Invasive candidiasis infection; deep dermatophytoses Table 4.4.2  Continued (continued)

346 SECTION 4  Immunological mechanisms Defects in innate immunity Defects in intrinsic and innate immunity Mutated gene/pathogenesis Associated features
7. Chronic mucocutaneous candidiasis (CMC) IL-​17RA deficiency Mutations in IL17RA; defective IL-​17RA signalling pathway; AR CMC; folliculitis Folliculitis 613953 IL-​17RC deficiency Mutations in IL17RC; defective IL-​17RC signalling pathway; AR CMC IL-​17F deficiency Mutations in IL17F; AD CMC; folliculitis STAT1 gain-​of-​function Gain-​of-​function mutations in STAT1; gain-​ of-​function STAT1 mutations that impair the development of IL-​17-​producing T cells; AD CMC; various fungal, bacterial, and viral (HSV) infections; autoimmunity (thyroiditis, diabetes, cytopenia); enteropathy ACT1 deficiency Mutations in ACT1, also called TRAF3IP2; fibroblasts fail to respond to IL-​17A and IL-​17
F, and their T cells to IL-​17E; AR CMC; blepharitis, folliculitis, and macroglossia 8. Toll-​like receptor (TLR) signalling pathway deficiency IRAK-​4 deficiency Mutations of IRAK4, a component of TLR-​ and IL-​ 1R-​signalling pathway; AR Bacterial infections (pyogenic) esp. S. pneumoniae, S. aureus, and Gram-​negative bacteria; poor acute-​phase responses MyD88 deficiency Mutations of MYD88, a component of the TLR and IL-​1R signalling pathway; AR Bacterial infections (pyogenic) esp. S. pneumoniae, S. aureus, and Gram-​negative bacteria; poor acute-​phase responses 9. Isolated congenital asplenia (ICA) Mutations in RPSA; RPSA encodes ribosomal protein SA, a component of the small subunit of the ribosome; AD Asplenia; bacteraemia (encapsulated bacteria) 10. Trypanosomiasis Mutations in APOL1; AD Trypanosomiasis Phenocopies of PID Genetic defect/​presumed pathogenesis Associated features Associated with somatic mutations Autoimmune lymphoproliferative syndrome (ALPS–​SFAS) Somatic mutation in TNFRSF6; defective lymphocyte apoptosis Increased CD4−CD8−double-​negative (DN) T α/β cells; increased number of CD5+ B cells, splenomegaly, lymphadenopathy, autoimmune cytopenias RAS-​associated autoimmune leukoproliferative disease (RALD) Somatic mutation in KRAS (gain-​of-​function) Splenomegaly, lymphadenopathy, autoimmune cytopenias, granulocytosis, monocytosis/​ALPS-​like RAS-​associated autoimmune leukoproliferative disease (RALD) Somatic mutation in NRAS (gain-​of-​function) Splenomegaly, lymphadenopathy, autoantibodies/​ALPS-​like Cryopyrinopathy, (Muckle-​Wells/​ CINCA/​NOMID-​like syndrome) Somatic mutation in NLRP3 Urticaria-​like rash, arthropathy, neurological symptoms Associated with autoantibodies Chronic mucocutaneous candidiasis (isolated or with APECED syndrome) Germline mutation in AIRE; autoAb to IL-​17 and/​ or IL-​22 Endocrinopathy, chronic mucocutaneous candidiasis/​CMC Adult-​onset immunodeficiency AutoAb to IFN-γ Mycobacterial, fungal, salmonella VZV infections/​MSMD, or CID Recurrent skin infection AutoAb to IL-​6 Staphylococcal infections/​STAT3 deficiency Pulmonary alveolar proteinosis AutoAb to GM-​CSF Pulmonary alveolar proteinosis, cryptococcal meningitis/​CSF2RA deficiency Acquired angioedema AutoAb to CI inhibitor Angioedema/​C1 INH deficiency (hereditary angioedema) Atypical haemolytic uremic syndrome AutoAb to complement factor H Atypical haemolytic uremic syndrome; spontaneous activation of the alternative complement pathway Disorders of homeostasis of inflammation (autoinflammatory syndromes) See Chapter 12.12.2 Inherited complement defects See Chapter 4.2 Source data from Picard C, et al. (2018). International Union of Immunological Societies: 2017 Primary Immunodeficiency Diseases Committee Report on Inborn Errors of Immunity. J Clin Immunol, 38, 96–128. Table 4.4.2  Continued

4.4  Immunodeficiency 347 include infections, such as HIV and measles; and cytotoxic and im- munosuppressive drugs (including biologic therapies), particularly those used in the management of transplantation, autoimmunity, and cancer. Physiological immune deficiency may occur at the extremes of age. Before investigating for a possible primary immunodeficiency disease, it is essential to consider the history, examination, and other investigations to exclude secondary immunodeficiency states. A stepwise approach to the diagnosis of immunodeficiency is shown in Table 4.4.4. Defects in anatomical or physiological barriers to infection One of the commonest predisposing causes of infection is a de- fect in the anatomical or physiological barriers to infection. Intact epithelial membranes, especially a stratified squamous epithelial surface such as the skin, constitute an extremely effective barrier to infection. Table 4.4.3  Causes of secondary immunodeficiency Causes of secondary immunodeficiency Defect Defects in anatomical and physical barriers to infection (see text for explanation) Various Malignancies of the B-​cell system Antibody Myelomatosis Non-​Hodgkin’s lymphoma Chronic lymphocytic leukaemia Therapeutic agents Biological agentsa Anti-​B cell agents: e.g. rituximab, ibrutinib Antibody Anti-​TNF agents Innate immunity and CMI Biologics blocking T-​cell costimulation or integrins CMI and/​or innate immunity Anticomplement C5 Terminal complement pathway Anticytokines (e.g. anti-​IL12; anti-​IL-​6, anti-​IL17, etc.) Innate immunity and CMI Cytotoxic drugs: alkylating agents, cytotoxic antibiotics, antimetabolites, vinca alkaloids, etoposide, etc. Myelosuppression and CMI Immunosuppressive drugs: corticosteroids, calcineurin inhibitors (e.g. ciclosporin), antiproliferative immunosuppressants (azathioprine, mycophenolate) CMI Drugs causing antibody deficiency: gold, penicillamine, sulphasalazine, carbamazepine, valproate, clozapine Antibody Radiotherapy CMI Metabolic/​nutritional deficiencies Renal failure CMI and innate immunity Liver failure CMI and innate immunity Protein calorie malnutrition CMI Vitamin A deficiency CMI Zinc deficiency CMI Transcobalamin-​II deficiency Antibody Multiorgan failure CMI and innate Increased loss of immunoglobulin Nephrotic syndrome Protein losing enteropathy Dystrophia myotonica Virus infections HIV CMI Measles CMI congenital rubella Antibody congenital CMV Antibody Age-​related prematurity CMI, innate, and antibody extreme old age CMI and innate CMI, cell-​mediated immunity. a This is a large and expanding area of therapeutics, therefore review properties of any Biological agent used to treat patient for possible immunosuppressive effect.

348 SECTION 4  Immunological mechanisms Table 4.4.4  Diagnostic algorithm for immunodeficiency disorders Type of immune disorder (IUIS classification) CID CID with associated features Predominantly antibody deficiency Immune dysregulation Phagocyte disorders Disorders of intrinsic/​ innate immunity Autoinflammatory disorders Complement disorders Presentation Recurrent/​chronic ENT/​respiratory infection • • ■ • • Failure to thrive in infancy, diarrhoea, opportunistic infection, eczema ■ • • Recurrent infections with pyogenic bacteria &/​or fungi +/​-​ granuloma, gut inflammation • • ■ • • Unusual/​unusually severe infections (including opportunistic pathogens) • • • • • Recurrent infections with one type of pathogen or stereotyped inflammatory response • • ■ • • Autoimmunity, chronic inflammation, and/​or lymphoproliferation • • ■ • • • Immunodeficiency with associated syndromic features • ■ • •

4.4  Immunodeficiency 349 Diagnostic work up First-​line tests All patients: thorough microbiologic evaluation including appropriate search for opportunistic pathogens and coinfection where—​may require cross-​sectional imaging, bronchoalveolar lavage, tissue biopsy, special culture conditions FBC + differential Lymphocyte subsets inc. naïve T cells, Class-Switched Memory B cells In vitro proliferation to mitogens Serum IgG, A, M, E Vaccine-​specific responses Biopsy skin rash in infants for evidence of GVHD Exclude HIV by PCR and serology Consider cardiac review where relevant Serum IgG, A, M, E Isohaemagglutinins Vaccine-​specific responses FBC + differential Consider assessing response to booster vaccinations As for CID plus: enumerate Tregs and double-​negative T cells vit B12 autoantibody screen including ANA, direct agglutination test thyroid function for HLH, measure ferritin, triglycerides, fibrinogen, soluble CD25 FBC + differential (serially to exclude cyclical neutropenia) Blood film for neutrophil morphology Neutrophil oxidative burst (NBT, DHR) Neutrophil surface phenotype (CD18/​11, CD15) FBC + differential Lymphocyte subsets inc. naïve T cells, Class- Switched Memory B cells Serum IgG, A, M, E Vaccine-​specific responses Mycobacterial infection: cytokine studies (IL12/​IFNg production and response), NOB Fungal susceptibility: anticytokine antibodies, thyroid function Recurrent bacterial sepsis: splenic ultrasound, blood film for Howell-​ Jolly bodies, complement function, CD62L shedding Viral susceptibility: exclude Combined Immunodeficiency see Chapter 12.12.2 CH50, AP50, C3, C4 Second line tests If abnormal, consider: T-​cell receptor repertoire (for T-​cell clonality) Tests for materno-​fetal engraftment in infants with features of GVHD MHC I and MHC II expression on lymphocytes Metabolic tests re ADA, PNP, TCN2 deficiency FISH/​CGH to detect del22q11 If abnormal: Lymphocyte subsets inc. naïve T cells, B-​cell phenotyping Special tests depending on clinical context, e.g. in a male, expression of BTK, CD40L, SAP; hair microscopy Assess target organs for damage Consider: tissue biopsy and detailed histology where relevant e.g. gut, skin, lung pulmonary function testing ophthalmologic evaluation endocrine assessment urinary sediment, protein:creatinine ratio specific tests as indicated, e.g. STAT phosphorylation, apoptosis assay, lymphocyte degranulation, perforin expression, anticytokine antibodies Consider: leukocyte subsets with detailed monocyte and dendritic cell phenotyping tissue biopsy and detailed histology where relevant Consult specialist laboratories for appropriate functional testing If only CH50 abnormal, investigate individual elements of classical pathway; if only AP50 abnormal investigate individual elements of Alt pathway; if both CH50 and AP50 abnormal investigate C3 and terminal pathway. If angioedema/​ abdominal pain, and low C4 investigate C1INH; also seek advice of specialist lab All patients: molecular genetic testing (single-​gene/​panel/​open-​ended depending on availability and clinical suspicion) ■ - Most frequent diagnosis. • - (very inclusive—​see main text for further details of discriminating features of individual immunodeficiency disorders)

350 SECTION 4  Immunological mechanisms The following defects predispose to infection: • Integumentary damage caused by burns, eczema, or trauma (including surgery) • Skull fracture, particularly damage of the cribriform plate, which may result in recurrent episodes of bacterial meningitis • Sinus tracts between deeper tissues and the skin surface • Presence of foreign bodies or avascular areas (e.g. within bone) • Obstruction to the drainage of hollow tubes and viscera (e.g. ob- struction of the biliary tract, urinary tract, or bronchi) • Impaired vascular perfusion of the tissues due to oedema, or angiopathy (including microvascular changes following diabetes mellitus) • Alteration of the normal commensal flora by broad-​spectrum antibiotic therapy • Damage from surgical instruments, perfusion lines, and catheters • Damaged tissues such as damaged cardiac valves Infections that recur in the same anatomical site are often due to defective anatomical or physiological barriers and hence should in- duce a diligent search for such factors. Causative organisms are pyo- genic bacteria such as staphylococci, commensal organisms from the skin or intestinal tract, and fungi, especially candida. Primary immune deficiencies—​combined immunodeficiency (T-​cell immunodeficiency) Primary combined immunodeficiencies are a genetically heteroge- neous group of disorders in which T-​cell hypofunction is accom- panied by variable degrees of humoral immunodeficiency. In the most severe type, a complete block in T-​cell development leads to catastrophic failure of the adaptive immune system, severe com- bined immunodeficiency (SCID). SCID usually presents in infancy, though with increased application of genomics it is evident that mo- lecular defects usually causing SCID may rarely present in later life. Other disorders may be compatible with preserved T-​cell numbers but impaired function, leading to variable degrees of immunodefi- ciency and correspondingly diverse clinical presentations. Figure 4.4.1 summarizes the immunodeficiencies that result from a block in lymphocyte development. Causes of combined immunodeficiency
(T-​cell immunodeficiency) These may be primary (congenital; see Table 4.4.2 for list of condi- tions) or secondary (see Table 4.4.3 for list of conditions). Clearly, HIV infection produces secondary T-​cell immunodeficiency:  the incidence of this disorder varies with geographical location and the presence of risk factors for acquiring HIV infection. Clinical phenotype of patients with T-​cell deficiency or combined immunodeficiency The key clinical features of combined immunodeficiency can be summarized as follows: • susceptibility to infection—​all pathogen types, but especially intracellular pathogens; opportunistic; severe; refractory/​ recurrent (see Table 4.4.1 and Fig. 4.4.1) • immune dysregulation—​ranging from Omenn’s syndrome to organ-​specific autoimmunity • increased risk of neoplasia—​especially lymphomatous and/​or vir- ally associated (e.g. human papilloma virus (HPV)-​associated car- cinoma, Epstein–​Barr virus (EBV)-​lymphoproliferative disease) • variable primary effects on other components of the immune system +/​–​ extrahaematological manifestations—​depending on the molecular defect The clinical phenotype of patients with impaired T-​cell dependent immunity is summarized in Table 4.4.1 and 4.4.4. Major categories of immunodeficiency exhibiting impaired T-​cell function are now described. Severe combined immunodeficiency (SCID) This syndrome is characterized by severe failure of adaptive im- mune responses because of a profound block T-​cell development. Patients with SCID exhibit a clinical and immunological pheno- type characterized by functional defects in both B and T cells. These are rare disorders, with an estimated incidence of 1 in 50 000 to 1 in 100 000 live births. Clinical features SCID presents in infancy, with failure to thrive and recurrent, se- vere, potentially life-​threatening bacterial, viral, or fungal infections. These infections may be caused by a broad range of common patho- gens, but often include persistent infections by organisms that usu- ally exhibit low-​grade virulence (e.g. Candida, cytomegalovirus). Diarrhoea, which is often due to viral infection, is common and associated with failure to thrive. Chronic lung infection may result from respiratory viruses such as respiratory syncytial virus, para- influenza virus, cytomegalovirus, and adenovirus, while interstitial pneumonia caused by Pneumocystis jirovecii is pathognomonic for T-​cell deficiency. Other common infections at presentation include oral candidiasis and regional or systemic spread of bacille Calmette-​ Guérin from the site of neonatal vaccination. Physical signs are chiefly those due to the presence of infection or complications of infection, including failure to thrive. The ab- sence of tonsils or other lymph nodes may be noted, and chest X-​ ray may reveal the absence of a thymus. A syndrome resembling graft-​versus-​host disease, with skin rashes, hepatosplenomegaly, and lymphadenopathy, may result from materno-​fetal engraftment, transfusion of non​irradiated blood or Omenn’s syndrome (see fol- lowing paragraphs). Immunologically, SCID is characterized by lymphopenia compared to age-​related absolute lymphocyte counts, the severe reduction or absence of major lymphocyte subsets, absent in vitro T-​cell proliferation to mitogens, and markedly reduced total and specific antibody levels. Immunological and molecular classification Based on the blood lymphocyte phenotype, patients with SCID can be divided into two broad groups (see Fig. 4.4.1): • T–​B+ SCID—​those who lack T cells but have normal or increased B-​cell numbers • T–​B–​ SCID—​those who lack both T and B cells Defects in four functionally related genes cause T–​B+ SCID. The commonest is X-​linked SCID, due to a defective IL2RG gene that

4.4  Immunodeficiency 351

=

=

= HSC CLP PRO NK PRE B1 PRO T im m B im m T PRE B2 PRE T CD4 T CD8 T NK trans B Thymus CD4 T CD8 T NK Blood

=

=

=

AK2 ADA RAG1/2 DCLRE1C LIG4 PRKDC CD3E CD247 IL2RG JAK3 IL7R IL2RG JAK3 BTK, BLNK IGHM CD79A/B IGLL1, TCF3 PIK3R1 CORO1 CD45 ZAP70 TAP1/2 CD45 CIITA RFX etc PNP, CXCR4 PRO B GATA2 CD3D PNP 2˚ lymphoid tissue naïve mature B MZ B foll B IgM+ plasma cell plasma cell class switched memory B cell BAFFR TWEAK TACI POLE class switch recombination defects: AID UND, PMS2, DNA repair defects MCM4

=

B-cell activation defects: ICOS, CD40, CD40LG, IL21R, CARD11, NIK, NEMO, IKBKB, HOIL1, HOIP, TI RAP, RAK4, MyD88 Fig. 4.4.1  Summary of immunodeficiencies resulting from a block in lymphocyte development. Haematopoietic stem cells differentiate in bone marrow into common lymphocyte precursors, from which NK, T, and B lymphocytes originate. γC, JAK-​3, IL-​7R deficiencies impair γC-​dependent cytokine signalling necessary for T-​cell and NK lymphocyte development. RAG1, RAG2, and DCLRE1C (Artemis) gene mutations impair V(D)J recombination of T-​cell receptor and immunoglobulin genes in pro-​T and pro-​B cells, respectively. HLA class II deficiency impairs development of CD4 T cells. ZAP70 kinase deficiency impairs CD8 T-​cell development and leads to the development of non​functional CD4 T cells. TAP 1/​2 deficiencies impair positive selection of CD8 T cells. µ heavy chain, Igα and β associated subunit, λ5, and BLNK deficiencies prevent the transition from pro-​B to pre-​B cells. BTK deficiency impairs B-​cell development. CD40L, AID (activation-​induced cytidine deaminase), and uracil-​DNA glycosylase (UNG) deficiencies prevent immunoglobulin class switch recombination. Modified from The Lancet, Vol. 357, Fischer A, Primary immunodeficiency diseases: an experimental model for molecular medicine, Pages 1863–​9. Copyright © 2001, with permission from Elsevier.

352 SECTION 4  Immunological mechanisms encodes the signal transducing γ-​chain common to the receptors for six cytokines (interleukins 2, 4, 7, 9, 15, and 21). The absence of response to these critically important cytokines explains the broad range of defects in specific B-​ and T-​cell function in these patients. Failure to respond to interleukins 7 and 15 results in the arrest of T and natural killer (NK) cell development at an early stage. Interaction of the common γ-​chain with the JAK-​3 tyrosine kinase is essential for signal transduction through the aforementioned cytokine recep- tors. Therefore JAK-​3 gene mutations result in an autosomal reces- sive form of SCID with a similar phenotype. Mutation of the α-​chain of the interleukin 7 receptor (IL7R) is a further cause of T–​B+ SCID but in this case NK cell development is preserved. About 50% of patients with T–​B–​ SCID have a mutation in one of the recombinase-​activating genes (RAG1 or RAG2). RAG1 and RAG2 are required to initiate the V, D, J gene rearrangements that generate a normal repertoire of T-​ and B-​cell antigen recep- tors. Without RAG1 and RAG2 function, T-​ and B-​cell develop- ment fails, giving rise to T–​ B–​ SCID. Hypomorphic mutations of RAG1 or RAG2 can cause the distinct entity Omenn’s syndrome (OMIM 603554). In this condition a few T-​cell clones develop and undergo secondary expansion in the periphery, leading to pathologic inflammatory infiltration of the skin and viscera, resembling graft-​versus-​host disease. Although patients with Omenn’s syndrome may have lymphocyte counts within the normal range, their T cells are oligoclonal and clinically they are severely immunodeficient. A few patients with T–​B–​ SCID have mutations in genes required for the repair of double-​strand breaks generated during VDJ re- combination (DCLRE1C, PRKDC, LIG4). These individuals are also highly radiation-​sensitive, which should be taken into account when designing conditioning regimens prior to HSCT. About 15% of cases of SCID are caused by adenosine deaminase (ADA) deficiency, which shows autosomal recessive inheritance. This enzyme is essential for the salvage of nucleotides within lymphoid cells. The lack of ADA results in the accumulation of toxic purine metabolites, and increased rates of lymphocyte death, through mechanisms that are incompletely understood. ADA defi- ciency results in profound lymphopenia with reduced T, B, and NK cells. Rare hypomorphic mutations of adenosine deaminase may cause a milder defect that presents in older patients. Importantly, ADA function can be supplied to deficient individuals in the form of pegylated enzyme replacement therapy as a temporizing measure. This is also one of the few conditions to have been successfully treated by gene therapy with excellent rates of survival and immune reconstitution, even compared with stem cell transplantation. None of these treatments prevents neurodevelopmental effects of ADA deficiency, which is associated with both cognitive and behavioural problems in survivors. Purine nucleoside phosphorylase (PNP) is another enzyme re- quired for purine salvage within lymphocytes. PNP deficiency causes a rare form of SCID which has a milder immunological but typically a more severe neurodevelopmental phenotype than seen in ADA deficiency. PNP deficiency is nevertheless usually fatal in childhood, unless treated with HSCT. A rare group of defects responsible for SCID impairs signal trans- duction through the T-​cell receptors. This includes a defect in the protein tyrosine phosphatase CD45 which perturbs signalling through both T-​ and B-​cell receptors. Mutation of the δ-​chain of the CD3 complex also causes SCID while defects in the γ-​ and ε-​chains of CD3 or the TCR-α chain may cause a milder phenotype. A mu- tation of the gene encoding ZAP70 (zeta chain associated protein 70), which interacts with the ζ-​chain of CD3, results in severe CD8 lymphopenia and profound immunodeficiency resembling SCID. In this condition, CD4 lymphocyte counts may be normal but their function is also reduced. A mutation of the ORAI1 gene encoding a subunit of the plasma membrane calcium channel CRAC, causes a rare form of SCID due to defective calcium entry into T cells which impairs T-​cell function. Recently, a T-​B+ NK+ form of SCID has been described in which a failure of T-​cell egress from the thymus is caused by a mutation of the gene CORO1A, encoding for the actin regulatory protein coronin 1A. Diagnosis The diagnosis of SCID is readily suspected in infants who fail to thrive and suffer from recurrent severe infections from an early age. The clinical features raising the suspicion of SCID are summar- ized earlier. SCID is a medical emergency, as patients can rapidly succumb to life-​threatening infections. Untreated SCID is invari- ably fatal, with most children dying in the first year of life, and the balance succumbing within the second year. Conversely, early stem cell transplantation results in long-​term survival in more than 90% of cases. The occurrence of intractable infections prior to transplant is associated with poorer outcome. For the aforementioned reasons, early diagnosis is essential and newborn screening for T lymphopenia has therefore been imple- mented or is planned in several countries. This capitalizes on ex- isting neonatal screening programmes by utilizing portions of the same dried blood spots for testing. Innovatively, the test involves amplification by polymerase chain reaction of T-​cell receptor ex- cision circles (TRECs). TRECs are small, circularized fragments of DNA that are generated in the process of TCR gene rearrangement and are normally found in a fraction of circulating T cells. SCID leads to a profound lack of TRECs, although these are also reduced in a variety of other T-​lymphopenic states. Babies with low TREC numbers are urgently referred for further assessment including flow cytometric characterization of peripheral lymphocyte subsets. The TREC screening test appears to perform well with high sensitivity and acceptable specificity. A detailed family history should enquire into consanguinity of parents, the occurrence of immunodeficiency in other family mem- bers, and deaths in early infancy within the pedigree. HIV infection may present with a similar clinical picture and needs to be excluded with appropriate tests. Initial tests used in the assessment of an individual with possible SCID are: • blood count and differential count • enumeration of blood lymphocyte populations • measurement of serum immunoglobulins Severe lymphopenia (absolute lymphocyte count <2.5 × 109/​ litre in the first year of life) is a characteristic feature seen in over 80% of patients with SCID. Hence SCID needs to be excluded in all infants with a lymphocyte count below the age-​related refer- ence range. The second stage is to enumerate blood lymphocyte subsets (T cells, B cells, and NK cells) using flow cytometry. These

4.4  Immunodeficiency 353 results should be interpreted using age-​matched reference ranges. The minimum panel of monoclonal antibodies recommended for lymphocyte phenotype determination is summarized in Table 4.4.5. The lymphocyte phenotypes typically associated with different mo- lecular variants of SCID are summarized in Table 4.4.6. The absence of lymphopenia does not completely rule out SCID. This can occur in SCID patients engrafted with transplacentally ac- quired maternal lymphocytes, in Omenn’s syndrome, or in T-​B+ SCID. In patients behaving as SCID despite apparently normal T-​cell numbers, a lack of naive T cells, oligoclonality of the T-​cell reper- toire, and poor in vitro T-​cell proliferation to mitogens help to con- firm a diagnosis of SCID/​Omenn’s. These tests are only available in specialized centres. HLA typing of the mother and baby will help to distinguish maternal engraftment from Omenn’s syndrome. Serum immunoglobulin levels are difficult to interpret in young infants. In SCID, IgM and IgA levels are usually low. The level of IgG, which is maternally derived, may be normal in early infancy but pro- gressively declines with time. In Omenn’s syndrome, IgE levels may be elevated. SCID is the probable diagnosis in infants who are less than 2 years of age and have (1) an absolute lymphocyte count less than 2.5 × 109/​litre; (2) CD3 cells less than 20% of the total lymphocyte count; and (3) proliferative responses to mitogens less than 10% of control values. Maternal engraftment is also an absolute indication of SCID. Once the diagnosis of SCID or Omenn’s syndrome is considered likely, additional investigations to identify the molecular phenotype of SCID are important, as these may guide the details of therapy, family counselling, and prenatal diagnosis. Such tests are available through nationally designated laboratory services and include metabolic studies (to identify adenosine deaminase or purine nu- cleoside phosphorylase deficiency), detection of proteins required for lymphocyte function by flow cytometry or Western blotting (e.g. to identify common γ-​chain defect), signalling assays (e.g. STAT5 phosphorylation by flow cytometric analysis), and mutation analysis of candidate genes. Management and prognosis Untreated SCID has a fatality rate of 100%. Patients suspected of SCID should be transferred to expert paediatric centres as soon as possible. Immediate management includes protective isolation, prophylaxis for Pneumocystis jirovecii pneumonia, a diligent search for existing infection and its treatment, as well as general supportive care. If infection is suspected but the microbiological diagnosis is uncertain, empirical antimicrobial therapy is usually indicated. Any blood transfusions should be irradiated and from cytomegalovirus-​ negative donors. Live vaccines are contraindicated. Immunoglobulin replacement should commence without delay. Once a diagnosis of SCID is confirmed, haemopoietic stem cell transplantation (HSCT) from an HLA-​identical family donor is gen- erally the treatment of choice. If a tissue-​matched family donor is not available, other donor choices include matched unrelated or a T-​cell-​depleted haplo-​identical family donor (typically a parent). In patients with SCID, HSCT can be achieved with little or no im- munosuppressive therapy, although long-​term immune reconstitu- tion may depend on myeloid engraftment and hence the degree of myeloablative conditioning. European data to 2005 indicate that 10-​year survival after trans- plants from HLA-​matched unrelated donors was 66%, while matched sibling donor transplants produced 84% survival. Treatment with HSCT before 3.5 months of age has produced 95% long-​term survival. Delay in treatment and (particularly) the occurrence of in- fection impairs outcome. Infection and graft-​vs.-​host disease are the main short-​term complications following stem cell transplantation. Review of European data between 1968 and 2005 indicates the pro- gressive improvement of outcome over time, which is mainly due to better prevention of graft-​versus-​host disease and the aggressive treatment of infection. Gene therapy for SCID Long-​term immune reconstitution is possible following gene therapy in patients with SCID caused by the common γ-​chain deficiency or ADA deficiency. This can be achieved by ex vivo gene transfer to haemopoietic stem cells isolated from the patient’s bone marrow. Table 4.4.5  Designations of monoclonal antibody combinations recommended for lymphocyte phenotyping Surface antigen recognized
by antibody Cells recognized CD3 All T cells CD3+CD4 T helper CD3+CD8 T cytotoxic CD16 and/​or CD56 NK cells CD19 or CD20 B cells MHC class II B cells, monocytes, activated T cells MHC class I All nucleated cells CD3+TCRαβ TCRαβ–​bearing T cells CD3+TCRγδ TCRγδ-​bearing T cells Table 4.4.6  Blood lymphocyte profile in different molecular forms of SCID particularly associated with lymphopenia SCID variant CD3+ CD4+ CD8+ B cells NK cells IL2RG (common γ-​chain), JAK3, Low Low Low Normal Low IL7RA, CD3D, CD3E, CD247, PTPRC, CORO1A Low Low Low Normal or high Normal or high RAG 1, RAG2, DCLRE1C (Artemis), PRKDC, LIG4, Cernunnos Low Low Low Low Normal ADA, AK2 Low Low Low Low Low MHC class II deficiency, LCK, MAGT1, UNC119 Normal Low Normal Normal Normal ZAP-​70, MHC I deficiency (TAP1, TAP2, TAPBP, B2M), CD8 Normal Normal Low Normal Normal Omenn syndrome (hypomorphic mutations) Low, normal, or high Variable Variable Usually low Normal

354 SECTION 4  Immunological mechanisms These gene-​reconstituted stem cells are retransfused into the patient, sometimes after mild conditioning. To date, gene therapy has been restricted to patients without an HLA-​matched family donor. Unfortunately, several cases of leukaemia occurred among γ-​ chain deficient patients treated in early trials of gene therapy. In these cases, the retroviral vector had integrated close to the LMO2 proto-​ oncogene in the leukaemic clone, leading to aberrant transcription and expression of LMO2. While this resulted in temporary discon- tinuation of gene therapy, safer treatment protocols have since been developed and trials continue with improved vectors. Other combined immunodeficiencies (CID) There are forms of combined immunodeficiency in which T-​cell de- velopment is preserved to a greater or lesser extent, yet T-​cell effector function is impaired. Sometimes this produces a clinical phenotype as severe as SCID—​as in the case of deficiencies of CARD11, IKBKB, ZAP70, and MHC class II. However, most non-​SCID disorders af- fecting T cells produce an immunodeficiency that is less severe, with onset typically delayed beyond infancy. In clinical practice this often means that patients progressively acquire end-​organ damage re- sulting from chronic or recurrent infection and/​or autoimmunity. Confident diagnosis of individual disorders in the absence of genetic testing is often difficult unless characteristic associated syn- dromic features are present (for example, skeletal dysplasia, facial dysmorphism, abnormal dentition, and so on). It is beyond the scope of this chapter to detail all forms of CID individually, but some generalizations about clinical behaviour will be made before describing selected illustrative disorders. Susceptibility to viruses is typically milder than in SCID and simple infections including respiratory viruses may be cleared. However, fatal primary infection with common exanthematous viruses (e.g. measles, varicella zoster virus (VZV)) can occur, and viral infections such as rotavirus or norovirus may persist abnormally. Patients com- monly fail to suppress herpes viruses such as CMV, herpes simplex virus (HSV), VZV, and EBV. Certain immunodeficiencies cause a particular predilection to EBV-​related lymphoproliferation, notably deficiencies of ITK, MST1, CD27, and CTPS1. EBV may also cause severe infectious mononucleosis or hairy leukoplakia of the tongue (as in HIV-​AIDS) in affected individuals. Persistent oral candidiasis in an adult, without predisposing fac- tors like broad-​spectrum antibiotic therapy, the wearing of den- tures, or the use of inhaled corticosteroids, and which recurs after antifungal treatment, is highly suspicious of T-​cell deficiency. In these patients Candida may affect the oesophagus and trachea as well. Interestingly, invasive candidiasis is not a typical feature of T-​cell deficiency whereas invasive infection caused by filamentous fungi (Aspergillus, Mucor) or Cryptococcus can occur in more severe forms of CID, as can interstitial pneumonia caused by Pneumocystis jirovecii. T-​cell deficient patients are highly susceptible to de novo infec- tion or reactivation of tuberculosis, which may be disseminated, extrathoracic, or atypical in presentation. In populations with low tuberculosis (TB) prevalence, other poorly pathogenic mycobac- teria may cause opportunistic infection, including Mycobacterium avium intracellulare and Bacille Calmette–​Guérin (BCG), which can be life-​threatening. Other intracellular bacteria such as Salmonella spp. may also establish persistent infection. Protozoal pathogens including Cryptosporidium and Giardia are on the differential diag- nosis for chronic gastrointestinal symptoms in these patients. Many with CID experience recurrent respiratory tract infections culminating in bronchiectasis, even though total immunoglobulins may be present in normal quantities. The quality of vaccine-​specific responses are often impaired, and susceptibility to pneumococcal disease is often a particular feature. Infection-​related malignancies may develop at excessive rate; for example, Epstein–​Barr virus-​induced non-​Hodgkin’s lymphoma, HPV-​related carcinoma, and Kaposi’s sarcoma (in which human herpes virus 8 is the cofactor). In addition, many disorders show an increased tendency towards lymphoma that is independent of EBV. There is an increased incidence of cutaneous malignancies in indi- viduals who are exposed to significant amounts of ultraviolet light (e.g. basal cell carcinoma and squamous cell carcinoma of skin). Skin malignancies are not common in northern latitudes but are typically seen in parts of the world with high year-​round sun exposure. Autoimmunity is an increasingly recognized manifestation of combined immunodeficiency, presumably reflecting impaired homeostasis of an immune system that may lack critical regula- tory components. The commonest manifestations are autoimmune cytopenias, particularly thrombocytopenia and autoimmune haemolytic anaemia, but neutropenia is also seen. Certain disorders (Wiskott–​Aldrich syndrome, hyper IgE syndrome (STAT3), STAT5b deficiency) are linked to atopic phenomena, especially dermatitis, but also occasionally food allergy (e.g. in DOCK8 deficiency). MHC class II deficiency The lack of expression of MHC class 2 on lymphocytes (MHC class 2 deficiency) leads to a combined immunodeficiency that clinic- ally resembles SCID. Autosomal recessive defects in various genes encoding for components of a transcription complex promoting the transcription of MHC class 2 genes can lead to this condition. CD4 T lymphocytes recognize antigen in the context of MHC class 2 genes expressed by antigen-​presenting cells. The absence of MHC class 2 therefore results in a failure of normal CD4 cell development and function. In turn this produces a severe failure of cell-​mediated im- munity. The consequent absence of CD4-​mediated help for B cells results in defective antibody responses. Wiskott–​Aldrich syndrome This X-​linked syndrome (OMIM 301000) is characterized by eczema and thrombocytopenic purpura with small, defective platelets and combined immunodeficiency. Patients usually present in infancy with a bleeding tendency, manifesting as petechiae, bruising, pro- longed bleeding from wounds, or bloody diarrhoea. Eczema can vary in severity. Antibody production to bacterial capsular polysacchar- ides is deficient and protein antibodies decline abnormally quickly. Patients therefore commonly develop recurrent sinopulmonary and middle ear infections. Progressive T lymphopenia develops with time, and T cells and NK cells display reduced functional capacity. Hence patients can develop opportunistic infections typical of T-​cell deficiency. Autoimmune conditions such as colitis, glomeruloneph- ritis, vasculitis, and autoimmune cytopenias occur in these patients. The risk of malignancies in Wiskott–​Aldrich syndrome patients has been estimated at 2% per year. Lymphomas are the most frequent tumours, most of which are induced by Epstein–​Barr virus.

4.4  Immunodeficiency 355 The gene that is defective in Wiskott–​Aldrich syndrome codes for the Wiskott–​Aldrich syndrome protein (WASP), which is a cytoplasmic component that regulates actin polymerization and cytoskeletal reorganization, required for normal platelet and leuko- cyte function. For example, WASP is involved in the formation of immunological synapses between cooperating T cells and antigen-​ presenting cells. Certain missense mutations of WASP cause X-​linked thrombocytopenia or X-​linked neutropenia. It is now rec- ognized that female carriers with skewed X-​chromosome inactiva- tion can occasionally be symptomatic. The diagnosis of Wiskott–​Aldrich syndrome is suspected on identifying thrombocytopenia with small platelets and confirmed in many cases by demonstrating the absence of WASP by Western blotting or flow cytometry. The severity of disease relates loosely to the degree of WASP function, its complete absence being associated with a severe phenotype. In the days before stem cell transplantation, the outlook for these patients was poor, with a median survival of 5–​7 years. HLA-​identical sibling-​derived HSCT is curative and associated with an approxi- mately 90% 5-​year survival. HLA-​matched unrelated transplants car- ried out before 5 years of age have a similar success rate. Above this age, individual risk assessment is needed. Immunoglobulin replace- ment therapy and antibiotics are supportive therapies. Splenectomy may help to raise platelet counts but compounds the existing immunocompromise and is associated with increased risk of sepsis both pre-​ and post-​transplant. LRBA deficiency (lipopolysaccharide (LPS) responsive
beige-​like anchor protein) LRBA is a member of the BEACH-​WD40 protein family and is ex- pressed in tissues including haematopoietic, neural, gastrointes- tinal, and endocrine cells. The repeated WD40 domain, located at the C-​terminal of LRBA, is highly conserved and participates in multiple cellular processes, including cytoskeleton assembly, signal transduction, vesicular trafficking, transcriptional regula- tion, chromatin dynamics, and apoptosis. In normal T cells, LRBA colocalizes with CTLA4 within recycling endosomes and the trans-​ Golgi network The disease phenotype caused by LRBA deficiency comprises a combination of enteropathy, autoimmunity, and immuno- deficiency, as well as lymphoproliferation. The enteropathy includes a spectrum of autoimmune conditions, an inflamma- tory bowel disease-​like condition and non​infectious diarrhoea; the autoimmunity phenotype includes autoimmune cytopenias; and the immunodeficiency phenotype includes combined im- munodeficiency (CID) and a common variable immune defi- ciency (CVID)-​like disease. Patients with CVID-​like disease can develop interstitial lung disease due to dense T-​cell infiltrates. A few patients have developed B-​cell lymphomas. There is no obvious genotype-​phenotype correlation as patients with the same mutation may have different clinical phenotypes or even be asymptomatic. An important function of LRBA is to support the expression of the T-​cell costimulatory molecule CTLA-​4 (cytotoxic T-​lymphocyte antigen-​4) by regulating its trafficking to the lysosomal compart- ment. It has therefore been postulated that the clinical manifest- ations of LRBA deficiency are due to underexpression of CTLA-​4. This is supported by studies showing that abatacept, a fusion pro- tein that mimics CTLA4 function, is associated with marked clinical improvement. Hyper IgM syndromes During primary antibody responses, B cells initially produce IgM. They later switch to the production of IgG, IgA, and IgE. This pro- cess is called immunoglobulin class switching and is associated with somatic hypermutation of the immunoglobulin variable-​region genes resulting in enhancement of antibody affinity for the stimu- lating antigens (affinity maturation). Reflecting the complexity of this process, several different molecular defects can lead to failure of immunoglobulin class switching and affinity maturation, as well as impaired generation of B memory cells. CD40L and CD40 deficiencies One of the key steps in the process of immunoglobulin class switching is the interaction of CD40 on the surface of B cells with the activation-​induced CD40 ligand (CD40L) protein on the surface of CD4 lymphocytes. Failure at this point may be caused by muta- tions in the CD40 ligand gene or the CD40 gene, which result in X-​ linked and autosomal recessive hyper IgM syndromes, respectively. CD40L deficiency can be diagnosed by demonstrating the absence of this protein on the surface of in vitro activated T cells by flow cytometry and confirmed by screening the CD40LG gene for mu- tations. Boys with defects in CD40L suffer from recurrent bacterial infections typical of antibody deficiency. However, they also suffer from opportunistic infections characteristic of T-​cell deficiency such as Pneumocystis jirovecii pneumonia (around one-​third present this way), cryptosporidiosis, toxoplasmosis, and non​tuberculous mycobacterial infection. These opportunistic infections can be ex- plained on the basis that CD40L on activated T cells is also involved in the activation of macrophages and dendritic cells. Many patients with CD40L deficiency develop progressive liver damage (sclerosing cholangitis), probably as a result of cryptosporidial infection of the bile ducts. Recurrent or persistent neutropenia and thrombocyto- penia occur in over one-​half of patients with CD40L deficiency. NEMO deficiency Hypomorphic mutations of the gene encoding the NFκB essen- tial modulator (NEMO, IKBKG), a component of the NFκB acti- vation pathway which is required for the B-​cell activation process (including signal transduction following CD40/​CD40L interaction), causes a further rare form of X-​linked combined immunodeficiency. This may present as hyper IgM syndrome, but there is usually strong clinical evidence of combined immunodeficiency, such as oppor- tunistic infections (pneumocystis, mycobacteria) and sometimes inflammatory bowel disease. Signalling via NF-​κB is also essential for ectodermal development and many (but not all) patients with NEMO defects have ectodermal dysplasia characterized by dental hypoplasia, reduced sweating, and hypoplastic hair. Autosomal dominant activating mutations in the related gene NFKBIA can cause a similar phenotype. DNA editing enzyme mutations Defects in the DNA editing enzymes activation-​induced cytidine deaminase (AICDA) and uracil-​DNA glycosylase (UNG) interfere

356 SECTION 4  Immunological mechanisms directly with the class-​switching process to cause hyper IgM syn- drome and are the only pure B-​cell defects currently known to cause hyper IgM syndrome. Homozygous mutation in the PMS2 compo- nent of the DNA mismatch repair machinery was identified as a rare cause of defective immunoglobulin class switching, resulting in low levels of serum IgG and IgA. PI3Kinase δ mutations Recently an activating heterozygous mutation in PI3Kinase δ was found to be responsible for about 10% of cases of hyper IgM syn- drome unexplained by one of the genetic defects just described. Clinically, this is characterized by recurrent respiratory infection with progressive lung damage, susceptibility to B-​cell lymphoma, nodular lymphoproliferation, and increased susceptibility to herpes viral infections. Laboratory evaluation reveals IgG2 subclass defi- ciency, normal or modestly elevated serum IgM levels, impaired specific antibody responses to bacterial capsular polysaccharides, and skewing of CD8+ T cells towards differentiation and senes- cence. Heterozygous splicing mutations in the PIK3R1 gene cause a similar syndrome. Immunoglobulin replacement therapy is required for patients with all forms of hyper IgM syndrome. Patients with CD40L defi- ciency require prophylaxis for Pneumocystis jirovecii pneumonia and precautions to prevent cryptosporidial infection, including boiling drinking water. Because of the high risk of developing severe liver disease, haemopoietic stem cell transplantation has been used to treat CD40L deficiency diagnosed in infancy but this practice is by no means universal. X-​linked lymphoproliferative syndromes Males with type I  X-​linked lymphoproliferative disease (XLP1), also called Duncan’s disease, have a mutation in an adaptor pro- tein called SAP (surface lymphocyte activation molecule associated protein). This regulates the activation of T lymphocytes and NK cells, and is particularly important in defence against herpes virus infections. Patients with XLP1 have defective NK and CD8 T-​cell cytotoxicity towards Epstein–​Barr virus-​(EBV-​)infected B cells and reduced numbers of certain innate-​like T cells known as natural killer T (NKT) cells. The persistence of virally infected cells together with ineffective responses by dysregulated CD8+ T cells drives the immunopathology of this condition. Most patients present with severe infectious mononucleosis with a high mortality (80%), usually caused by a hepatic necrosis induced by activated cytotoxic (CD8) T cells. Other consequences of EBV infec- tion in these patients include haemophagocytic lymphohistiocytosis, aplastic anaemia, the development of B-​cell non-​Hodgkin’s lymphoma, and/​or progressive dysgammaglobulinaemia. The out- look of EBV-​infected patients is poor, with most dying in childhood unless treated with stem cell transplantation. Mutations in the gene XIAP which encodes for the protein X-​linked inhibitor of apoptosis causes a second form of X-​ linked lymphoproliferative syndrome. Similar to XLP1, EBV-​ triggered haemophagocytic lymphohistiocytosis (HLH), and hypogammaglobulinaemia are major features, but XLP2 is distinguished clinically by frequent splenomegaly, occurrence of colitis, and lack of lymphomatous transformation. Flow cytometric testing can reveal lack of SAP or XIAP expression and has become an important screen in males with severe EBV-​ related disease. Thymic defects Hemizygous deletion of chromosome 22q11 (del22q11.2) causes a complex syndrome including cardiac malformation, thymic hypoplasia, palatal abnormalities with associated velopharyngeal dysfunction, hypoparathyroidism, and facial dysmorphism, known as DiGeorge syndrome or thymic aplasia (OMIM 188400). 22q deletion has an incidence of about 1 in 2500 live births, but the clinical phenotype is highly variable. Some patients with 22q deletion have normal thymic development (and hence normal T-​cell mediated immunity) but have cardiac, pharyngeal, and a var- iety of other defects associated with the velocardiofacial (VCF) or Shprintzen syndrome (OMIM 192430). These abnormalities arise from defective development of the third and fourth branchial arches during fetal development. Only about 20% of those with 22q deletion show evidence of re- duced number and function of T cells. The degree of T lymphopenia is modest in most affected infants, and a near normal repertoire and the function of T cells is acquired by 2 years of age; infections char- acteristic of T-​cell deficiency are therefore uncommon. A minority (<1%) exhibit profound T lymphopenia (CD3 count <0.5 × 109/​litre) and manifest a SCID-​like phenotype, with opportunistic infections (‘complete’ DiGeorge syndrome). Such patients have been treated with HLA-​matched stem cell transplants or thymic transplants with variable success. The diagnosis of 22q deletion should be considered in any child with congenital heart disease, velopharyngeal abnormalities, or neo- natal hypocalcaemia. The 22q deletion that is seen in 95% of patients with DiGeorge/​velocardiofacial syndrome can be readily detected by cytogenetic studies employing fluorescent in-​situ hybridization. In most (96%) affected individuals the 22q deletion is de novo and in the remaining 4% it is inherited from a parent. There is exten- sive phenotypic overlap between 22q11 deletion and CHARGE syndrome, the latter often resulting from heterozygous mutations in CHD7. TBOX 1 (TBX1) is a gene which maps to the centre of the DiGeorge syndrome chromosomal region on 22q11.2. It is one member of the so-​called TBOX genes, which are transcription factors involved in the regulation of developmental processes. TBX1 mutations have been identified in patients with the clinical phenotypes that are seen in the del22q11.2 syndrome, including abnormal facies, cardiac de- fects, thymic hypoplasia, velopharyngeal defects, and hypoparathyr- oidism. This suggests that haploinsufficiency of the TBX1 gene may be responsible for significant components of the phenotype of the 22q deletion syndrome. DNA repair defects associated with immunodeficiency Ataxia telangiectasia Cerebellar ataxia, oculocutaneous telangiectasia, growth retard- ation, variable immunodeficiency, and autosomal recessive in- heritance are typical features of ataxia telangiectasia (OMIM 208900). Affected individuals exhibit increased sensitivity to ion- izing radiation and radiomimetic drugs, and 80% of patients show increased susceptibility to malignancy, especially leukaemias and lymphomas. IgA deficiency, with or without IgG subclass

4.4  Immunodeficiency 357 deficiency, and defective responses to bacterial capsular polysac- charides are common. Patients therefore often develop recurrent sinopulmonary infections. Lymphopenia and impaired T-​cell function may also be detected. Chromosomal translocations cor- responding to the locations of immunoglobulin heavy chain and T-​cell receptor loci are commonly detected in T cells of ataxia tel- angiectasia patients. The product of the affected gene, ATM, is required for detecting double-​stranded breaks in DNA prior to their repair. This explains the radiation sensitivity, abnormal immune cell development and function, and the cytogenic abnormalities that are frequently detected in ataxia telangiectasia. Some 95% of affected individ- uals have elevations in serum α-​fetoprotein, which is helpful for diagnosis. There is unfortunately no specific treatment for this condition and most patients die by the third decade of lymphoreticular malignancy or complications of neurological disease. Other DNA repair defects associated with immunodeficiency Several other, rare defects in the process of non​homologous DNA double-​strand break repair cause similar syndromes of immuno- deficiency, genetic instability including sensitivity to ionizing ra- diation, and neurodevelopmental delay. The Nijmegen breakage syndrome (OMIM 251260)  is caused by mutation of the NBN gene encoding a protein that acts as a substrate for ATM and which is also critical for sensing damage to DNA. Both Nijmegen Breakage Syndrome and DNA-​ligase 4 deficiency (OMIM 601837), which also causes defective DNA repair, result in growth retard- ation with disproportionate microcephaly and immunodeficiency. Cernunnos deficiency is a related disorder caused by mutations of NHEJ1. Mutation of the MRE11A gene, which encodes for another component of the DNA damage-​sensing machinery, causes a syn- drome similar to ataxia telangiectasia (OMIM 604391), but without mutations in the ATM gene. Autosomal dominant hyper IgE syndrome Autosomal dominant hyper IgE syndrome (OMIM 147060)  is a condition characterized by recurrent bacterial (S.  aureus, Gram-​ negative bacteria) and fungal infections of skin, lymph nodes, lungs, bones, and joints; dermatitis; facial dysmorphic features; delayed shedding of primary dentition connective tissue defects, especially cardiovascular (aneurysms); and osteopenia. These patients have elevated serum IgE, levels, eosinophilia, and impaired acute-​phase responses during infections. Patients with this disorder have heterozygous mutations in the gene encoding the signal transducing protein STAT3. These mutant proteins reduce the DNA binding of the phosphorylated STAT3 dimer in response to interferon-​α, IL-​10, and IL-​6. This results in a combination of functional cytokine defects: reduced response to IL-​6 explains the defective acute-​phase response, and the defective response to IL-​10 explains the overproduction of IgE. STAT3 is es- sential for the generation of T-​helper 17 cells, which produce the cytokines IL-​17 and IL-​22 that are required for the secretion of the bactericidal peptides called β-​defensins by epithelial cells of the skin and lungs, as well as for neutrophil mobilization and recruit- ment to the sites of infection. This may in part explain the increased incidence of severe bacterial and fungal sepsis, especially involving the lungs. Primary immune deficiencies—​ predominantly antibody deficiencies Antibody deficiency diseases are characterized by a decrease in the levels of serum immunoglobulins to below the fifth centile for age. The reduction may be in all classes of immunoglobulins or a single isotype. Clinical features associated with antibody deficiency are summarized in Table 4.4.4. Disorders causing antibody deficiencies Common variable immune deficiency (CVID) Patients with CVID are a heterogenous group, the diagnosis being based on the exclusion of other known causes of antibody deficiency. CVID is the commonest primary immunodeficiency disease with an estimated incidence of 1 in 10 000 to 1 in 50 000. It affects both sexes equally and can present at any age, although the modal presentation is in the second or third decade of life. The underlying molecular defect in most CVID patients is unknown, although the number of single-​gene defects identified is increasing with the application of next generation sequencing methodologies (see next), and 10–​15% of cases can be attributed to a specific molecular defect involved in B-​cell maturation and differentiation. Many cases are sporadic while others are familial, with autosomal recessive or dominant modes of inheritance. There is clinical variation within affected pedigrees, with the phenotype ranging from selective IgA deficiency to CVID. These defects are summarized in Table 4.4.2. Suspected antibody deficiency should be confirmed with: 1. Measurement of serum IgG, IgA, IgE, and IgM (IgG subclasses (see section discussing IgG subclass deficiency, next)) and com- parison with age-​specific normal ranges to determine if these levels are below the fifth centile. 2. Serum protein electrophoresis and, if required, immunofixation for the exclusion of paraproteinaemia, with the possibility of malignancies of the B-​cell system (myelomatosis or B-​cell non-​ Hodgkin’s lymphoma) excluded by appropriate investigations. 3. Response to immunization with T-​cell dependent (tetanus toxoid, haemophilus B conjugate) and T-​cell independent (poly- valent Pneumococcal polysaccharide, if >2 years of age) vaccines should be assessed. Diagnosis and differential diagnosis As with other major immunodeficiencies, diagnostic criteria for CVID have been agreed by a consortium of European and American immunologists (Box 4.4.1). The condition is a clinically defined syndrome characterized by susceptibility to infection accompanied by a reduction of serum IgG below the fifth centile for age, and with evidence of impaired specific antibody production in response to natural microbial exposure or vaccination. Serum IgA is reduced in most patients with CVID, while IgM is often but not invariably re- duced. Since CVID is a diagnosis of exclusion, patients with normal or elevated serum IgM should be evaluated for hyper IgM syn- dromes, and X-​linked agammaglobulinaemia should be excluded in male patients with antibody deficiency and B lymphopenia. It is also essential to exclude secondary causes of antibody deficiency (Table 4.4.3). The clinical features of antibody deficiency are sum- marized in Box 4.4.2.

358 SECTION 4  Immunological mechanisms X-​linked agammaglobulinaemia This is caused by a defect in a cytoplasmic tyrosine kinase desig- nated Bruton’s tyrosine kinase (BTK), which results in the arrest of B-​cell maturation at the pre-​B-​cell stage. As a consequence, there is peripheral B lymphopenia associated with profound antibody de- ficiency. The gene for BTK is encoded on the X chromosome and affected males usually develop recurrent infections typical of anti- body deficiency, commencing at around 6 months of age when ma- ternal immunoglobulin has been catabolized. They may also fail to handle certain viral diseases (notably enteroviral encephalitis) and enteric infections (e.g. giardiasis) for reasons that are incompletely understood. Characteristic diagnostic features include profound reduction of all immunoglobulin isotypes (below the fifth centile for age) and absence of isohaemagglutinins and responses to childhood vaccines. Numbers and function of T lymphocytes are normal. Demonstration of the absence of BTK protein in monocytes or platelets by flow cytometry or Western blotting, or a demonstration of a pathogenic mutation in the BTK gene, confirm the diagnosis but are not essential. Patients with X-​linked agammaglobulinaemia do not develop systemic granulomatous disease, as is seen in CVID. In female car- riers the chromosome carrying the BTK mutation is preferentially lyonized during B-​cell development. During the characterization of the BTK gene defect it has been recognized that the clinical pheno- type may vary, even within the same family. Some affected males may therefore present at a later age and the condition should be con- sidered in all males with antibody deficiency, especially in the pres- ence of B lymphopenia. The outlook is good provided X-​linked agammaglobulinaemia is diagnosed early, before organ damage is evident, and patients are treated with optimum immunoglobulin replacement therapy and antibiotics as required. Autosomal recessive antibody deficiencies with B lymphopenia Seven autosomal recessive gene defects have been identified as resulting in antibody deficiency associated with severe B lymphopenia. These are mutations in the µ heavy chain gene (IGHM), the gene encoding the surrogate light chain which is util- ized by the pre-​B-​cell receptors (IGLL1), and signalling compo- nents of the B-​cell receptor complex, namely Igα and Igβ (CD79A and CD79B, respectively) and the signal transducing/​scaffold pro- tein called B-​cell linker protein (BLNK). A homozygous truncating variant in the PIK3R1 gene also causes hypogammaglobulinaemia Box 4.4.1  European Society of Immunodeficiency diagnostic criteria for CVID Probable Male or female patient who has a marked decrease of IgG (at least 2 SD below the mean for age) and a marked decrease in at least one of the isotypes IgM or IgA, and fulfils all of the following criteria: 1 Onset of immunodeficiency at greater than 2 years of age 2 Absent isohaemagglutinins and/​or poor response to vaccines 3 Defined causes of hypogammaglobulinemia have been excluded Possible Male or female patient who has a marked decrease (at least 2 SD below the mean for age) in at least one of the major isotypes (IgM, IgG, and IgA) and fulfils all of the following criteria: 1 Onset of immunodeficiency at greater than 2 years of age 2 Absent isohaemagglutinins and/​or poor response to vaccines 3 Defined causes of hypogammaglobulinemia have been excluded Box 4.4.2  Clinical features associated with antibody deficiency 1 Recurrent infections caused by encapsulated bacteria, for example Streptococcus pneumoniae or Haemophilus influenzae type B (HIB). Sites involved are the upper and lower respiratory tract, middle ear, meninges, bones, and joints. Most patients with antibody deficiency suffer from repeated sinopulmonary infections which eventually result in structural damage, and bronchiectasis is the most important cause of morbidity in these patients. Non​typeable Haemophilus influenzae commonly cause exacerbations of sinopulmonary infections. Less common respiratory pathogens in patients with antibody deficiency include S. aureus and Gram-​negative bacteria such as Pseudomonas spp. Infections by fungi, intracellular bacteria (e.g. mycobacteria) or parasites are not usually a problem in these patients. 2 Viral infections are generally not a problem in pure antibody deficiency diseases, except for the rare occurrence of enteroviral infections. ECHO viruses or Coxsakie viruses can cause meningoencephalitis or dermatomyositis-​like conditions. Poliomyelitis associated with oral polio vaccine has been rarely reported in patients with antibody deficiency. 3 Arthritis has been reported in a few patients. This may be septic caused by HIB, S. pneumoniae, or mycoplasma/​ureaplasma, or aseptic, re- sembling seronegative rheumatoid arthritis. 4 Diarrhoea and malabsorption may occur due to chronic infection with intestinal pathogens including giardia, campylobacter, salmon- ella, or cryptosporidium, or as a consequence of bacterial overgrowth in the small intestine. Chronic diarrhoea is often associated with a mild colitis and a minority may have Crohn’s-​like inflammatory bowel disease with ileitis and occasional strictures. A few patients may have intestinal villous atrophy with a non​specific inflammatory infiltrate of the mucosa and submucosa, and a minority of these will respond to a gluten-​free diet, although antibody-​based screening tests for coeliac disease will be negative. In common variable immunodeficiency (CVID) the following features may be seen 5 Intestinal villous atrophy in CVID is often caused by chronic norovirus genotype 2 infection. Patients with antibody deficiency associated with this condition may have nodular submucous lymphoid hyper- plasia throughout the small intestine and occasionally the large intes- tine. This is usually clinically silent, although occasionally these lesions may bleed or cause obstruction. 6 Granulomatous lesions occurring in the lungs giving rise to a sarcoid-​ like condition with impaired gas transfer and secondary fibrosis. They may also affect other organs such as the liver, spleen, kidneys, or lymph nodes. The aetiology of this condition is unknown. 7 Autoimmune disorders are seen in approximately one-​fifth of patients with CVID. These include autoimmune haematological disorders such as haemolytic anaemia, autoimmune thrombocytopenia, and pernicious anaemia, or neurological diseases such as Guillain–​Barré syndrome, autoimmune endocrinopathies (e.g. thyroid disease), and (rarely) a lupus-​like syndrome. 8 Splenomegaly can be seen in up to 30% of patients with CVID; in many this is due to infiltration with sarcoid-​like granulomata. 9 Malignancies:  there is an increased incidence of non-​Hodgkin’s lymphomas and gastric neoplasms in patients with CVID. The inci- dence of gastric carcinoma may be related to atrophic gastritis and Helicobacter pylori infection.

4.4  Immunodeficiency 359 with severe B lymphopenia. Heterozygous Ikaros deficiency (IKZF1) and autosomal recessive hypomorphic mutations in SLC39A7 (ZIP7) were also recently linked to absent B cells. All these conditions are rare and share many clinical features with X-​ linked agammaglobulinaemia and CVID. Physiological antibody deficiencies During the last trimester of pregnancy, maternal IgG is actively transported across the placenta to the fetus. At full term, neonates are born with IgG levels approximating to or even higher than the adult normal range. In contrast, preterm babies are relatively IgG deficient at birth, to a degree that correlates with the degree of pre- maturity. Maternally derived immunoglobulins are metabolized after birth and the IgG levels reach a nadir around 4 to 6 months of age. Serum IgG levels begin to rise after this due to increase in synthesis by the neonate, and reach approximately 70% of adult levels by 12 months. During the first 6 months of life, therefore, the neonate is protected by maternally transferred immunoglobulins. Protection by maternal antibody explains why many children with inherited antibody deficiencies do not develop infections until 4 to 6 months of age. Human infants, including preterm babies, have normal antibody responses to protein and protein-​polysaccharide conjugate vaccines (e.g. Haemophilus B conjugate vaccine), hence primary immun- ization can start at 2 months of age. In contrast, children less than 2 years of age are unable to produce effective antibody responses to bacterial capsular polysaccharides. Antipolysaccharide antibody responses progressively mature after 2 years of age and it may take up to 5 to 7 years before the responses are quantitatively and qualita- tively equivalent to those of adults. Transient hypogammaglobulinaemia of infancy In some infants there is a delay in the onset of de novo immuno- globulin synthesis and as a result serum IgG levels show a pro- longed trough lasting up to 18 to 36 months of age. These infants can be differentiated from patients with primary antibody defi- ciency by their capacity to respond to immunization with T-​cell dependent vaccines (tetanus, Haemophilus b conjugate vaccine) and their ability to produce blood group isohaemagglutinins. No treatment is required if affected infants are asymptomatic, but antibiotic prophylaxis is warranted if there are severe or recurrent bacterial infections. Replacement immunoglobulin is only very rarely required. Although this is a self-​limiting disorder, infants should be followed up until immunoglobulin levels are normal to differentiate them from children with primary immunodeficiency disease. Selective antibody deficiency with normal immunoglobulins Some individuals with recurrent respiratory tract infections fail to respond to specific microbial antigens. The most common de- fect is an inability to respond to bacterial capsular polysaccharides that lasts beyond early childhood. Protein antibody responses are characteristically preserved. The prevalence of this condition is not known. Although most such individuals are asymptomatic, some develop recurrent sinopulmonary infections. The diagnosis is established by demonstrating normal IgG and IgM levels, accompanied by a failure to respond to immunization with some antigens, but with normal responses to others. Tetanus and the Haemophilus b (Hib) conjugate vaccine can be used to assess T-​cell dependent responses. Measurement of serotype-​specific re- sponses to the pneumococcal polysaccharide vaccine (Pneumovax) is used to assess thymus-​independent antibody responses. Pneumococcal conjugate vaccine stimulates T-​cell dependent anti- body responses. In countries employing routine immunization of infants with the conjugate pneumococcal polysaccharide vaccine, antibody responses to five or more serotypes contained only within the polyvalent pneumococcal polysaccharide vaccine need to be as- sessed. Such serotype-​specific pneumococcal antibody assays need to be calibrated with an international reference standard (Food and Drug Administration SF 89) and the patient’s serum preabsorbed with C-​polysaccharide shared by all pneumococcal strains and 22F polysaccharide, which is cross-​reactive. Interpretation of pneumococcal antibody responses is difficult because of the lack of age-​specific normal ranges. Furthermore, even healthy individuals may show reduced responses to individual serotypes. Pure polysaccharides are poor immunogens in infants less than 2 years of age, but response to at least 50% of the serotypes tested is the norm between 2 and 5 years, while normal adults re- spond to about 70% of the capsular polysaccharides when immun- ized with the pneumococcal polysaccharide vaccine. Haemophilus b and pneumococcal conjugate vaccines are powerful immunogens, and failure to respond to a full course of these vaccines should raise the suspicion of a defect of antibody production. A consensus group in the United States of America has published provisional criteria for interpreting postimmunization responses to pneumococcal polysaccharide vaccines, defining a normal response as achieving an antibody level of at least 1.3 µg/​ml against each serotype, or a greater than fourfold increase over baseline values, although the evidence base for such diagnostic consensus criteria are limited. Patients with selective polysaccharide antibody deficiency respond to and may benefit from conjugate vaccines. Antibiotic prophylaxis is sufficient for the management of most infection-​prone patients with selective antibody deficiency. A few patients failing these meas- ures may need a trial of immunoglobulin replacement therapy. Antibody deficiency associated with thymoma Antibody deficiency is an uncommon complication of thymoma, known as Good’s syndrome. The presenting feature may be oppor- tunistic infections, including recurrent bacterial infections, and autoimmune neutropenia, haemolytic anaemia, and red cell aplasia may occur. Laboratory findings are complete absence of or very low numbers of B cells, and low serum IgG and IgM antibody levels. Plain radiographs may miss a thymoma and a CT scan of the chest may be required. These tumours can be locally invasive and thym- ectomy is recommended, although the immunodeficiency is not re- versed by this procedure. IgA deficiency This condition has an approximate incidence of 1 in 700 in white persons. It is rare in Africans and Japanese. Most individuals remain healthy, but long-​term prospective studies indicate that a few de- velop recurrent sinopulmonary infections. Most infection-​prone patients have concomitant IgG2 subclass deficiency and a selective inability to respond to pure capsular polysaccharides. IgA deficiency is associated with an increased incidence of atopy, coeliac disease, and a range of autoimmune diseases including

360 SECTION 4  Immunological mechanisms arthritis, a lupus-​like syndrome, autoimmune endocrinopathies, and autoimmune cytopenias. Individuals with complete IgA defi- ciency (serum levels less than 0.07 g/​litre) are at risk of developing anti-​IgA antibodies on receiving blood products, and such patients are at risk of transfusion reactions following the administration of subsequent blood or its fractions. IgA deficiency can coexist in families with other members affected by CVID. Mutations in TNFRSF13B (which encodes the transmem- brane activator and CAML interactor protein, TACI) can cause IgA deficiency in some family members, while others develop CVID. IgG subclass deficiency Serum IgG is comprised of four subclasses—​IgG1, -​2, -​3, and -​4—​ in order of the relative abundance of these isotypes in the serum. IgG subclass deficiency is diagnosed when there is a reduction in the serum IgG subclass concentration two standard deviations below the normal value for age, despite the total IgG level being normal. CVID is more likely if the total IgG level is reduced. The lack of an internationally accepted reference preparation makes IgG sub- class assays difficult to standardize. Furthermore, there are genetic variations that influence IgG subclass levels among different ethnic groups, and age-​ and population-​related normal bounds are not always available. Functional assessment of antibody production through vaccine challenge is therefore often used to determine the clinical significance of low IgG subclass levels. As with IgA deficiency, many individuals with IgG subclass de- ficiency are asymptomatic, although some are prone to recurrent sinopulmonary and other infections. Most with sinopulmonary infec- tions exhibit impaired capacity to mount specific antipolysaccharide antibodies against antigens like the pneumococcal capsule. This is most often seen in individuals with IgG2 deficiency, with or without concomitant IgA deficiency. Most infection-​prone patients with IgG subclass deficiency can be managed with antibiotic therapy or prophylaxis. Immunoglobulin replacement should be limited to those with recurrent severe sinopulmonary infections despite antibiotic prophylaxis. Such pa- tients usually have specific antibody deficiency, especially to polysac- charides, hence (in the United Kingdom) there is a strong consensus that assessing specific antibodies is useful in determining the clinical significance of IgG subclass deficiency in infection-​prone patients. Management of antibody deficiency: Immunoglobulin replacement therapy Immunoglobulin replacement therapy through the intravenous (IVIG) or subcutaneous (SCIG) routes is the mainstay of therapy for antibody deficiency. Different products are licensed for IVIG and SCIG therapy, and these are not interchangeable. IVIG and SCIG have been shown to be equivalent in terms of safety and ef- ficacy. All licensed immunoglobulin products have similar efficacy, safety, and tolerability, hence product selection depends on avail- ability. However, once patients are stabilized on one preparation, this should not be changed except for sound medical reasons. Several methods for delivering immunoglobulin have been de- veloped. Intravenous immunoglobulin is generally administered every 3 weeks. SCIG is traditionally infused once or twice a week, using a small infusion pump. It can also be delivered by adminis- tering a fractionated daily dose by a slow subcutaneous push using a syringe and needle. Recently, an immunoglobulin replacement product, to be used by subcutaneous infusion facilitated by a pre- ceding hyaluronidase infusion, has been licensed. This product enables the whole monthly dose to be infused subcutaneously, al- lowing subcutaneous therapy to be infused at 3–​4 weekly intervals. With adequate training and regular supervision, most patients can administer immunoglobulin replacement therapy at home. Current practices of prescreening donors and multiple antiviral steps employed by manufacturers have eliminated the risk of trans- mission of HIV and hepatitis B and C. Rare hepatitis C outbreaks occurred in the 1990s before the current multistage viral inactiva- tion steps were introduced. Creutzfeldt–​Jakob disease (classical and new variant) could theoretically by transmitted by immunoglobulin therapy, but the risk has been estimated to be exceedingly low. Adequacy of replacement therapy is judged by clinical well-​ being (freedom from infections and prevention of their complica- tions) and preinfusion (trough) levels in the middle of the normal range (approximately 8 g/​litre). Based on these criteria, replacement therapy needs to be individualized for each patient by altering the dose or frequency of administration. Dosage of immunoglobulin replacement therapy Recent publications have addressed the appropriate dosage of im- munoglobulin replacement therapy in patients with primary anti- body deficiency. One study analysed data from patients with CVID collected over 20 years and showed that the range of trough IgG levels preventing breakthrough infection in individual patients ranged from 5 to 17 g/​litre, with the doses of immunoglobulin re- quired to prevent infection ranging from 0.2 to 1.2 g/​kg per month. A meta-​analysis of published literature provided evidence that the incidence of pneumonia can be progressively reduced by immuno- globulin replacement therapy in patients with primary antibody de- ficiency by achieving higher IgG trough levels. The goal of treatment should therefore be to reduce breakthrough infection rather than to achieve a particular trough IgG level, with individual tailoring of dosage of immunoglobulin replacement therapy. Typical dosage for immunoglobulin replacement therapy are shown in Box 4.4.3. Adverse effects of immunoglobulin replacement therapy About 10% of patients experience mild reactions during or imme- diately after IVIG therapy, including headaches, malaise, backache, nausea, and myalgia. These can usually be overcome by a combin- ation of reducing the infusion rate, antihistamines, and antipyretics. Anaphylactic reactions requiring cessation of therapy and adren- aline (epinephrine) are rare, but commoner during the first few in- fusions or in the presence of intercurrent infections. These can be almost eliminated by administration of the initial infusion at a slow rate and postponing IVIG therapy until infections have resolved on antibiotic therapy. Rarely, anaphylactic reactions may be due to pa- tients with severe IgA deficiency (serum levels less than 0.07 g/​litre) producing anti-​IgA antibodies. Box 4.4.3  Dosage of immunoglobulin replacement therapy • IVIG—​from 400 to 1200 mg/​kg every 4 weeks. • SCIG—​from 100 to 300 mg/​kg once a week, or half the dose adminis- tered twice a week; alternatively, a fractionated daily dose is delivered by slow subcutaneous push.

4.4  Immunodeficiency 361 Acute kidney injury and thromboembolic disease have also been occasionally reported, especially following high-​dose IVIG therapy. Premedication with paracetamol, antihistamines, and/​or hydro- cortisone, or changing the immunoglobulin product, often help in patients who develop repeated adverse reactions. Apart from local pain and swelling, adverse reactions are rare with SCIG therapy. Switching to SCIG may be an option for patients who fail to tolerate IVIG. Supplementary management of antibody deficiency Breakthrough infections can occur, even with optimum immuno- globulin replacement therapy. There should be a low threshold for treating infections with antibiotics and recurrent infections, especially when associated with structural lung damage, may re- quire long-​term antibiotic prophylaxis. Amoxicillin, co-​amoxiclav, clarithromycin, azithromycin, doxicycline, and ciprofloxacin are useful agents for prophylaxis. Postural drainage of lung secretions and appropriate treatment of concomitant bronchial asthma is important. A subset of patients with common variable immunodeficiency (CVID) develops granulomatous and lymphocytic interstitial lung disease (GLILD), a restrictive lung disease associated with early mortality. The optimal therapy for this condition is uncer- tain, but recent small studies have shown improvement with a combination of rituximab (monoclonal antibody to CD20) with azathioprine. Patients with serious lung disease, gastrointestinal disease, or impaired liver function should be managed with multidisciplinary input from relevant organ-​based specialists. Patients should be encouraged to join support groups for education and counselling as well as practical help with social problems. Referral for genetic counselling should be considered in patients with a familial disorder or a known gene defect. Prognosis Prospective studies have shown that optimal immunoglobulin re- placement therapy decreases the frequency of infections and re- duces the incidence of sepsis, especially by encapsulated bacteria. Recipients are likely to have a normal lifespan if this is instituted before structural lung damage is established. However, long-​term studies in Italy have demonstrated that some patients with X-​linked agammaglobulinaemia may continue to develop lung damage des- pite optimum immunoglobulin replacement therapy. The cause for this is unclear. CVID patients with systemic granulomatous disease or interstitial lung disease exhibit reduced survival com- pared to those without these complications. The occurrence of non-​ Hodgkin’s lymphoma (2–​7%) and gastric carcinoma (approximately 1%) reduce survival. Diseases of immune dysregulation Complex regulatory mechanisms ensure that innate and adaptive immune responses are held in check within the healthy immune system. Their importance is revealed by a series of distinct pheno- types in which prominent immune dysregulation predominates over susceptibility to infection. Haemophagocytic lymphohistiocytosis (HLH) HLH describes a life-​threatening systemic illness in which exces- sive but ineffective immune activation leads to a stereotyped pat- tern of fever, splenomegaly, and laboratory abnormalities including cytopenias and hyperferritinaemia. The Histiocyte Society has pro- posed a consensus set of diagnostic criteria to assist in the prompt recognition of HLH, which is a medical emergency. Both congenital (‘primary’) and acquired (‘secondary’) forms are recognized, al- though genetic factors are likely to contribute to both. Familial HLH is a set of autosomal recessive diseases that are as- sociated with a failure of cytotoxicity. The onset may be triggered by infection. Many of the cases are caused by mutations in the perforin gene, the genes Munc13-​4 and 18-​2, and in syntaxin 11 or its binding protein. Others are associated with partial oculocutaneous albinism (Chédiak–​Higashi syndrome, Griscelli syndrome, Hermansky–​ Pudlak syndrome), but there remain some patients for whom no underlying genetic diagnosis can be found. HLH may also be a presenting feature of other forms of im- munodeficiency, particularly those that predispose to severe EBV infection (XLP1, XIAP deficiency (see section on X-​linked lymphoproliferative syndromes); ITK deficiency, CD27 deficiency), but also others such as class II MHC deficiency. Secondary HLH is a macrophage activation syndrome with haemophagocytosis as a result of immunological activation trig- gered by a variety of conditions including infection, rheumatoid dis- orders, malignancies, and metabolic disorders. Familial HLH is fatal without curative therapy. Survival and cure depend on initial and continuation therapy to control HLH followed by early HSCT, preferably while in remission. First-​line treatment consists of a regimen containing steroids, ciclosporin, etoposide +/​–​ intrathecal methotrexate. In patients who fail to respond or relapse, salvage therapy may be attempted using T-​cell directed biologics. Similar approaches are required for patients with most other forms of immune deficiency presenting as HLH. Patients with secondary HLH may also need active management at first as not all cases resolve spontaneously. Treatment may then need to be adapted depending upon the underlying cause of the disease. Syndromes with autoimmunity Autoimmune lymphoproliferative syndrome (ALPS) An increasing number of monogenic disorders are being rec- ognized as causing autoimmunity, often in association with lymphoproliferation. Nomenclature is somewhat confusing as the term ‘autoimmune lymphoproliferative syndrome’ (ALPS) is re- served for a particular subset of disorders, most of which result from impaired lymphocyte apoptosis. Consensus diagnostic criteria for ALPS require both clinical evidence of lymphoproliferation (spleno- megaly, lymphadenopathy) and an excess of CD4-​CD8-​TCRαβ+
T lymphocytes. Cytopenias are the commonest manifestation of autoimmunity, but others including a lupus-​like picture can be seen. Susceptibility to infection is part of the primary picture in some dis- orders. The most frequent molecular lesion, heterozygous germline mutation of FAS, is incompletely penetrant and there is some evi- dence that a ‘second hit’ is required to produce disease. Nonetheless, a highly significant (up to 250-​fold) increase in lymphoma inci- dence has been documented and affects asymptomatic carriers as well as individuals with clinical ALPS. Sirolimus can be an effective

362 SECTION 4  Immunological mechanisms steroid-​sparing agent, while combinations of high-​dose IV im- munoglobulin and rituximab may help to relieve cytopenias. Immunodysregulation, polyendocrinopathy, enteropathy, X-​linked (IPEX) IPEX is a severe disorder in which the function of regulatory T cells (Treg) is impaired by mutations in FOXP3. This gene encodes a tran- scription factor that is critical to Treg differentiation and activity. Affected boys often present with neonatal diabetes along with ex- tremely severe autoimmune enteropathy, eczema, and a variety of other autoimmune phenomena such as cytopenias. Laboratory evalu- ation may show impaired expression of FOXP3 by cells destined to become Tregs as shown by their high levels of the high affinity IL2 receptor, CD25, and lack of the IL7 receptor, CD127. Other conditions affecting regulatory T cells Other lesions that impair Treg function may produce a similar spec- trum of autoimmunity, albeit the tempo of disease is often slower. Among these are deficiencies of CTLA4 (also known as ALPSV), LRBA and IL2RA, and gain-​of-​function (GOF) mutations in STAT3. Conservative management of these disorders using immuno- suppressive medication can be extremely challenging despite the availability of agents such as sirolimus and abatacept/​belatacept that target the relevant cells and pathways. Stem cell transplantation has curative potential and has become the treatment of choice for IPEX syndrome, while experience of transplantation for CTLA4 defi- ciency and STAT3 GOF continues to accumulate. Disorders affecting the IL-​10 pathway Early onset, severe colitis is the major feature in disorders of the IL10 pathway, including deficiencies of IL-​10 itself and either component of the IL10 receptor. Associated features may include recurrent respira- tory infection, arthritis, and lymphoma. The likelihood of a monogenic origin for inflammatory bowel disease is high among children with onset before 5 years of age and special investigations are warranted. Interferonopathies The IUIS classification places type I interferonopathies among the im- mune dysregulatory disorders. These are a heterogenous group of dis- orders characterized by inappropriate activation of type 1 interferon signalling. The phenotypic spectrum is broad, ranging from mild cu- taneous disease to severe neurological disorders, and the determin- ants of this variability are unknown. The predominant pathology is autoinflammatory in nature, with autoimmune overlap in certain types, sometimes amounting to familial lupus. The fundamental problem driving inflammation is usually inappropriate signalling via innate antiviral recognition pathways, often because of a failure to catabolize endogenous nucleic acids. Demonstration of a type I inter- feron stimulated gene signature can be diagnostically helpful in such patients. Although treatment remains challenging at the present time, early results with JAK inhibitors are encouraging. Phagocyte deficiencies Neutropenia The commonest phagocyte deficiency seen in clinical practice is neu- tropenia, which results in increased susceptibility to a broad range of pyogenic organisms and fungi. Commensal organisms including skin and intestinal bacteria often cause septicaemic illnesses in neutropenic patients. Invasive candidiasis and occasionally other fungal infections may also be seen. Neutrophils are particularly im- portant for maintaining the integrity of mucous membranes, hence inflammation of mucous membranes (e.g. ulceration of mouth and perioral tissues and perianal inflammation and excoriation) can be features of neutropenia. A neutrophil count of less than 0.5 × 109/​ litre is associated with a high risk of life-​threatening bacterial sepsis. Several inherited monogenic defects lead to severe congenital neutropenia as an isolated finding (e.g. ELANE, JAGN1, WAS GOF) or in conjunction with syndromic features (e.g. HAX1, SBDS). It is important to remember that neutropenia can also complicate primary immunodeficiencies of the adaptive im- mune system such as CD40L deficiency or X-​linked agamma- globulinaemia. Severe congenital neutropenias may respond to granulocyte-​colony-​stimulating-​factor therapy, but with age comes an increasing risk of myelodysplasia and transformation to acute myeloid leukaemia. Defects in bacterial killing Functional defects of neutrophils are rare. The best-​characterized condition is chronic granulomatous disease, with an incidence of about 1 in 100 000 births. Neutrophils and macrophages of these pa- tients show impaired killing of ingested bacteria. This is due to faulty postphagocytic activation of the NADPH oxidase complex, the role of which is to produce superoxide (O2–​) and generate a milieu within the phagosome that activates bactericidal enzymes cathepsin and elastase. In the X-​linked form (75% of all cases) this is due to a de- fect of the 91-​kD chain of the cytochrome b (gp91phox), whereas the rarer autosomal recessive form may be either due to deficiency of the 22-​kD chain of cytochrome b (p22phox) or cytosolic cofactors called p47phox and p67phox, respectively. Chronic granulomatous disease typically presents with infec- tions in infancy, but inflammatory phenomena may predominate and initial presentation in adulthood is well documented. Patients typically develop infections with S. aureus or Gram-​negative bac- teria (Burkholderia cepacia, Salmonella, Serratia, enteric bacteria). Invasive fungal infections (Aspergillus) can be life-​threatening. Nocardia is another pathogen seen in chronic granulomatous dis- ease. Unusual environmental bacteria of low-​grade virulence may be isolated from blood and lymph nodes. Characteristic sites of in- fection include skin, lymph node or deep subcutaneous abscesses, or visceral abscesses involving liver, spleen, or lung. Oral and perioral ulceration and gingivitis are common. These patients also develop granulomas in various tissues:  granulomatous obstruction of the gastrointestinal tract or the urinary tract may occur, and granuloma- tous infiltration of the lung may rarely be seen. Hepatosplenomegaly due to granulomatous involvement of these organs may also be a feature. Colitis resembling Crohn’s disease is seen in approximately 15% of cases. The diagnosis is based on the inability of affected neutrophils to oxidize a dye called nitro blue tetrazolium and change it from yellow to a blue-​black colour. Modifications of this principle using newer methods such as the oxidation of the fluorescent dye dihydrorhodamine, which can be detected by flow cytometry, are reliable and sensitive for establishing the diagnosis. Management consists of antimicrobial prophylaxis and prompt diagnosis and treatment of infections. Co-​trimoxazole at 5 mg/​kg

4.4  Immunodeficiency 363 divided into two doses per day significantly reduces bacterial infec- tions, and daily itraconazole (100 mg/​day for patients <50 kg body weight or 200 mg/​day for heavier individuals) reduces Aspergillus infections. US studies have shown a reduction in severe infections with prophylactic interferon-​γ at 50 µg/​m2, three times a week, via subcutaneous injection, but clinical experience is not clear-​cut and this practice has not been widely adopted in Europe. Invasive fungal infections are difficult to treat despite the avail- ability of an expanding range of antifungals such as voriconazole and posaconazole. Granulocyte transfusions may be beneficial in those with severe, refractory infections. Steroids may also play a role, particularly for refractory visceral infection such as liver abscess, but also for chronic granulomatous inflammation, but these com- pound the existing immunodeficiency. Stem cell transplantation is becoming the standard of care based on excellent medical outcomes and improved health-​related quality of life. Gene therapy has pro- duced temporary physiological and clinical improvement for a few months in a few patients, and clinical trials are ongoing. Defects in leucocyte adhesion and migration To confer protection from infection, circulating leucocytes need to migrate along chemotactic gradients across capillary endothelium into sites of infection. As a prelude to this, Lymphocyte function-​ associated antigen 1 (LFA1) on leucocytes needs to bind tightly to the ligand intercellular adhesion molecule 1 (ICAM-​1) on activated endothelial cells. Leucocyte adhesion deficiency type 1 is caused by deficiency of CD18, which is a subunit component of three leucocyte surface re- ceptors called CD11a/​CD18 (LFA1), CD11b/​CD18 (complement receptor 3), and CD11c/​CD18 (complement receptor 4). In leuco- cyte adhesion deficiency type 2, Sialyl-​Lewis X, which is expressed on the surface of leucocytes and acts as the ligand for E selectin expressed on endothelial cells, cannot be synthesized. This is due to autosomal recessive mutation of a GDP-​fucose transporter re- sulting in a failure of fucosylation of proteins within the Golgi ap- paratus. Without this interaction the initial adhesion of leucocytes to endothelial cells, a prelude to diapedesis, fails. Leucocyte ad- hesion deficiency types 1 and 2 thus exhibit impaired endothelial adherence, chemotaxis, and diapedesis of neutrophils and other leucocytes, which are held back in the circulation and cannot reach the sites of infection. Patients with leucocyte adhesion deficiency characteristically manifest delayed cord separation and periumbilical sepsis during early infancy. Other features are recurrent pyogenic infections, per- sistent marked leucocytosis (>15 × 109/​litre) due to the inability of leucocytes to migrate from the bloodstream into the tissues, and poor wound healing, with the development of pyoderma-​like ul- cers that may eventually heal with paper-​thin scars. Pus fails to form during infections due to failure of neutrophils to enter sites of infec- tion. These inherited disorders of neutrophil function are charac- teristically associated with gingivitis and periodontal disease, again indicating the particular importance of normal neutrophil function for the maintenance of a healthy dental/​gingival interface. Patients with leucocyte adhesion deficiency type 2 have facial dysmorphism and developmental delay. Diagnosis of these conditions is by flow cytometry of blood leucocytes to detect CD18 or CD15 (Sialyl-​Lewis X) deficiency, respectively. In the complete form of leucocyte adhesion deficiency type 1, and in type 2, outcome is poor with early death from sepsis. Rare patients with a partial form of type 1 and a milder phenotype may survive to adulthood. Stem cell transplantation is curative in leucocyte adhesion deficiency type 1 and should be considered early. Oral fucose supplementation can result in clinical improvement in leucocyte adhesion deficiency type 2. For leucocyte adhesion defi- ciency type 3, see Table 4.4.2. For information on rarer primary immunodeficiency diseases with impaired phagocyte function, see the sources listed in the ‘Further reading’ section. Defects in intrinsic and innate immunity Many disorders are now recognized as predisposing to infec- tions caused by a narrow spectrum of pathogens. Frequently this reflects the non​redundancy of pathogen-​specific innate im- mune mechanisms of sensing or antimicrobial activity. These conditions may manifest as susceptibility to invasive infections caused by pyogenic bacteria, predominant susceptibility to viral infection, susceptibility to fungal infections, or susceptibility to mycobacteria. Some of these recently described defects are outlined next. Predominant susceptibility to invasive infections with pyogenic bacteria Innate immune responses to pathogens are initiated by recogni- tion of pathogen associated molecular patterns by cell surface and intracellular ‘pattern recognition receptors’, for example Toll-​like receptors. The interleukin receptor-​associated kinase-​4 mediates signalling via most Toll-​like receptors and members of the IL-​1 re- ceptor superfamily. Individuals with homozygous mutations of the IRAK4 gene develop recurrent, life-​threatening, pyogenic sepsis. They are especially susceptible to pneumococcal, S.  aureus and P.  aeruginosa infection. Interestingly, the incidence and severity of infections decreases by adolescence, with improvement in out- come. Mutations in the gene encoding the protein myeloid differen- tiation primary response gene 88 (MYD88), which is also required for signal transduction following Toll receptor engagement, causes a similar clinical syndrome. A combination of antibiotic prophylaxis and immunoglobulin supplementation has been advocated for chil- dren with these disorders. Predominant susceptibility to viral infection UNC93B is a protein of the endoplasmic reticulum involved in the activation of Toll-​like receptors. Mutations in UNC93B results in de- fective interferon-​α and -​β production in response to herpes simplex and other viruses, and patients develop herpes simplex encephalitis between 3 months to 6 years of age. Heterozygous dominant-​negative mutations in the gene-​encoding Toll receptor 3 (TLR3) have rarely been identified in patients with herpes simplex encephalitis. TLR3 is expressed in the central ner- vous system where it helps to initiate interferon-​α and -​β responses to viral double-​stranded DNA. TRIF, also known as TICAM1, is a Toll/​IL1 receptor (TIR) domain-​containing adaptor molecule, that specifically interacts with TLR3 and activates nuclear factor kappa-​B (NFKB), thus inducing interferon-​β production. Cells lacking tumour necrosis

364 SECTION 4  Immunological mechanisms factor (TNF) receptor-​associated factor 3 (TRAF3) are defective in type I interferon responses activated by several different Toll-​like re- ceptors. TRIF mutations, as well as autosomal dominant–​negative mutations of TRAF3, cause susceptibility to develop herpes simplex encephalitis. TANK-​binding kinase 1 (TBK1) is a kinase at the crossroads of multiple type I interferon (IFN)-​inducing signalling pathways. Partial TBK1 deficiency caused by heterozygous mutations in this gene also causes increased susceptibility to herpes simplex encephalitis. Signal transduction via receptors to interferons -​γ, -​α, and -​β in- volves the participation of the signal transducing molecule STAT1. IFNγ-​R-​mediated signalling results in dimerization of phosphoryl- ated STAT1 molecules, which migrate to the nucleus and induce gene transcription. Signalling via interferon-​α and -​β receptors involves the formation of a complex between STAT1, STAT2, and a third pro- tein called interferon stimulated gene factor 3-​γ (ISGF3-​γ). Complete (homozygous) defects of STAT1 result in impaired responses to interferons-​ γ, -​α, and -​β, resulting in susceptibility to disseminated mycobacterial infections, as well as severe herpes simplex virus infec- tion. Partial STAT1 deficiency, which interferes with STAT1 dimer- ization required for signal transduction via interferon-​γ receptors, produces increased susceptibility to mycobacterial infections alone. In these patients the cellular responses to interferon-​α and -​β are in- tact, thus preserving antiviral immunity. By contrast, autosomal reces- sive deficiency of STAT2 or IFNAR produces the reciprocal phenotype by selectively impacting innate interferon signalling. Affected patients can suffer dissemination of live attenuated vaccine viruses. Another immunodeficiency characterized by susceptibility to a specific viral infection is the Warts hypogammaglobulinaemia in- fections myelokathexis (WHIM) syndrome (OMIM 193670). This is characterized by severe warts, hypogammaglobulinaemia, and neu- tropenia due to retention of neutrophils in the bone marrow. This is an immunodeficiency caused by a gain-​of-​function mutation in the gene encoding the CXCR4 chemokine receptor, with the mutant re- ceptors showing increased responsiveness to its ligands and leading to a failure of chemotaxis and leucocyte trafficking. Predominant susceptibility to fungal diseases. Chronic mucocutaneous candidiasis (CMC) is a syndrome consisting of recurrent and/​or refractory infections of the skin, nails, and oral and genital mucosa with candida species in the absence of a recog- nized inherited or acquired T-​cell immune deficiency. Recognized clinical associations of this condition are summarized in Table 4.4.7. Recent findings have helped to elucidate its pathophysiology and molecular basis. CMC is caused by conditions that result in defi- ciency of IL 17, neutralization of IL-​17 by autoantibodies, or lack of response to IL-​17. In approximately 50% of patients with isolated or autoimmunity-​associated CMC, gain-​of-​function mutations in the signal transducing molecule STAT1 have been identified. This abnor- mality is associated with secondary inhibition of IL-​17 production. Specific susceptibility to candida infection is a feature of another group of patients who may develop invasive candidiasis affecting the brain as well as recurrent mucocutaneous candidiasis. These patients have autosomal recessive mutations in the gene encoding the caspase recruitment domain-​containing protein, CARD9, which is required for intracellular signalling downstream of Dectin-​1, a pattern recognition receptor for fungal carbohydrates. Activation of this signalling pathway results in the production of proinflammatory cytokines, including IL-​ 1β, IL-​6, and IL-​23. CARD9 deficient patients also have reduced IL-​17-​ producing T cells that also contribute to mucosal immunity. Mendelian susceptibility to mycobacterial disease (MSMD) Primary and secondary immunodeficiencies leading to severely impaired T-​cell function result in increased susceptibility to myco- bacterial infections, including those caused by poorly patho- genic mycobacteria (non​tuberculous mycobacteria) and bacillus Calmette–​Guérin (BCG). However, a few individuals are specifically susceptible to mycobacteria, which may occur in a disseminated or fatal form in the absence of other evidence of immune deficiency. This condition has been called Mendelian susceptibility to mycobac- terial disease (OMIM 209950). Causal genetic defects Genetic analyses of affected kindreds have, to date, identified mu- tations in thirteen disease-causing genes, including two X- linked (CYBB and NEMO) and thirteen autosomal (IFNGR1, IFNGR2, STAT1 (LOF), IL12B, IL12RB1, ISG15, IRF8, TYK2, RORC, JAK1 (LOF) , SPPL2a, IL-12RB2 and IL-23R) genes. Mutations in JAK1 and RORC have been described as responsible for syndromic MSMD. MSMD-causing genes affect IFN-γ dependent immunity, in terms of either IL-12/IL-23/ISG15-dependent induction of IFN-γ or IFN-γ induced down-stream, cellular responses (Fig. 4.4.2). Genetic lesions of dendritic cell development and function also pro- duce prominent mycobacterial susceptibility, although usually this is accompanied by other evidence of immunodeficiency. Several gene defects responsible for Mendelian Susceptibility to Mycobacterial Disease have been documented: Table 4.4.7  Conditions associated with chronic mucocutaneous candidiasis Clinical condition Pathogenesis of CMC Autoimmune polyendocrinopathy syndrome type 1
due to mutation in AIRE Neutralizing autoantibodies to IL-​17 family of cytokines and to IL-​22 Thymoma Neutralizing autoantibodies to IL-​17 family of cytokines Hyper IgE syndrome (STAT3 mutation) Low TH-​17 cells, which produce IL17A and IL22. Hence deficiency of the cytokines IL-​17A and IL-​22 IL12p40 or IL-​12 receptor b1 deficiency Low IL-​17A-​producing T cells Caspase recruitment domain 9 (CARD9) deficiency Low IL-​17A-​producing T cells Autosomal recessive IL-​17 receptor deficiency Abolishes cellular responses to IL17-​A and IL17-​F homo-​ and heterodimers Autosomal dominant IL-​17F deficiency Reduced activity of IL-​17F homo-​ and heterodimers resulting in partial IL-​17 deficiency CMC with isolated hypothyroidism Activating mutation of STAT1 gene resulting in secondary reduction of IL-​17 family of cytokines

4.4  Immunodeficiency 365 Phagocytes/DC ISG15 TH1/NK TNF-α IL-12 IL-23 IL 18-R1 IL 18-R2 IL18 p35 IL 12-Rβ2 IL 12-Rβ1 IL 23R TYK-2 TYK-2 JAK-2 STAT-4 STAT-4 STAT-4 JAK-2 NK / (T) ? TNF-α IFN-γ + + + IL 12-Rβ1 IKK IKK IRF8 (GATA2) TRAF TRAF Y/NEMO y/NEMO α β p40 p40 p19 Mycobacterium CYBB gp91phox GTP MΦ p22 phox P membrane O2 O2– • NADPH NADP p67phox p40 phox Rac Phagosome CYBB Lysosome STAT-1 IL-12 IL-23 IL-18 α β IFN-γ IFN-γR2 IFN-γR1 STAT-1 JAK-1 JAK-2 p47 phox TNFα Granulocytes Type I IFN’s ? TLR Fig. 4.4.2  Genetic defects predisposing to MSMD. A highly simplified diagrammatic representation of the key cytokine receptor interactions relevant for immunity against intracellular bacteria, derived from observations in gene knock-​out mice. Stimulation of macrophages/​dendritic cells by mycobacteria by infection and via Toll receptors results in secretion of IL-​12 which acts on antigen stimulated CD4 T cells (which express IL-​12 receptors). IL-​12 partitions responding CD4 T cells to develop along the Th1 pathway and secrete interferon-​γ. Interferon-​γ homodimers activate (a) macrophages enhancing their antimicrobial pathways and (b) T cells and NK cells in an autocrine fashion, via IFN-​γR1/​R2 dimers. Type I cytokine deficient patients indicate the relevance of these pathways for human immunity.

366 SECTION 4  Immunological mechanisms • Recessive null mutations in the gene encoding the interferon-​ γ receptor (IFN-​γR1) chain—​these can either abolish receptor expression or their binding of interferon-​γ. Dominant IFN-​ γR1 deficiency is due to the truncation of the intracellular do- main of the receptor chain, resulting in the accumulation of non​functional ­receptors which interfere with the function of the residual normal receptors. Recessive mutations of the gene encoding the IFN-​γR2 signalling chain are responsible for com- plete or partial IFN-​γR2 deficiency. • Null recessive mutations of the IL-​12RBI gene encoding the IL-​12 receptor chain, IL-​12RβI—​these abrogate the cell surface expres- sion of this chain, which is shared by IL-​12 and IL-​23 receptors, re- sulting in the inability to respond to the cytokines IL-​12 and IL-​23. Mutation of the IL-​12/​23 receptor-​associated tyrosine kinase, Tyk 2, also results in defective signal transduction via IL-​12 receptors. • Inability to produce IL-​12 and IL-​23, due to deletion within the gene encoding the inducible chain of IL-​12 (IL12B) which is shared by IL-​12 and IL-​23. • Partial or complete defects in the signal transduction molecule STATI, which is required for signalling via the interferon-​γ receptor. In addition, acquired interferon-​γ deficiency due to the produc- tion of neutralizing autoantibodies to this cytokine has been identi- fied in patients with disseminated mycobacterial infection. This may also result in infections caused by other intracellular pathogens such as Penicillium marneffei. These defects need to be sought by immunological and molecular methods in patients with refractory or disseminated mycobacterial infections in the absence of an underlying cause such as HIV in- fection, immunosuppressive therapy, or a recognized primary T-​cell immunodeficiency. Clinical features The severity of the clinical phenotype depends on the genotype. Patients with complete IFN-​γR1 or R2 deficiencies develop dissem- inated mycobacterial infections caused by BCG or non​tuberculous mycobacteria, present in early childhood, and have a high mortality. The lesions in these patients are characteristically multibacillary and associated with impaired granuloma formation. In contrast, partial IFN-​γR1 deficiency, complete IL-​12B defi- ciency (resulting in IL-​12 and IL-​23 deficiency), and IL-​12/​IL-​23 re- ceptor deficiency are usually associated with milder mycobacterial infections presenting at a later age. The dominant form of partial STAT1 deficiency, with impaired biological responses to interferon-​γ, appears to primarily affect antimycobacterial defences. In contrast, recessive, complete STAT1 deficiency with impaired responses to interferon-​γ and type 1 inter- ferons leads to mycobacterial infections and to fatal herpes viral in- fections which present in infancy. In addition to this, extraintestinal or systemic relapsing infections caused by non​typhoid Salmonella species are the most common infection occurring in patients with defects in the IL-​12/​23 system. X-​linked susceptibility to mycobacterial infection can occur due to mutations of the CYBB gene encoding the p91-​phox component of the phagocyte oxidase complex. Defective NFκB activation caused by X-​linked hypomorphic mu- tations of the NFκB essential modulator gene (IKBKG) compromises the function of Toll-like​, IL-​1, and tumour necrosis factor α (TNFα) receptors, and also increases susceptibility to severe mycobacterial infections. Patients with inherited defects in the phagocyte NADPH oxidase system are highly susceptible to Salmonella infections but exhibit only slightly increased susceptibility to mycobacteria. Autosomal recessive deficiency of interferon regulatory factor 8 (IRF8) causes a severe immunodeficiency with absent monocytes and dendritic cells, characterized by early onset disseminated BCG infection and a myeloproliferative blood picture. Autosomal dom- inant IRF8 deficiency is associated with more subtle abnormalities of the dendritic cell compartment and mycobacterial susceptibility. Monocytopenia and dendritic cell deficiency, associated with myco- bacterial, viral, and histoplasma infections, as well as susceptibility to pulmonary alveolar proteinosis and myelodysplasia, is caused by heterozygous mutations in the GATA2 gene. RORC deficiency has been identified in patients with susceptibility to both mycobacteria and candida infections. Mycobacterial infections in patients with IL-​12B, IL-​12B1, and dominant partial IFN-​γR deficiency that are refractory to chemo- therapy may respond to interferon-​γ supplementation. Interferon-​γ is of no use in complete IFNγ-​R1 or -​R2 deficiency, where the out- come is often poor despite antimycobacterial chemotherapy, and HSCT should be considered at an early age. Complement deficiencies Defects in the complement pathway resulting in immunodeficiency are described in Chapter 4.2. The role of genomics in primary immunodeficiency The foregoing text has highlighted significant molecular diagnostic challenges arising from the tremendous genetic heterogeneity of primary immunodeficiency, the overlapping and pleiomorphic na- ture of disease presentation, and the existence of contrasting allelic disorders with alternative mutation types. Nonetheless, correct as- certainment of genetic diagnosis can bring significant benefits for patients, based on better knowledge of disease mechanism, natural history, and inheritance model. In turn, these insights inform treat- ment selection, prediction of prognosis, and genetic counselling, respectively. Furthermore, the molecular dissection of primary im- munodeficiency offers important scientific opportunities for im- proved understanding of the immune system in health and disease, and the potential to develop novel targeted therapies in the context of ‘precision medicine’. In this context, it is not surprising that the primary immuno- deficiency community has been quick to embrace next generation sequencing as both a clinical diagnostic and a research tool. This technology enables massively parallel sequencing of very large num- bers of DNA fragments. These can be derived from an entire genome (‘whole genome sequencing’, WGS) or enriched for regions of interest by hybridization to probes corresponding to particular disease genes (‘targeted panel’) or indeed the entire coding fraction of the genome (‘whole exome sequencing’, WES). Currently available platforms re- quire many overlapping fragments to be sequenced individually but

4.4  Immunodeficiency 367 simultaneously. The individual sequences are then aligned with the reference genome and with each other to stitch together a patchwork covering the entire region of interest. Subsequent bioinformatic ana- lysis is an increasingly streamlined process using open source or proprietary software to implement a series of filtering steps. In the case of rare monogenic disease these are designed to focus attention on the most likely pathogenic variant(s) by eliminating common, silent, or predicted tolerated variants from consideration. Targeted primary immune deficiency panels have been designed and implemented in many diagnostic centres and offer the ability to screen a large number of disease genes in a rapid and efficient fashion. Variants are typically identified with a high degree of confi- dence, although it is still standard practice to confirm medically ac- tionable findings by conventional (dideoxy) sequencing. However, as with any genetic test, the significance of variants within disease genes may not immediately be apparent, particularly in the case of missense mutations, hence laboratories and clinicians must be ready to interrogate pathogenicity further where relevant by pro- tein/​functional assays and/​or further genetic testing within affected families. Whole exome and whole genome sequencing have powerful po- tential to enable disease gene discovery. Since 2010 these methods have contributed to an explosion of knowledge amounting to more than 50 new primary immune disorder (PID) genes. This rate of gene discovery, while clearly not sustainable indefinitely, none- theless emphasizes the extreme heterogeneity of PID and suggests that comprehensive targeted gene panels are likely to remain unob- tainable for some years to come. WGS offers several other advan- tages including its ‘one size fits all’ applicability to the full range of genetic disorders, improved coverage of disease genes when com- pared with targeted capture approaches, and potential to reveal pathogenic variants within non​coding space. As a result, several healthcare organizations are exploring the clinical diagnostic im- plementation of WGS, and ultimately this may become the inves- tigation of choice for patients who present with severe, unusual, or recurrent infections. FURTHER READING Amaya-Uribe L, et al. (2019). Primary immunodeficiency and auto- immunity: a comprehensive review. J Autoimmun, 99, 52–72. Bonilla FA, et  al. (2015). Practice parameter for the diagnosis and management of primary immunodeficiency. J Allergy Clin Immunol, 136, 1186–​205, e1–​78. Bousfiha A, et al. (2018). The 2017 IUIS Phenotypic Classification for Primary Immunodeficiencies. J Clin Immunol, 38, 129–​43. de Vries E (2012). Patient-​centred screening for primary immuno- deficiency, a multi-​stage diagnostic protocol designed for non-​ immunologists: 2011 update. Clin Exp Immunol, 167, 108–​19. Gennery AR, et  al. (2010). Transplantation of hematopoietic stem cells and long-​term survival for primary immunodeficiencies in Europe:  entering a new century, do we do better? J Allergy Clin Immunol, 126, 602–​10.e1. Heimall J (2019). Genetic testing to diagnose primary immunodefi- ciency disorders and to identify targeted therapy. Immunol Allergy Clin North Am, 39, 129–40. Kwan A, Puck JM (2015). History and current status of newborn screening for severe combined immunodeficiency. Semin Perinatol, 39, 194–​205. Lucas M, et al. (2010). Infection outcomes in patients with common variable immunodeficiency disorders:  relationship to immuno- globulin therapy over 22  years. J Allergy Clin Immunol, 125, 1354–​60 e4. Ochs HD, et al. (eds) (2007). Primary immunodeficiency diseases: a mo- lecular and genetic approach. pp. 669–​87. Oxford University Press, New York. Orange JS, et al. (2010). Impact of trough IgG on pneumonia incidence in primary immunodeficiency: a meta-​analysis of clinical studies. Clin Immunol, 137, 21–​30. Orange JS, et al. (2012). Use and interpretation of diagnostic vaccin- ation in primary immunodeficiency: a working group report of the Basic and Clinical Immunology Interest Section of the American Academy of Allergy, Asthma & Immunology. J Allergy Clin Immunol, 130(3 Suppl), S1–​24. Parvaneh N, Filipovich AH, Borkhardt A (2013). Primary immuno- deficiencies predisposed to Epstein–​Barr virus-​driven haemato- logical diseases. Br J Haematol, 162, 573–​86. Picard C, et al. (2018). International Union of Immunological Societies: 2017 Primary Immunodeficiency Diseases Committee Report on Inborn Errors of Immunity. J Clin Immunol, 38, 96–128. Smith T, Cunningham-Rundles C (2019). Primary B-cell immuno- deficiencies. Hum Immunol, 80, 351–62. Stiehm ER, et al. (2010). Therapeutic use of immunoglobulins. Adv Pediatr, 57, 185–​218. Wood P, et al. (2007). Recognition, clinical diagnosis and management of patients with primary antibody deficiencies: a systematic review. Clin Exp Immunol, 149, 410–​23. Websites European Society for Immunodeficiencies. Clinical Diagnostic Criteria for Primary Immunodeficiencies. https://esid.org/Working-Parties/ Clinical-Working-Party/Resources/Diagnostic-criteria-for-PID2 National Center for Biotechnology Information. Online Mendelian Inheritance in Man (OMIM). https://www.omim.org Orphanet. https://www.orpha.net/consor/cgi-bin/index.php

4.5 Allergy 368

4.5 Allergy 368

ESSENTIALS Allergy is common and becoming commoner: it now affects about one-​third of the UK population. This is being driven by environ- mental changes, which are also leading to an increase in both the complexity and severity of the condition. In addition to the trad- itional allergic disorders—​asthma, rhinitis, and eczema—​multisystem allergic disease and reactivity to several allergens are now common; new allergies have appeared, including those due to foods, drugs, and diagnostic agents; and anaphylaxis is increasing. In contrast, new cases of latex allergy have become uncommon. Where possible, patients with significant allergy should be re- ferred to an allergy specialist who can provide expertise not offered by—​and complementary to—​that of other specialties. Identifying and managing allergic causes of disease leads to reduction or resolution of its manifestations. Aetiology and pathogenesis Mechanism—​allergy in its classical form occurs following inter- action of allergen with specific IgE antibody bound to high-​affinity IgE receptors on mast cells, which results in mast cell activation, degranulation, and mediator release, but the same clinical pres- entation can occur as a result of IgE independent mast cell de- granulation (e.g. idiopathic anaphylaxis or angioedema). A normal subject has no specific IgE to common allergens and a low or normal total serum IgE level: production of specific IgE antibody requires a change in immunoregulation leading to sensitization (atopic state), with some sensitized subjects progressing to de- velop clinical allergy. Allergens—​common allergic triggers include (1)  inhaled allergens—​house dust mite, pollens, and animal danders are the commonest causes of allergic asthma, rhinitis and eczema; (2) foods—​commonly egg, milk, peanuts, and tree nuts; mainly cause acute reactions of varying severity (e.g. urticaria, angioedema, or anaphylaxis); (3) drugs—​particularly antibiotics, aspirin, non​steroidal anti-​inflammatory drugs, and drugs given during general anaes- thesia, although allergy to other drugs/​agents including insulin, radiocontrast media, and chlorhexidine is being described; (4) bee and wasp stings; (5) latex rubber—​now less common. Clinical features and diagnosis Clinical presentation—​this can be in various guises acute or chronic, with common manifestations being (1)  allergic rhinitis—​timing of symptoms indicates the causative allergen; (2) non​allergic rhinitis—​ some have aspirin sensitivity, rhinosinusitis, nasal polyps, and asthma; (3) conjunctivitis; (4) asthma—​timing or circumstances of symptoms and exacerbations gives clues to aetiology; (5) eczema; (6) urticaria and angioedema—​severe tongue swelling is a medical emergency and most often drug induced (especially angiotensin-​converting en- zyme inhibitors) or idiopathic (non-​IgE mediated); (7) anaphylaxis—​ presents with acute dyspnoea or hypotension/​collapse, usually with cutaneous features such as erythema or urticaria. History taking—​a good history is the key to diagnosis: too often the underlying allergic trigger is not identified and disease which could be ameliorated by allergen avoidance continues unchecked; aware- ness of drug and latex allergy are essential. In some conditions exclu- sion of allergy is important. Clinical investigation—​(1) serum tryptase—​may be transiently ele- vated for up to 4 h following an acute reaction, but peaks at 1–​2 h; (2) skin prick tests or serum-​specific IgE assays—​many patients have positive tests without symptoms, hence performing them in the ab- sence of appropriate clinical information is a common source of error; (3) intradermal and challenge tests—​performed by allergy spe- cialists, mainly for diagnosis of drug and food allergy. Prevention and treatment Prevention—​there are no widely applicable proven methods for pri- mary prevention of allergy. Emerging data suggests that early intro- duction of foods (e.g. peanut), in infants may prevent food allergy, particularly in those at high risk of allergy. Acute or chronic disease—​management requires (1) allergen avoid- ance; and may also involve (2) pharmacotherapy—​including non​ sedative antihistamines and topical corticosteroids (nasal sprays, inhalers, and creams); and, less commonly, (3) immunotherapy (de- sensitization)—​should be offered to patients with poorly controlled allergic rhinitis or venom anaphylaxis. Anti-​IgE therapy should be considered for severe asthma or urticaria refractory to standard therapy. Other monoclonal antibodies are being introduced. 4.5 Allergy Pamela Ewan

4.5  Allergy 369 Anaphylaxis—​first-​line treatment is intramuscular adrenaline (epi- nephrine). All patients should subsequently be referred to an allergy specialist for diagnosis and management: allergen or trigger avoid- ance (e.g. food or drug), reduces further episodes and should be combined with an adrenaline autoinjector for early self-​treatment. Introduction Allergic disorders are wide ranging, and include asthma, eczema, rhinitis, anaphylaxis, angioedema, and urticaria. Some disorders will always be allergy driven (e.g. food, venom, or latex allergy), whereas others (e.g. asthma or rhinitis) may be allergic or non-​ allergic, but the role of allergy is increasing. Allergy has increased in prevalence, severity, and complexity over the last four decades, with a resulting burden on patients and cost to health services. Failure to make an allergy diagnosis adversely affects manage- ment and outcome. Avoiding a food or a drug can completely ameliorate disease, yet many allergic disorders are treated with pharmacotherapy only. Allergy practice involves both IgE-​mediated (classical allergy) and non-​IgE-​mediated disorders. In the latter group—​which in- cludes certain types of anaphylaxis, angioedema, and rhinitis—​the signs and symptoms mimic IgE-​mediated allergy because release of mast cell mediator occurs, but IgE is not involved. Historical perspective Early descriptions of allergy exist. Hay fever was described in 1873 by Charles Blackley, who demonstrated that pollen was the cause by applying pollen grains to his nose and eye in winter and reproducing the symptoms. Passive transfer of sensitivity to the skin by injecting serum from a fish allergic person to a non​allergic subject was dem- onstrated by Prausnitz and Kustner in 1921 but it was not until 1967 that the serum factor (reagin) was shown to be a new class of im- munoglobulin, IgE, by the Ishizakas in the United States of America and then by Bennich and Johansson in Sweden in 1971. Aetiology and pathogenesis Type I hypersensitivity (allergic) reactions The term ‘allergy’ is used variably. It is often used synonymously with the type I IgE-​mediated reaction described by Gell and Coombs. Interaction of allergen with specific IgE antibody bound to high-​ affinity IgE receptors (FcεRI) on mast cells results in mast cell acti- vation, degranulation, and mediator release (Fig. 4.5.1). Histamine and other mediators including leukotrienes and prostaglandins cause vasodilation, smooth muscle contraction, mucosal oedema, and secretions. Non-​IgE-​mediated reactions Mast cell activation and mediator release may occur independent of IgE antibody. Certain drugs and physical stimuli (e.g. pressure on the skin, cold, or exercise) do this in susceptible individuals. However, it can also occur without a recognized trigger. Mechanisms are poorly understood. Steps in the development of allergy A normal subject has no specific IgE to common allergens and a low or normal total serum IgE level—​the non​atopic state. Production of specific IgE antibody requires a change in immunoregulation with a switch from the Th0/​Th1 state to a Th2 dominant state. This may result from failure of function of T regulatory cells, which produce interleukin-​10 (IL-​10) and transforming growth factor β. Th2 cells secrete the cytokines IL-​4 and IL-​13 which result in B cell switching to IgE production (atopic; Fig. 4.5.2). The atopic state often has no associated symptoms: this is known as sensitization. A proportion of sensitized subjects progress to develop clinical al- lergy (allergic). Prevalence Atopy Atopy is defined as the presence of specific IgE to one or more common allergens. Specific IgE can be detected by skin prick testing. The incidence of atopy in the general population is high, many studies previously showing rates of about 40%, but this appears to be rising. In a study in the United States of America, the third National Health and Nutrition Examination Surveys conducted between 1988 and 1994, 54% of the population tested had positive skin prick tests to 1 or more of 10 common allergens (i.e. they were atopic). This was an increase compared to the findings in an earlier study (1970 to 1980), with prevalences two to five times higher for the six allergens common to both studies. Mast cell Allergen Specific IgE Mediator release Histamine Fig. 4.5.1  The type I allergic reaction. Reproduced from the BMJ, Pamela W. Ewan, 316(7142):1442–​5, © 1998, with permission from BMJ Publishing Group Ltd. Nonatopic Atopic or sensitized Clinical allergy Specific IgE positive Symptomatic Specific IgE positive Asymptomatic Specific IgE negative Asymptomatic Fig. 4.5.2  Stages to the development of allergy.

370 SECTION 4  Immunological mechanisms Sensitization as a predictor of allergy Various studies suggest that sensitization is a predictor of later al- lergy. In some infants, sensitization to egg precedes and predicts the development of eczema and/​or peanut allergy. Early sensitization to food allergens in the first year of life is also a predictor of subsequent sensitization to inhalant allergens, which in turn predicts the inci- dence of asthma and hay fever in young adulthood. Clinical allergy It is not possible to provide precise prevalence data for allergy overall. There are incomplete or missing data for some allergic dis- orders (e.g. drug allergy and anaphylaxis); allergy is involved in a subset of certain diseases (e.g. eczema or asthma); and different manifestations of allergy occur in one individual (multisystem al- lergic disease). A picture can be built up, however, with minimum estimates for number of people affected. In the United Kingdom, about 18 million (39% of children and 30% of adults) have been diagnosed with one or more of asthma, eczema, and rhinitis. A considerable proportion of this is allergic in origin (e.g. 26% of the population has allergic rhinitis). Serial surveys show that the prevalence of asthma, rhinitis, and ec- zema has increased about threefold over three decades, and this is thought to be due to an increase in the prevalence of allergy. Comorbidity is common and increases the likelihood of allergy being involved. Food allergy occurs in about 3% of adults and 4% of children—​ 1.8  million people in the United Kingdom. Nut allergy, where there are more accurate data, occurs in 1–​2% of children in de- veloped countries and a growing number of adults as this allergy emerged in the mid-​1990s in more than 460 000 individuals in the United Kingdom. Venom allergy occurs in about 2% of the population. There are incomplete epidemiological data on anaphylaxis. The lifetime risk of symptoms suggestive of anaphylaxis in the general population as reported in surveys of the public, is at least 1.6%. Results of several studies in Europe suggest an incidence of 1.5–​7.9 per 100 000 person-​years and a prevalence estimated at 1 in 300. United Kingdom data shows that the number of hospital admissions for anaphylaxis rose by 615% from 1992 to 2012 (Fig. 4.5.3). The same picture is seen in developed countries worldwide. The absolute numbers do not reflect prevalence as only a minority is admitted, most being managed in emergency departments, however the trend is clear. Admission rates and fatalities from drug and venom al- lergy predominate in older people. Food-​induced anaphylaxis is commonest in children and fatal food reactions in the teens and early twenties. Taking individual causes, food, and venom allergy cause anaphylaxis in a few million people in the United Kingdom. Since 1990, hospital admissions for food allergy, urticaria, and angioedema have also increased. Important areas where there are few data are drug allergy and angioedema. Even for penicillin allergy, where there are most data, prevalence data is incomplete. About 5.9  million people in the United Kingdom are labelled as penicillin-​allergic, yet only about of 10% of these are truly allergic. There are no data on prevalence of sensitivity to aspirin, NSAIDs, or other analgesics—​an increasing problem. There are several new allergies (e.g. to fruits and vegetables and sesame), where information is incomplete. Complex or multisystem allergic disease is now common. Allergic asthma, rhinitis, and ec- zema commonly coexist, and food allergies occur mainly in patients with these disorders. Latex allergy is now uncommon, following the introduction of non​powdered latex gloves. Aetiology: Allergens The allergic trigger varies with the disorder and to some extent with the age of the patient. • Inhaled allergens are the commonest cause of allergic asthma, rhin- itis, and eczema, especially house dust mite, pollens, and animal danders. These three allergens are the most common causes of al- lergy in the United Kingdom. Alternaria and Cladosporium are important causes of acute seasonal severe asthma (and/​or rhinitis) in late summer. Eczema in adults or older children may be driven by house dust mite allergy. 10 8 6 4 2 0 1995 2000 2005 2010 Male lCD change 4-hr wait target introduced Female Year Hospital admissions for anaphylaxis per 100 000 population 0.10 0.05 0.00 Male Female 1995 2000 2005 2010 Year Fatalities due to anaphylaxis per 100 000 population (a) (b) Fig. 4.5.3  Rising hospital admissions for anaphylaxis. Reproduced from Turner PJ, et al. (2015). Increase in anaphylaxis-​related hospitalizations but no increase in fatalities: an analysis of United Kingdom national anaphylaxis data, 1992–​2012. J Allergy Clin Immunol, 135, 956–​63.e1.

4.5  Allergy 371 • Foods commonly responsible for allergy include egg, milk, peanuts, and tree nuts. Others are fish, shellfish, fruits, vegetables, sesame, seeds, and soya. These will mainly cause acute reactions of varying severity (e.g. urticaria, angioedema, or anaphylaxis). In toddlers, foods, particularly egg or cow’s milk, are important triggers for eczema. • The important drugs are antibiotics, aspirin, non​steroidal anti-​ inflammatory drugs (NSAIDs) and drugs given during gen- eral anaesthesia, especially the neuromuscular blocking agents. Insulin, opiates, and vaccines are rarer causes of systemic allergic reactions. Local anaesthetic rarely causes allergy, although it is commonly perceived. Diagnostic dyes, chlorhexidine, intra- venous colloid, and radio contrast media occasionally cause anaphylaxis. • Bee and wasp venoms cause systemic allergic reactions of varying severity including anaphylaxis. • Latex rubber causes a variety of symptoms including urticaria, angioedema, asthma, rhinitis, and anaphylaxis. Prevention Primary prevention Environmental factors play an important role in the development of allergy. The hygiene hypothesis suggests that early exposure to microbial infection is protective, driving Th1 responses (e.g. chil- dren exposed to endotoxin from farm animals were less likely to develop allergic disease). Avoiding exposure to food allergens in early life (maternal diet during pregnancy and lactation and the infant’s diet), was sug- gested in 1998 as a means of preventing allergy, but the effect is not established. The lack of evidence was confirmed in a gov- ernment committee review in 2016. Emerging data suggests the opposite, that early introduction of foods (peanut and egg), par- ticularly in higher-​risk infants may induce tolerance and prevent food allergy. Secondary prevention Treatment with oral antihistamine has been shown to prevent or delay the development of asthma in infants with atopic dermatitis sensitized to grass pollen or house dust mite. Immunotherapy for rhinitis can prevent the development of asthma. Clinical features The history is the key to reaching an allergy diagnosis (Box 4.5.1). This is supported by appropriate tests, particularly those for specific IgE. Knowledge of allergens, their seasons, sources, and disorders they may cause, as well as presentations and patterns of disease, is essential. Enquiry should be made into the timing of symptoms and the effect of allergen exposure (there may be multiple manifestations of allergy). Some of the ‘allergic’ diseases, including asthma, rhinitis, eczema, and anaphylaxis, may be IgE-​mediated or non-​IgE-​mediated. This distinction needs to be drawn. In the allergic group, the allergic cause should be identified. The likelihood of allergy is increased • in children and young adults; • if multiple systems are involved (e.g. asthma, rhinitis, eczema, food allergy). Allergic rhinitis Symptoms include rhinorrhoea, nasal congestion or obstruction, and sneezing. The dominant symptom varies with the allergen. Pollen allergy (hay fever or seasonal allergic rhino-​conjunctivitis) mainly causes sneezing, nasal itch, and profuse watery secretions as well as itchy watering eyes. The timing of the symptoms indicates the causa- tive allergen (e.g. tree pollen allergy occurs in spring, grass pollen in early summer, and late summer and autumn symptoms are due to shrub or weed pollens or the moulds Alternaria and Cladosporium). In perennial allergic rhinitis, nasal congestion and secretions are the main features. There may be a history of triggers responsible for exacerbations, as well as remissions when away from the allergen. House dust mite allergic patients are often worse on waking (fol- lowing exposure from mattress and bedding overnight), or after cleaning, and better at altitude (dust mite does not survive above 1000 m). Animal allergy is usually evident from exacerbations on contact, or remissions away from home if a pet is kept there. Highly allergic subjects react to exposure to small amounts of hair on the clothing of others, without direct animal exposure. In horse allergy, a few hairs can cause severe periorbital oedema or asthma. Non​allergic rhinitis In non-​IgE-​mediated rhinitis, symptoms are perennial but intermit- tent and variable. A subgroup has aspirin sensitivity, rhinosinusitis, nasal polyps, and asthma. This is often severe, with marked nasal obstruction, and difficult to control. Conjunctivitis This mainly occurs in association with rhinitis, especially due to pollen but also to animals and dust mites. In a small number of patients with hay fever, isolated conjunctivitis and periorbital oedema occurs. Severe disease can result in conjunctival oedema and impaired vision. Asthma Allergy plays an important role in most asthma in children and young adults. The importance of identifying the allergic trigger is that avoid- ance can significantly modify disease and reduce drug consump- tion. The timing and circumstances of symptoms and exacerbations Box 4.5.1  Key knowledge and actions for the physician • Awareness of disorders where allergic aetiology should be considered • Management of allergic medical emergencies—​anaphylaxis (IM adren- aline first line)—​tongue or laryngeal oedema • Measure acute serum tryptase in suspected anaphylaxis • After suspected anaphylaxis provide adrenaline autoinjector • Avoiding the triggering agent in drug, food, or latex allergy • Interpretation of specific IgE results to avoid diagnostic error (positives often indicate sensitization and not clinical allergy) • Document and check for drug allergy; distinguish drug allergy from intolerance • Have established referral route to allergy

372 SECTION 4  Immunological mechanisms gives clues to aetiology. In pollen asthma, symptoms are seasonal. Alternaria allergy results in acute severe attacks of asthma at har- vest time (July and August in the United Kingdom), when allergen is released resulting in sudden peak levels. Many patients have had hospital admissions with asthma at that time of the year. Accurate allergy diagnosis means that prevention measures can be put in place. Other causes are animals: exposure to cat, dog, or horse may induce acute wheeze. In a subset this can be life-​threatening. House dust mite allergy is a major cause of perennial asthma. Eczema Atopic eczema in children most commonly affects the flexures and neck. In severe cases it may be widespread. It is more difficult in eczema to identify triggers from the history. Sometimes exacerbations are ob- vious (e.g. after contact with animals or if both eczema and rhinitis are exacerbated by dust exposure). House dust mite allergy is an important trigger. Scratching at night will rub allergen into the skin, driving the disease. Food allergy causes eczema in children. A problem is that many patients with eczema have specific IgE antibody to multiple allergens, many of which are not clinically relevant. If there is no clear history of flares with allergens, interpretation of allergy tests can be difficult. A trial of allergen exclusion over a few weeks is then required for diagnosis. Urticaria and angioedema These may occur separately or together. Urticaria is common. Acute episodes may be allergic (see earlier) but chronic urticaria, defined as daily symptoms lasting for 6 weeks, is rarely allergic. Urticaria con- sists of itchy wheals, raised lesions with pale centres and surrounding erythema. Wheals are of varied size and usually occur in crops on the limbs or trunk but can be extensive. Lesions tend to be short lived, but as one crop fades another appears. In giant urticaria, lesions the size of the palm of the hand occur. These are more oedematous and take longer to resolve. When urticaria and angioedema coexist, urticaria is usually dominant with occasional episodes of angioedema. It is important in chronic urticaria to determine aetiology. The usual assumption is that this is allergic, and foods are often impli- cated, leading to restricted diets, but most urticaria is non-​IgE medi- ated (idiopathic). Ruling out allergy is important. Some is related to auto-immunity. Some is physical, triggered by heat, cold, exercise, pressure on the skin, or contact with water. In cold urticaria, chilling of the skin (e.g. by putting the hands in cool water or exposure to cold wind) causes erythema and pruritus. After more prolonged exposure, angioedema occurs. If a large surface area is involved, as in sea swim- ming, hypotension and loss of consciousness occur. Drugs, especially NSAIDs and aspirin, are an important cause of acute attacks. Infection commonly triggers urticaria, especially in children. Angioedema The commonest sites for angioedema are the lips and eyelids. This type of angioedema often occurs with urticaria but may occur alone. Angioedema may also involve the tongue and larynx, pharynx, or uvula. Swelling is often unilateral. Tongue swelling usually occurs alone, without swelling at other sites or urticaria. Angioedema of the whole tongue can cause respiratory obstruction, cyanosis, or respiratory arrest. Severe tongue swelling is a medical emergency. Tongue swelling is mostly drug induced (especially angiotensin converting enzyme inhibitors) or idiopathic (non-​IgE mediated). If the patient is taking an angiotensin-​converting enzyme inhibitor, the first step should be to stop the drug. Angiotensin converting enzyme inhibitor induced angioedema is thought to be due to activation of the kallikrein-​kinin system with bradykinin generation. The diagnosis is made from the history combined with exclusion of other causes. There is no confirmatory laboratory test. Onset is either within weeks of starting medication or, confus- ingly, after an interval of many months or years. It is a class effect. Angiotensin II receptor antagonists are usually tolerated. Most iso- lated angioedema is non​allergic. There is no erythema or pruritus. Allergic (IgE-​mediated) angioedema When angioedema is allergic, other features are usually present. Horse, other animal and pollen allergy cause periorbital oedema but usually with conjunctivitis and/​or conjunctival oedema; there may also be rhinitis and asthma. Food allergy commonly causes perioral angioedema but there will also be perioral urticaria and oral prur- itus and systemic features, such as abdominal pain or vomiting, may occur. In latex allergy the angioedema will be periorbital or perioral if there has been local rubber contact (e.g. rubber goggles, swim- ming cap, or blowing up a balloon). Pruritus, erythema, and urti- caria will be present. Hereditary angioedema (HAE) and C1 inhibitor deficiency C1 esterase inhibitor deficiency is a rare autosomal dominant dis- order (hereditary angioedema, HAE) but may sometimes be acquired (acquired angioedema, AAE). HAE is a distinct disorder, due to the deficiency or dysfunction of the complement protein C1 inhibitor. In acquired angioedema, there is depletion of C1 inhibitor due to com- plement activation by monoclonal or auto-​antibodies. C1 inhibitor is a key regulator of the Factor XII/​kallikrein proteolytic cascade that leads to bradykinin production. The deficiency results in unopposed activation of the kallikrein system, leading to generation of brady- kinin, which causes increased vascular permeability and oedema. The deficiency also results in activation of the classical complement pathway, with generation of the peptides C3a and C5a, which cause increased capillary permeability and smooth muscle contraction. In type 1 HAE (which accounts for about 85% of patients), there is a low level of C1 inhibitor in serum (immunochemical assay low); whereas in type 2, there is a functional defect and the immunochemical assay for C1 inhibitor is normal, but the functional assay is extremely low. In HAE there is a usually a family history, but spontaneous mutations arise. These various forms have similar clinical features and man- agement. There is another form of HAE, extremely rare and difficult to confirm, having the clinical features of HAE often with a family history but no complement abnormalities (HAE with normal C1 in- hibitor), sometimes associated with mutations in the gene for factor XII. AAE mainly occurs in older patients, has no family history, and is associated with an underlying, usually lymphoproliferative, dis- ease. Treatment of the underlying condition results in a return to normal of the C1 inhibitor level. Angioedema affects the skin or mucosa and occurs at one or more of three sites: cutaneous, intestinal, and laryngeal. There is no ur- ticaria. Patients present with peripheral swellings, typically flitting, involving different sites in different attacks—​hence distinct from idiopathic or allergic angioedema which is restricted in site, usually lips or eyelids. The hand, limbs, and genitals are often affected. If the face is involved the swelling is more extensive and not limited to lip or eye. Intestinal mucosal oedema, which causes partial intestinal

4.5  Allergy 373 obstruction, presents with abdominal pain and vomiting. The least common but most severe manifestation is laryngeal oedema, which is life-​threatening and can be fatal. Attacks are self-​limiting, lasting 48 to 72 h, and of variable severity. Although the angioedema re- solves spontaneously without treatment, laryngeal oedema can cause asphyxia and severe attacks the gastrointestinal tract mucosal oedema result in subacute intestinal obstruction. Attacks are inter- mittent, with many months between episodes when patients are well. The diagnosis of HAE should be suspected from the characteristic clinical picture which is distinct from other causes of angioedema. The different pattern and distribution of angioedema combined with attacks of unexplained abdominal pain, differentiate this from idiopathic angioedema. However, diagnostic delay of many years is usual, and these patients have commonly had repeated emer- gency attendances or admissions and sometimes laparotomies. Confirmatory investigations are low C4 and C1 inhibitor levels. Treatment should be managed by a specialist. Prophylactic treat- ment is with the attenuated androgen, danazol, which increases the hepatic production (by the normal gene) of C1 inhibitor. Only a mar- ginal increase in C1 inhibitor is required to control the disease and levels of C1 inhibitor remain profoundly low. There have been con- cerns about side effects of danazol, compounded by the use of unneces- sarily high doses. Low or moderate doses of danazol prevent attacks in many patients, thus limiting side effects. The approach should be to gain control then reduce the dose, titrating this down until a few mild breakthrough attacks occur. Some patients are controlled on as little as 100 mg twice weekly. Determining the minimum effective dose limits or avoids side effects. This is the first-​line approach in most patients, but danazol cannot be used in prepubertal children or pregnancy. Tranexamic acid can be used in children but is much less effective than danazol. Fortunately, HAE may not present until the teens. There are several treatments for acute attacks. This may be by replace- ment with intravenous C1 inhibitor concentrate derived from plasma. A newer recombinant C1 inhibitor concentrate is available but in trials, effectiveness was not established in patients with laryngeal attacks. Drugs that target the downstream pathways involved in C1 inhibitor deficiency are alternatives for acute attacks and include the bradykinin B2 receptor antagonist, icatibant and ecallantide, a recombinant plasma kallikrein inhibitor (not licensed in the United Kingdom), both for subcutaneous injection. All of these are high cost. Icatibant comes in a prefilled syringe suitable for self-​injection and home use. There is a delay of a few hours to the onset of symptom reduction, so it is dangerous for patients with laryngeal attacks to remain at home and they must imme- diately attend the emergency department after self-​injection. In some patients C1 inhibitor appears to be more effective than icatibant. There are encouraging efficacy results from trials of a new kallikrein inhibitor, lanadelumab which is now licensed. This is a recombinant fully human monoclonal antibody and the first therapeutic agent to allow sustained inhibition of kallikrein and limit bradykinin generation. Home therapy with icatibant or intravenous C1 inhibitor concen- trate is being used to give earlier control and avoid emergency de- partment visits. This approach may encourage some patients to stop danazol prophylaxis due to fears about side effects putting them at more risk of life-​threatening reactions. Patients should have fast track arrangements with their local emergency department and, as this is a rare disease, carry an information sheet/​treatment plan. A proposal is for prophylaxis with C1 inhibitor or icatibant (e.g. twice weekly) in patients having frequent attacks, but this occurs in very few patients. Anaphylaxis Anaphylaxis is an acute severe systemic reaction of rapid onset. There is no universally agreed definition. There are many features, not all of which need be present (Box 4.5.2). One of the two severe features, respiratory difficulty, or hypotension, should be present. Severe dyspnoea is due to laryngeal oedema, often described as a sensation of the throat closing up, or acute asthma. Hypotension presents as weakness, difficulty standing, collapse, or loss of con- sciousness. Cutaneous features are usually present. Rarely profound hypotension with loss of consciousness is the only feature, mostly after bee or wasp stings or intravenous drugs. The main causes of anaphylaxis are foods, drugs, and venom al- lergy or idiopathic reactions. Food accounts for around 90% of childhood anaphylaxis, whereas in adults, food, venom, and drug allergy and idiopathic reactions each account for approximately a quarter of cases. In food-​induced anaphylaxis, respiratory symp- toms are the dominant severe feature. In contrast, in venom allergy or allergy to an intravenous drug, hypotension predominates. Thus, the clinical picture varies with the cause of the anaphylaxis. Non-​IgE-​mediated (idiopathic) anaphylaxis is becoming more common. This usually presents in a different way and with a slightly slower evolution. It often begins with pruritus of the palms and soles, then progresses to general pruritus, erythema, and urticaria, with diarrhoea, abdominal pain, and hypotension. Respiratory symp- toms are uncommon. Food allergy Different foods cause different patterns of disease: with differences in severity, clinical features, comorbidities, and likelihood of reso- lution or persistence. Severity varies from mild, usually facial and oral urticaria/​oedema, to anaphylaxis (Table 4.5.1). Egg and cow’s milk are the commonest food allergies in infants and toddlers. Egg allergy Egg allergy is mostly mild or moderate. Symptoms include perioral or facial erythema, urticaria, and angioedema, often with vomiting. Diarrhoea is rare. In more severe egg allergy asthma and anaphylaxis occur. Egg allergy can be partial and at presentation the child may Box 4.5.2  Clinical features of anaphylaxis • Erythema • Pruritus • Urticaria • Angioedema • Laryngeal oedema • Asthma • Nausea, vomiting, abdominal cramps • Sense of impending doom • Fainting, light-​headedness • Collapse • Loss of consciousness • Fits (rare) • Incontinence (rare)

374 SECTION 4  Immunological mechanisms tolerate a low allergen dose (e.g. egg as a baked ingredient, but not whole cooked egg). Most egg allergy in infants and young children resolves by or before school age. Management includes exclusion of egg from the diet and review to decide on timing and pace of gradual egg reintroduction. In the minority where disease persists, this can be severe, as can adult-​or teenage-​onset egg allergy. The measles, mumps, and rubella (MMR) vaccine does not contain egg protein and can safely be given. Inactivated (injected) influenza vaccines that are egg-​free or contain egg protein, at very low levels, can be given to most patients with egg allergy. Children from age 2 years with egg allergy should be vaccinated with the nasal influenza vaccine. Yellow fever vaccine contains egg thus specialist advice is re- quired. A desensitization protocol and incremental dose regime have been trialled and shown to be effective in a small number of patients. Cow’s milk allergy Mild to moderate cow’s milk allergy causes similar symptoms to egg al- lergy, but gastrointestinal symptoms with vomiting, abdominal cramps, and diarrhoea are more common. Most milk allergy in infancy resolves early. Where disease persists it can be severe, tiny quantities of milk protein causing anaphylaxis. Non IgE-​mediated reactions against cow’s milk are described as cow’s milk protein intolerance. Clinical presen- tation overlaps with milk allergy, but gastrointestinal symptoms and eczema predominate. This usually resolves by the age of 12 months. Nut allergy Nut allergy has the propensity to cause severe life-​threatening reactions. In the mid-​1990s, when this disorder first appeared in significant num- bers, about two-​thirds of cases were severe with airway involvement, either laryngeal oedema or asthma. Peanuts account for most food-​ induced fatal and near-​fatal reactions. In these airway obstruction and asphyxia occur. The diagnosis of peanut allergy therefore causes anxiety. Although about two-​thirds of individuals now have a history of mild disease there is the risk of reactions becoming more severe. Peanut is a legume, not a nut; it is thus botanically distinct from tree nuts—​nonetheless, allergy to both in an individual is frequent. Peanut is the most common ‘nut’ to cause reactions. Of the tree nuts, Brazil nuts, almonds, hazelnuts and cashew nuts most frequently cause allergy. Brazil nuts and cashew nuts result in the highest pro- portion of severe reactions. Most peanut-​allergic subjects are not al- lergic to pulses such as peas and beans. Nut allergy mostly begins in childhood. The average age of onset of peanut allergy is 2 years. Allergy to tree nuts then appears progres- sively during childhood, but some patients remain ‘monoallergic’. Allergy presenting in older children or adults is more likely to be due to a tree nut. There is a strong association with atopy: 96% are atopic and about two-​thirds have asthma, rhinitis, or eczema. Kiwi fruit allergy This is one of several newer food allergies. Kiwi allergy began to appear after the introduction of kiwi fruits to the United Kingdom food market, and the incidence gradually rose in parallel with con- sumption. The main symptoms are perioral urticaria and oral mu- cosal oedema, but anaphylaxis with laryngeal oedema may occur. Oral allergy syndrome This is new disorder, emerging over the last two decades and now very common. It is allergy to fruits and sometimes vegetables or nuts in patients with tree or grass pollen allergy (spring or summer hay fever). The primary sensitisation is to pollen and the major al- lergens in certain fruits show structural homology with the major birch pollen allergen. Stoned fruits (e.g. apple and peach) are the commonest cause. Symptoms are oral, palatal, and pharyngeal itch and mucosal oedema. The food is tolerated when well–​cooked as the allergens are heat labile. This is thought to be a mild disorder, but over time a small subset with more marked symptoms has emerged. Severe allergy to fruit is increasingly recognized. This appears to be a distinct disorder where the IgE is directed against different allergen components to those in oral allergy syndrome. Similarly, primary nut allergy, potentially more severe, should be distinguished from oral al- lergy syndrome to a nut. The differential diagnosis can be supported by component resolved diagnosis, measuring the cross-​reacting subset of specific IgE (PR-10 proteins) found in OAS and the IgE to storage pro- teins found in primary allergy. For example, in peanut allergy, IgE to the PR-10 peanut protein, ara h8, is found in oral allergy syndrome whereas IgE to ara h2, the seed storage protein, is found in primary peanut allergy. Fish and shellfish These causes urticaria, angioedema, and vomiting. Severe anaphyl- actic reactions occur. Fish and shellfish allergy often occur separ- ately and within fish allergy, not all fish types may be involved. Hymenoptera venom allergy Bee or wasp (yellow jacket) stings are an important cause of anaphylaxis but cause allergic reactions of varied severity, from urticaria through to anaphylaxis. Hypotension is common in severe venom reactions. Wasp sting allergy is more common in the United Kingdom. Bee sting allergy mainly occurs in beekeepers and their relatives (i.e. those frequently stung). The pattern of reactions varies but need not be progressively worse. A factor favouring a less severe reaction subsequently is a long interval between stings. Rarely, these stings can be fatal. A risk factor for very severe reactions is a raised baseline tryptase, indicating suscepti- bility to mast cell activation. For further information see Chapter 10.4.2. Drug allergy Evidence from national patient safety incident reports and from re- search shows that a large number of NHS patients with known drug allergies are re-​exposed to these drugs in error each year (of 18 000 preventable drug incidents, 80% were in patients with known allergy). Many people have been inaccurately diagnosed and recorded as ei- ther having or not having a drug allergy. While re-​exposure to a drug Table 4.5.1  Varying presentations of acute allergic reactions to foods according to severity Severity Clinical features Mild Cutaneous features only: pruritus, erythema, urticaria, or mild angioedema Moderate The above, plus more severe angioedema and/​or vomiting, abdominal pain, and/​or mild dyspnoea or tightening of throat Severe The above, plus respiratory difficulty (laryngeal oedema or asthma) and/​or hypotension (less common)

4.5  Allergy 375 has not caused harm in the majority of people, a minority of these incidents have caused harm or death. One of the commonest errors is administration of co-​amoxiclav to a penicillin allergic patient. Better recognition and documentation of drug allergy is essential. Diagnosis can be difficult and many reactions are falsely labelled as drug allergy. Penicillin, muscle relaxants, insulin, and other hor- mones act via IgE-​mediated mechanisms, whereas NSAIDs and acetyl-cholinesterase inhibitors, produce angioedema and ana- phylaxis or adverse reactions by non-​IgE-​mediated mechanisms. Opiates can cause IgE and non-Ige mediated reactions. However, this is a complex area and antibiotics including β lactams, may also cause reactions through other mechanisms (e.g. T-​cell mediated as one possibility). IgE-​mediated reactions to β-​lactam antibiotics), re- sult in rash, angioedema, or anaphylaxis. Less severe reactions in- volve rash which may be maculopapular or urticarial, and mainly follow oral administration. Reactions usually occur after one dose or 1 to 2 days into treatment. Anaphylaxis occurs after parenteral ad- ministration (within minutes of intravenous administration) but is described, rarely, after oral treatment. Always check for drug allergy before administering an intravenous drug. With the increased use of antibiotics, more patients are developing allergy. Some patients are allergic to more than one class of antibiotic. Investigation is not simple and for the most part blood tests are not helpful. Serum IgE to penicillin has such poor predictive value (sen- sitivity 0–​25%) it should not be used alone. Specialist allergy referral is required and investigations include skin prick tests, intradermal tests (with late reading if T-​cell mediated reactions are suspected) and drug challenge. The latter is often required in penicillin allergy, as up to 30% of patients cannot be identified on skin testing alone. Most penicillin allergic patients tolerate aztreonam or carbapenems (e.g. all of a series of 212 patients) but rare cross reactivity between penicillins and these drugs has been reported. Sensitivity to aspirin and NSAIDs presents with urticaria and angioedema or with asthma and laryngeal oedema. The time of onset in relation to drug admin- istration depends on the route and formulation: 30 to 60 min after oral non​enteric coated preparations and several hours after slow-​ release preparations. Intravenous or rectal administration results in more rapid onset, often 15 to 30 min. Anaphylaxis during anaesthesia may be due to the anaesthetic agent, usually a neuromuscular blocking agent. However, increasingly other drugs administered are responsible. These include antibiotics, NSAIDs, opiates, chlorhexidine, diagnostic agents, and colloid. If a drug reaction is suspected, it is important to document the de- scription, all drugs being taken at the time, and the time of onset in relation to duration of drug administration, as detailed in the NICE drug allergy guideline (Table 4.5.2). Latex allergy Latex allergy causes reactions of varying severity. Most are mild to moderate, with contact erythema, urticaria, and angioedema. This can remain localized or become generalized as the allergen is ab- sorbed. Most reactions occur in medical settings, from latex gloves or equipment. Thin, stretchy rubber products such as surgical gloves are most allergenic, whereas black solid rubber is inert and causes few reactions. Exposure through contact with dentists’ gloves causes local symptoms in the mouth or face. Absorption from surgeons’ gloves may occur through the peritoneum or mucosal surfaces (e.g. vaginal examinations during labour) resulting in anaphylaxis. In daily life exposure may be from condoms, household gloves, swimming caps, and so on. Blowing up balloons leads to perioral angioedema. Inquiry into the effect of these exposures can elicit whether a pa- tient has latex allergy. Almost all patients with latex allergy are atopic and have other allergies such as asthma, rhinitis, and eczema. There is a strong association with hand eczema, and broken skin increases Table 4.5.2  Approach to drug allergy (adapted from NICE guideline 2014) Assessment Document signs, time of onset, suspected drug, other drugs Immediate reactions (early onset c.1 h) may include: erythema, urticaria, angioedema, hypotension and/​or bronchospasm Non​immediate (onset after 3–​6 days) reactions without systemic symptoms red macules or papules (exanthema-​like) Non​immediate reactions with systemic involvement (e.g. DRESS, Stevens–​Johnson syndrome, toxic epidermal necrolysis) Acute investigation Measure serum tryptase Documentation and sharing information Document allergy/​suspected allergy in medical records Complete allergy box Share drug allergy status With patient, in GP letters, discharge summaries Patient wrist bands Distinguish drug allergy from adverse drug reactions Check drug allergy status before prescribing or administering any drug Non​specialist management Stop drug Treat reaction Document Refer to specialist (if indicated) Allergy specialist Provide written advice on: Drug allergy Investigations used to confirm diagnosis; which drugs to avoid, and drugs safe for future use

376 SECTION 4  Immunological mechanisms absorption of latex. About 50% have food allergy due to cross-​reacting allergens. This association was originally made for banana, avocado, and melon, but a wide variety of foods may be involved. A latex al- lergic subject need only avoid foods to which allergy has been proved. However latex allergy has become uncommon since the intro- duction of non​powdered latex gloves. Non​latex gloves (e.g. nitrile) are also used for many healthcare tasks. In the past, latex allergic subjects were often healthcare workers—​medical, nursing, dental, and ambulance staff—​presumably sensitized through their in- creased exposure to latex. The commonest reaction is a local reaction on the hands with pruritus and urticaria. Longer exposure results in increased symptoms, including angioedema. In operating theatres where powdered latex gloves are used, repeated glove change leads to an aerosol of allergen in the powder, causing rhino-​conjunctivitis. These symptoms can be linked to occupational exposure. Other groups at increased risk of developing latex allergy include children who have undergone repeated surgery (e.g. for spina bifida). Until the 1980s latex allergy was rare, with only a few case reports. It then became common, especially in healthcare workers, probably because of the increased use of rubber gloves. There were deaths from anaphylaxis due to rectal absorption of allergen from latex rubber catheters for barium enemas. New cases are now rare, fol- lowing the introduction of non-powdered latex gloves. Latex allergic patients require strict latex avoidance when under- going medical procedures or surgery; latex-​free equipment must be used. Most catheters and many other medical products are now non-​ latex, but the vaginal probe used in gynaecological ultrasonography may be covered with a condom and reactions have occurred. It is important to distinguish latex allergy from contact dermatitis due to a type IV reaction to chemicals used in rubber manufacture. This presents with hand eczema, and is diagnosed by a patch test to these chemicals. It is not dangerous and if the patient is admitted strict latex avoidance is not required although contact with rubber should be avoided. Most Trusts have latex policies providing advice on latex avoidance for patients. Healthcare workers with latex al- lergy are usually able to continue their employment. Differential diagnosis Anaphylaxis may present with collapse and loss of consciousness or severe dyspnoea and cyanosis. It is important to consider myocar- dial infarction, pulmonary embolus, diabetes, or severe asthma. The presence of urticaria or angioedema is helpful. Hypotension is usu- ally accompanied by tachycardia. It is usually straightforward to diagnose the disorder (e.g. asthma or anaphylaxis). The issue is to determine the cause: whether allergy is playing a role and if so which allergy(-​ies). Clinical investigation Acute reactions Tryptase In acute severe reactions, including anaphylaxis, it is valuable to take a timed blood sample for serum tryptase. This is elevated transiently, so the sample should be taken within 1 to 2 h of onset. In very severe anaphylaxis it may still be elevated up to 4 h, so if the optimal 1–​2 h time point is missed a later sample can still be useful. This confirms mast cell activation and degranulation. Tryptase is often but not al- ways elevated in anaphylaxis. Later investigation A detailed allergy history is the key. Knowledge of allergens, the disorders, and symptomatology they cause is essential. Tests do not substitute for this; they can only be used as an adjunct to history, to confirm or refute suspected allergy. Specific IgE: Skin prick tests or serum-​specific IgE assays Tests for specific IgE are best done by skin prick testing, but can also be measured in serum. The latter is commonly referred to as the radio-​allergosorbent test (RAST) after the original test although this is now available as other assays including enzyme-​linked immuno- sorbent assay (ELISA). Skin prick tests are superior, but mainly available in specialist settings. The problem with these tests is interpretation, as many subjects have positive tests without symptoms. To aid interpretation, 95% predictive levels have been identified for some allergens, levels above which there is a high probability of clinical allergy. Although helpful, this does not resolve the problem, as for individual allergens, different predictive values are proposed depending on the cohort of patients (age, disease, and so on). In addition, there remains a grey area where the test is positive but below the predictive level, where some patients are allergic and others not. A simple rule of thumb is that about half of those with positive tests in the low/​moderate range are sensitized but not allergic, but this varies with the population being studied. The key is the clinical history as test results in isolation can be misleading. If the reaction is non-​IgE mediated, tests for specific IgE are ir- relevant. For most of these reactions there are no confirmatory la- boratory tests. This applies to some drug allergies (e.g. NSAIDs and aspirin), where the reaction is a result of leukotriene generation. Intradermal tests There are performed by specialists, and can be useful in drug allergy to detect specific IgE when the skin prick test is negative or in T-cell mediated reactions. Challenge tests These are useful if the diagnosis cannot be reached from history and skin tests. They are used mainly for diagnosis of drug and food allergy, and to determine if resolution has occurred. Challenge testing should only be undertaken in a specialist allergy unit because of the risk of anaphylaxis. Criteria for diagnosis For allergic disorders where there is a trigger, whether IgE-​mediated or not, diagnosis is made by history and confirmatory tests, which may be skin prick tests (or serum-​specific IgE), intradermal test, or challenge. For idiopathic anaphylaxis, angioedema, and so on, diag- nosis is made by exclusion of other causes. Treatment Allergen avoidance Treatment involves pharmacotherapy and avoidance of allergen or trigger. In the case of food or drug allergy, avoidance can completely

4.5  Allergy 377 stop further acute episodes or chronic disease (e.g. eczema). Avoidance of animals will significantly reduce or prevent allergic asthma and rhinitis. House dust mite avoidance measures can only reduce, not eliminate, exposure, but can impact on symptoms. Drug avoidance is easier to achieve, but patients need education on over-​the-​counter medication and on drug groups to avoid. Drug al- lergy should be recorded in medical records. For some foods, avoid- ance is difficult to achieve because the food is an ingredient, often hidden, or listed under an obscure name. Patients therefore need de- tailed advice. However, data from large studies on nut allergy shows that if patients receive education on avoidance, significant reduction in further episodes with a 60-​fold reduction in severe attacks can be achieved (Fig. 4.5.4). In contrast, if the advice is ‘just avoid nuts’, fur- ther reactions because of inadvertent ingestion are frequent. Pharmacotherapy Key drugs are non​sedative antihistamines (quick onset with once daily dosage) and topical corticosteroids, as nasal sprays, inhalers, and creams. Larger than standard doses of antihistamines may be required for difficult urticaria. For allergic rhinitis and conjunctivitis, step-​up therapy tailored to severity is used, as for asthma. Mild disease can be controlled by oral antihistamines, and moderate by nasal cortico- steroid ± oral antihistamines. Steroid nasules or nose drops alone or alternating with nasal steroid sprays are used in severe perennial rhin- itis and nasal polyps. Cromoglicate or nedocromil eye drops are used long term, occasionally with steroid eye drops for short periods for severe conjunctivitis. Intramuscular adrenaline is the drug of choice for anaphylaxis, followed by intravenous chlorpheniramine and hydrocortisone. Further treatment with oxygen, nebulized salbutamol, and intra- venous fluids may be required. If an acute reaction is evolving, with generalized urticaria and angioedema, but not clearly anaphylaxis, treatment can be begun with intravenous chlorpheniramine and hydrocortisone. Patients who have suffered anaphylaxis should carry an adrenaline autoinjector and be trained in its use, unless the allergen can be absolutely avoided. Tongue swelling, depending on severity, requires oral antihista- mine, soluble oral prednisolone, or intramuscular adrenaline. Early self-​treatment with these drugs usually controls the attack. Montelukast is helpful as an adjunct to other therapy in certain subgroups with asthma, angioedema, urticaria (including exercise induced), and nasal polyps with rhinosinusitis. Immunotherapy Allergen immunotherapy, or desensitization, is a different approach to treatment. It alters the immune response, down-regulating and ideally ‘switching off’ the allergy. Conventionally it is given sub- cutaneously. Incremental doses of allergen are given at 1 to 2 weekly intervals until the top dose is reached, then maintenance therapy for 3 years. For pollen, shorter courses of preseasonal treatment are avail- able. Sublingual immunotherapy is increasingly used for a range of allergens in most of Europe and is licensed for pollen in the United Kingdom. A tablet is taken daily for 3 years and only the first dose has to be given in hospital, making it more accessible to patients. There are fewer side effects than with subcutaneous immunotherapy. Cochrane meta-​analysis shows immunotherapy is effective for seasonal allergic rhinitis (subcutaneous) and allergic rhinitis due to pollen or other allergens (sublingual). Immunotherapy is highly ef- fective for venom anaphylaxis. Issues are patient selection and safety. There is an incidence of severe allergic reactions to immunotherapy and deaths occurred in the 1980s, so immunotherapy should only be given by specialists with appropriate expertise. Recent data has demonstrated it is possible to desensitize to foods, including peanut, using oral immunotherapy, with efficacy con- firmed in a systematic review and meta-​analysis. A licensed prepar- ation is expected soon. Anti-​IgE and other biologicals Monoclonal antibody therapy is available for severe asthma and has been shown to reduce repeated hospital admission. Expense and restrictive criteria limit its use. Anti-​IgE is also approved for severe urticaria. While giving control this is usually lost on stopping the drug. as this therapy does not affect the natural course of the disease, so repeat courses are re- quired. In both conditions, anti-IgE therapy can transform patients lives. Other biologicals include an anti-IL4/IL13 for severe eczema and anti-IL5 monoclonal antibodies, which target and deplete eosino- phils for use in severe eosinophilic asthma. Health economics There is a significant burden of allergic disease, but much of this is not diagnosed or coded as allergy. The true size of this burden, the cost to the patient or to the health service, is unknown. In the United Kingdom direct health service costs for managing allergic problems are estimated at over £1 billion/​year. However, the failure to diagnose allergy means disease is left unchecked, resulting in unnecessary cost to the National Health Service. Diagnosis of drug and food allergy stops further manifestation of the disease—​anaphylaxis, acute allergic reactions, or eczema in a child. Other allergen avoidance leads to con- trol of symptoms and lower drug consumption, again saving cost. Incorrect labelling as allergy also has economic consequences. Only 10% of the 5.9  million people in the United Kingdom la- belled as allergic to penicillin are in fact allergic. Costly alternative antibiotics are prescribed unnecessarily, causing further problems with prolonged hospital stays and increased incidence of C. difficile and vancomycin-​resistant enterococcus. Accurate diagnosis in the subset with high or specific antibiotic needs is cost effective. Areas of uncertainty • Requests for allergy tests, usually serum-​specific IgE, without al- lergy knowledge to interpret the results is a common source of 0 25 50 75 Index reaction Follow up reaction Severity of reaction % Severe Moderate Mild Fig. 4.5.4  Effect of a management plan in reducing subsequent reactions to nuts. Reproduced from Ewan PW, Clark AT (2005). Efficacy of a management plan based on severity assessment in longitudinal and case-​controlled studies of 747 children with nut allergy: proposal for good practice. Clin Exp Allergy, 35, 751–​6. With permission from Wiley-​Blackwell.

378 SECTION 4  Immunological mechanisms confusion and often results in the wrong diagnosis being made. This is because a ‘positive’ test does not necessarily correlate with disease. Thus, a positive specific IgE alone, without the history, cannot be assumed to indicate allergy. A common misconception is that the level of specific IgE correlates with severity. There are levels of specific IgE above which clinical allergy is likely. • It is not known if sublingual immunotherapy is as effective as sub- cutaneous immunotherapy, and further studies are needed on its safety profile. • Oral immunotherapy for food allergy is a promising research area with efficacy demonstrated for peanut allergy and licensed prod- ucts expected. • The mechanism of idiopathic allergic-​type reactions is not known. Better understanding of mast cell biology is needed. These reac- tions may reflect altered mast cells stability. • There are gaps in the epidemiology of allergy, especially for and drug allergy. Future developments Research into new forms of allergen immunotherapy, including oral im- munotherapy for food allergy, are likely to lead to changes in practice. A systematic review has shown that oral immunotherapy for certain foods is effective. Further monoclonal antibodies and drugs targeted against cytokines and other mediators, are likely to become available. FURTHER READING Anagnostou K, et al. (2014). Assessing the efficacy of oral immuno- therapy for the desensitisation of peanut allergy in children (STOP II):
a phase 2 randomised controlled trial. Lancet, 383, 1297–​304. Blumenthal KG, Peter JG, Trubiano JA, Phillips EJ (2019). Antibiotic allergy. Lancet, 393, 183–98. Chu DK, et al. (2019). Oral immunotherapy for peanut allergy (PACE): a systematic review and meta-analysis of efficacy and safety. Lancet, 393, 2222–32. Clark AT, Ewan PW (2008). Good prognosis, clinical features and circumstances of peanut and tree nut reactions in children treated by a specialist allergy center. J Allergy Clin Immunol, 122, 286–​9. Du Toit G, et al.; LEAP Study Team (2015). Randomized trial of peanut consumption in infants at risk for peanut allergy. N Engl J Med, 372, 803–​13. Francuzik W, Dolle S, Worm M (2018). Risk factors and treatment of refractory anaphylaxis - a review of case reports. Expert Rev Clin Immunol, 14, 307–14. Gupta R, et al. (2007). Time trends in allergic disorders in the UK. Thorax, 62, 91–​6. Longhurst HJ (2017). Kallikrein inhibition for hereditary angioedema. N Engl J Med, 376, 788–​9. Mirakian R, et al. (2015). Management of allergy to penicillins and other beta-​lactams. Clin Exp Allergy, 45, 300–​27. Muraro A, et al. on behalf of the EAACI Food Allergy and Anaphylaxis Guidelines Group (2014). Anaphylaxis:  guidelines from the European Academy of Allergy and Clinical Immunology. Allergy, 69, 1026–​45. NICE (2014). Clinical Guideline. Drug Allergy: Diagnosis and Manage­ ment. https://​www.nice.org.uk/​guidance/​cg183 Soar J, et  al. (2008). Working Group of the Resuscitation Council (United Kingdom). Emergency treatment of anaphylactic reactions—​ guidelines for healthcare providers. Resuscitation, 77, 157–​69. Yu JE, Lin RY (2018). The epidemiology of anaphylaxis. Clin Rev Allergy Immunol, 54, 366–74. Zeerleder S, Levi M (2016). Hereditary and acquired C1-​inhibitor-​ dependent angioedema: from pathophysiology to treatment. Ann Med, 48, 256–​67.

4.6 Autoimmunity 379

4.6 Autoimmunity 379

ESSENTIALS Autoimmune diseases occur when a sustained, specific, adaptive im- mune response is generated against self-​components, and results in tissue damage or dysfunction. They probably affect more than 3% of Western populations, more commonly women than men, and have peak incidence in the third to sixth decades. Aetiology and pathogenesis These can usefully be described in terms of (1)  susceptibility—​ inherited or acquired defects in pathways required to maintain tolerance to self-​antigens render the individual susceptible to dis- ease initiation; (2) initiation of autoimmunity—​interaction between susceptibility genes and environmental events initiate an immune response directed at self-​antigens; (3)  transition—​targets of im- mune response change and are amplified, with clinical symptoms assuming a recognizable phenotype; and (4) propagation—​specific immune response to self-​antigens causes damage of tissues, with the release of more antigens that further drive the immune response. Although a single immune effector pathway may predominate in generating tissue dysfunction and damage in some autoimmune diseases, it is much commoner for multiple effector pathways to participate in generating the final phenotype. Those pathways which generate tissue damage or dysfunction include autoantibody binding to target cells, immune complex-​mediated activation of complement and Fc receptor pathways, cytokine pathways, as well as lymphocyte-​mediated cytotoxicity of target cells. The nature and sites of tissue damage determine the pathological and clinical fea- tures of specific diseases. Tissue-​specific autoimmune diseases Immune-​mediated damage is restricted to a particular tissue or organ that specifically expresses the targeted antigen (e.g. (1) Graves’ disease—​autoantibodies bind to and stimulate the TSH receptor, re- sulting in thyrotoxicosis; (2) myasthenia gravis—​autoantibodies target the acetylcholine receptor at the neuromuscular junction, resulting in muscular weakness and fatigue due to the inefficient transmission of the acetylcholine signal; (3) type 1 diabetes—​a cytotoxic T-​cell re- sponse to the β cells of the pancreatic islets results in destruction of the insulin-​producing cells). Systemic autoimmune diseases Typically characterized by simultaneous damage in multiple tissues (e.g. kidney, lung, skeletal muscle, nervous system, and skin). Unlike autoantibodies in tissue-​specific autoimmune diseases, which target tissue-​specific antigens, the autoantibodies in systemic autoimmune diseases are frequently directed against intracellular molecules ex- pressed ubiquitously in multiple tissues (e.g. (1)  aminoacyl-​tRNA synthetases—​targeted in autoimmune myositis and associated inter- stitial lung disease; (2)  small nuclear ribonucleoproteins—​targeted in systemic lupus erythematosus; (3) topoisomerase-​1—​targeted in scleroderma). Each of these molecules is expressed in all cells, where they play critical roles in essential cellular processes (e.g. protein translation, mRNA splicing, and DNA replication and remodelling, respectively). Non​sustained autoimmune diseases Organ or tissue damage and dysfunction tend to be self-​limited and resolve after the first attack, and are very unlikely to recur (e.g. epi- demic Guillain–​Barré syndrome). These diseases typically occur in the setting of infection, and are associated with cross-​reactive im- mune responses that recognize both components of the infecting organism as well as the target tissue. Introduction The effector mechanisms that the immune system utilizes to destroy extracellular pathogens, or cells that either harbour intracellular for- eigners (e.g. mycobacteria or viruses) or become malignant, must be appropriately targeted if indiscriminate damage to normal host tissue is to be avoided. Under most inflammatory circumstances, some bystander tissue damage is unavoidable. In most situations, this damage is self-​limited, due to efficient clearance of the exogenous antigen source and appropriate downmodulation of the immune response. Tissue damage in autoimmune diseases differs funda- mentally from bystander damage, in that the host immune system is specifically activated and driven by self-​components, focusing damaging immune effector pathways on host tissues expressing those components, in an autoamplifying and self-​sustaining way. 4.6 Autoimmunity Antony Rosen

380 SECTION 4  Immunological mechanisms The danger inherent in initiating a self-​sustaining, specific immune response directed against components of self-​tissues is intuitively apparent, since antigen clearance under these circumstances is ne- cessarily associated with complete tissue destruction. It is now clear that an autoimmune component is a feature of many human diseases. Indeed, there are some estimates that auto- immune diseases afflict more than 3% of Western populations, and imposes a significant personal and economic burden on individ- uals and nations. This chapter will illustrate many of the principles unifying various autoimmune states, and will present a conceptual framework within which to understand their aetiology, pathogen- esis, and pathology. The rapid advances in knowledge being made in this group of disorders predict that disease mechanisms will soon be more clearly understood, and will greatly impact therapeutics. Epidemiology Autoimmune diseases may affect individuals at all stages of life. In general, diseases have a predilection for beginning after the second decade, with peak incidence in the third to sixth decades. In many instances, there is a preference for the female gender, with the mag- nitude of this sex difference varying among the different diseases. Thus, for the systemic autoimmune diseases (e.g. systemic lupus erythematosus (SLE), rheumatoid arthritis, Sjögren’s syndrome, scleroderma, and autoimmune myositis), and autoimmune thyroid disease, the female:male (F:M) ratio is approximately 4–​9:1, while for type 1 diabetes, multiple sclerosis, and myasthenia gravis, the female predominance is much less prominent (F:M ratio <2:1). The exact mechanisms underlying this female predominance remain unknown, but this striking biological difference provides a major clue to pathways underlying susceptibility to autoimmunity. Recent studies describing possible gender-​related differences in Toll-​like receptor (TLR) expression may be relevant in this regard (see fol- lowing section). Aetiology An important theme related to the development of various auto- immune diseases has emerged in recent years. One of the most unexpected observations came from studies of patient popula- tions in whom blood samples had been stored for a period of years prior to the onset of clinical disease (military cohorts or stored blood bank samples), allowing investigators to address whether autoantibodies are first generated coincident with clinical disease, or precede this. In a landmark study by Harley and colleagues in SLE, clear evidence was obtained showing that the relatively ‘non-​ specific’ antinuclear autoantibodies and antiphospholipid anti- bodies generally precede the diagnosis of SLE, often by a period of several years. These investigators also showed that phenotype-​ specific autoantibodies in SLE (e.g. anti-​Sm, anti-​RNP) occurred around the time of onset of clinical disease, suggesting that dif- ferent autoantibody specificities were marking different phases in the disease. Similar observations have been made in rheumatoid arthritis, where anticyclic citrullinated peptide antibodies pre- date clinical symptoms, whereas more specific antibodies (e.g. antivimentin) only occur when disease becomes established. It is therefore operationally useful to divide autoimmune diseases into separate kinetic phases: 1. Susceptibility—​predisease, in which inherited or acquired de- fects in pathways required to maintain tolerance to self-​antigens render the individual susceptible to disease initiation; 2. Initiation of autoimmunity—​the interface of susceptibility genes and unique events associated with changes in the struc- ture of autoantigens (e.g. mutation or novel post-​translational modification), which initiate an immune response directed at self-​antigens—​this phase is generally not accompanied by clin- ical symptoms; 3. Transition—​during which the targets of immune response change and are amplified, and during which clinical symp- toms assume a recognizable phenotype. This generally occurs subacutely over weeks to months, and frequently begins with non​specific symptoms and signs. Examples include the fatigue and constitutional symptoms that predate diagnosis of SLE and rheumatoid arthritis. This transition phase has important impli- cations for early diagnosis and intervention. 4. Propagation—​a self-​amplifying phase in which the specific im- mune response to self-​antigens causes damage of tissues, with the release of more antigens, which further drive the immune response. It is important to note that this last, amplified phase does not manifest initially fully developed, but rather evolves over time towards the diagnostic phenotype. Both genetic and environmental factors play important roles in ini- tiation and propagation of autoimmune diseases. They probably play their central roles by regulating the activation, function, and targets of the host immune system. There is also evidence that sto- chastic processes play an important role in disease initiation, greatly complicating studies to define the causes and mechanisms of auto- immune disease (see next). Genetic factors Although autoimmune diseases in humans are genetically com- plex, significant advances in understanding have occurred over the past several years. In some cases, advances have come from the study of autoimmunity with mendelian patterns of inheritance (e.g. APECED, IPEX, C1q deficiency—​see definitions to follow). Advances have also come from genetic association studies of various autoimmune phenotypes (e.g. rheumatoid arthritis, SLE, type 1 dia- betes). Together, the studies stress that multiple genes interact in ren- dering an individual susceptible to autoimmunity, and highlight a critical role for pathways of tolerance induction, immunoregulation, and setpoints/​thresholds for immune signalling in avoiding emer- gence of autoimmunity. A reciprocal role of target tissue pathways (e.g. antigen structure/​expression) in regulating autoimmunity has also been recognized. The following are some general prin- ciples regarding the genetics of autoimmunity that have emerged in recent years. Certain major histocompatibility complex (MHC) class II alleles are associated with disease susceptibility One of the most striking genetic associations with autoimmunity resides in the MHC, an area on chromosome 6 in humans which is highly enriched in genes that participate directly and indirectly in the immune response. The strength of association of different

4.6  Autoimmunity 381 autoimmune phenotypes with MHC class II genes in this area is very robust (odds ratios in the 3–​8 range). For example, patients with rheumatoid arthritis have an increased frequency of HLA DR4. HLA DR4 (initially defined serologically) encompasses numerous different alleles that have been defined by sequencing. Interestingly, not all subtypes of HLA DR4 are associated with an increased fre- quency of rheumatoid arthritis, but those alleles that are associ- ated with rheumatoid arthritis share a short amino acid sequence (QKRAA) at positions 70 to 74 of the β chain of the HLA DR mol- ecule. This sequence, termed the ‘shared epitope’, is located along the peptide-​binding groove of HLA DR4 which presents peptides to the antigen receptor of T cells. Interestingly, this same ‘shared epitope’ is present in many HLA DR1-​positive individuals with rheumatoid arthritis. A similar principle appears to hold for patients with type 1 diabetes, where there is a strong association of disease with specific DQβ genotype. Since MHC class II molecules function as a scaffold for presenta- tion of specific peptides to CD4T cells (see next), it is possible that this MHC-​encoded susceptibility to disease reflects the ability of these alleles to present unique self-​peptides to autoreactive T cells. The presence of significant linkage disequilibrium within the MHC region (i.e. large stretches of DNA do not undergo recombination, generating functional cassettes of associated genes) also creates the potential for the disease-​association of particular MHC alleles to be influenced by additional genes on the extended haplotype in affected individuals. Studies to define these other genes, and the mechanisms whereby they influence development of autoimmunity, are challen- ging, and are ongoing in many diseases. Incomplete thymic tolerance induction predisposes to autoimmunity Significant insights into basic mechanisms can derive from the study of rare human phenotypes. This has been true for auto- immunity, where several monogenic disorders have defined im- portant pathogenic principles. Autoimmune polyendocrine syndrome type I (APS1; OMIM 240300), also called autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), is a rare disease in which patients develop multiple autoimmune diseases, often beginning in childhood. The syndrome is character- ized by striking autoimmunity directed against multiple different target tissues, including parathyroids, adrenals, pancreatic β cells, parietal cells, thyroid, liver, and gonads. Numerous autoantigens have been defined as targets of autoimmunity in APS1, and in- clude enzymes specifically expressed in various endocrine tissues (e.g. steroid 21-​hydroxylase—​specific for adrenal cortex; steroid 17a-​hydroxylase—​found in adrenal cortex and gonads, GAD65—​ found in pancreatic islets, and thyroid peroxidase). The genetic basis of APS1 was mapped to a gene on chromosome 21q22.3, sub- sequently termed AIRE (for autoimmune regulator). AIRE expres- sion is highest in the thymus, where it is expressed in medullary thymic epithelial cells. Significant evidence has now been obtained that AIRE is a transcriptional regulator, which regulates expression in thymic epithelial cells of various peripheral autoantigens nor- mally expressed exclusively in endocrine target tissues. Thus, AIRE appears to regulate the ectopic expression in the thymus of tissue-​ restricted autoantigens, and provide an antigen source against which to establish central tolerance. Several AIRE-​deficient mouse models were subsequently generated; these animals developed various autoimmune endocrine phenotypes, resembling those found in human APS1. Impaired clearance and tolerance induction by apoptotic cells: Susceptibility defect in systemic autoimmunity Although little is known in humans about the thymic pathways of tolerance induction to ubiquitously expressed autoantigens, there is accumulating evidence to suggest that in the periphery, apoptotic cells play an important role in providing a source of autoantigens against which the organism becomes tolerant. Apoptotic cells are generally very efficiently cleared by phagocytic cells; these events are normally associated with the production of anti-​inflammatory cytokines and result in tolerance induction. Interestingly, early components of the classical complement pathway (e.g. C1q and C4) and C-​reactive protein are required for efficient apoptotic cell clearance, with production of interleukin (IL)-​10 and transforming growth factor β (TGFβ). It is of particular note, therefore, that homozygous C1q deficiency is associated with a striking suscep- tibility to SLE, suggesting that rapid, efficient, tolerance-​inducing clearance of apoptotic cells may play a similar role to AIRE ex- pression in the thymus in preventing subsequent emergence of autoimmunity to ubiquitously expressed autoantigens. Additional support for this model comes from studies of milk fat globule-​EGF factor 8 (MFG-​E8), a glycoprotein secreted from macrophages that is required for the efficient attachment and clearance of apoptotic cells by macrophages and immature dendritic cells. MFG-​E8 is also expressed in tingible-​body macrophages at the germinal centres of secondary lymphoid tissues. Interestingly, many unengulfed apop- totic cells are present in the germinal centres of the spleen in MFG-​ E8-​deficient mice, which develop a striking lupus-​like phenotype. Other examples exist in which defects in clearance of apoptotic cells are associated with development of systemic autoimmunity (e.g. Mer deficiency). Together, the data strongly suggest that efficient, anti-​inflammatory clearance of apoptotic cells plays a central role in tolerance induction and prevention of autoimmunity. Defective production of regulatory T cells Although pathways exist that (1) regulate autoantigen expression at sites of tolerance induction, and (2) guide autoantigens towards tolerance-​inducing outcomes, these pathways alone are clearly in- sufficient to prevent the emergence of autoimmune disease. This fact is highlighted by the emergence of autoimmunity when regu- latory T-​cell differentiation is abnormal in humans with IPEX syn- drome (immune dysregulation, polyendocrinopathy, enteropathy, X-​linked syndrome; OMIM 304790). IPEX is a rare X-​linked reces- sive disorder, which is characterized by type 1 diabetes, thyroiditis, atopic dermatitis, and inflammatory bowel disease, and is caused by mutations in the FOXP3 gene. FOXP3 is a member of the forkhead family of transcription factors, and is essential for the development of regulatory T cells (Tregs), which regulate the activation and dif- ferentiation of effector T cells at many different levels. It is therefore likely that induction of tolerance is incomplete under most circum- stances, and that self-​sustaining autoimmunity is normally limited by Treg function. Signalling thresholds and susceptibility to autoimmunity Several modulators of T-​cell signalling have also been defined as im- portant susceptibility determinants in autoimmunity. For example,

382 SECTION 4  Immunological mechanisms CTLA4 polymorphisms are associated with increased risk of a var- iety of autoimmune diseases, including type 1 diabetes, Graves’ disease, SLE, and rheumatoid arthritis. Similarly, a functional poly- morphism in PTPN22 has been identified as a major risk factor for several human autoimmune diseases, including SLE, rheuma- toid arthritis, and type 1 diabetes. Although the exact mechanisms underlying susceptibility to autoimmunity remain unclear, in both cases the polymorphisms appear to regulate the balance of stimu- latory and inhibitory signalling in effector and regulatory T cells, favouring effector T-​cell activation. The genetic studies in autoimmunity therefore highlight that there are many barriers to the development of autoimmunity, including effective tolerance induction in the thymus and periphery, tightly regulated immune signalling, and homeostatic pathways of immunoregulation to limit self-​responses should these occur. There are also cassettes of immune response genes encoded in the MHC which appear to be more likely to capture specific self-​antigens and generate a response to them. It is likely that the genetic suscepti- bility to autoimmunity in outbred humans represents an integrated threshold involving genes that regulate these various pathways, upon which environmental and stochastic events act to accomplish disease initiation and propagation. Environmental factors Twin studies in human autoimmune diseases showing that indi- viduals with an identical genotype may be variably affected by dis- ease (concordance rates vary widely, from 15 to 50%, in identical twins with SLE or rheumatoid arthritis), demonstrating that en- vironmental insults and stochastic events likely play a significant role in the development of autoimmunity. Recent studies have also strongly implicated circumstances that generate neoantigens (e.g. somatic mutations in cancer) as important initiators of the specific immune response in some autoimmune diseases; this is discussed further next. In terms of potential environmental insults that may play a role in autoimmune disease, there is evidence of a role for infections, ir- radiation, and exposure to drugs and toxins. For example, exacerba- tions of SLE can follow sunlight exposure, and there are numerous reports that disease initiation may have a similar association with ultraviolet irradiation in rare patients. Numerous infections have been postulated to play a role in disease initiation across the spec- trum of human autoimmune diseases. In rare cases, the association between antecedent infection and subsequent development of dis- ease is evident (e.g. coxsackievirus infection-​induced autoimmune myocarditis, acute rheumatic fever following streptococcal infec- tion, Epstein–​Barr virus infection, and childhood SLE). In most autoimmune diseases, however, it has not been possible to confirm such environmental connections with any certainty. This does not prove that a causal connection does not exist in these instances, but rather reflects several features of the diseases that greatly complicate the firm establishment of such a connection: • kinetic complexity of the autoimmune diseases—​since develop- ment of autoimmunity occurs in several distinct phases, and once the propagation phase begins, establishment of a recognizable disease phenotype often takes months, evidence of the initiating insult may have disappeared by the time the environmental com- ponent is sought for the first time; • several different environmental insults may induce a similar response; • the environmental force may be extremely frequent in the popula- tion, but may only induce autoimmune disease in a unique subset of individuals with appropriate susceptibility genes. How various forces influence initiation of autoimmune diseases is not yet known for most autoimmune diseases, but several plausible mechanisms have been advanced. These include • the disruption of cell and tissue barriers, allowing previously se- questered antigens access to a previously ignorant immune system (see next); • inducing novel pathways of antigen presentation; • alteration of the structure of self-​antigens; and • molecular mimicry. Some of these mechanisms are dealt with in more detail next. Pathogenesis Although extraordinarily complex in detail, the adaptive immune response operates by a set of relatively simple principles: (1) the immune system has the capacity to discern molecular structure in extremely fine detail; (2) it has a uniquely adapted set of signalling systems that computes the amount of antigen; (3) it responds in a binary way to contextual information, that is, seeing an antigen in the setting of a dangerous context (e.g. infection or cancer) initiates an immune response, whereas seeing the antigen in the absence of such costimulatory signals leads to tolerance. Numerous studies over the past two decades have underscored that the sustained autoimmune response is extremely similar to adaptive immune responses directed against foreign pathogens, except that the driving antigens in autoimmune disease are self-​molecules. For example, autoantibodies in most autoimmune diseases display evi- dence of isotype switching (e.g. from IgM to IgG or IgA), and show features of having undergone affinity maturation through somatic hypermutation. These properties of autoantibodies require the ac- tivity of antigen-​specific CD4+ T cells, and have therefore focused much attention on defining the mechanisms whereby self-​reactive T cells are activated in autoimmunity. Since this is such a central issue in the understanding of autoimmunity, and since there are nu- merous mechanisms employed by the normal individual to prevent activation of autoreactive T cells, it is important to briefly review the mechanisms that the normal immune system uses to maintain toler- ance against self-​proteins. Central and peripheral tolerance To prevent the survival of lymphocytes that will likely encounter their cognate antigens in healthy self-​tissues, with potential auto- immune destruction of tissues, the immune system spends sig- nificant energy on testing the specificity of all receptors generated during antigen-​independent development of lymphocytes initially in the thymus, and subsequently in the periphery. When the T-​cell receptor generated through somatic recombin- ation recognizes a peptide–​MHC complex in the thymus with high affinity/​avidity, cells expressing this receptor are negatively selected (since they are likely self-​reactive, and will recognize their cognate

4.6  Autoimmunity 383 antigens at additional peripheral sites). These self-​reactive cells undergo apoptosis in the thymus, and never make it into the per- iphery. In contrast, those T-​cell receptors that have some affinity for the selecting MHC molecule, but not for the peptide contained in the groove, likely will recognize foreign peptides, and are positively selected. This process of establishing tolerance to self-​proteins in the thymus is called ‘central tolerance’. T cells exiting the thymus there- fore include cells that can recognize peptides within the scaffold of the MHC molecule used to select that T cell, but have not encoun- tered their specific peptide in the thymus. Since not all self-​antigens are expressed in the thymus, there is still a chance that exiting T cells will encounter a self-​peptide–​MHC complex in the periphery for which they have high affinity. Since cells that have left the thymus no longer have the developmental context that likely denotes a self-​peptide (i.e. recognition with high affinity of a peptide–​MHC complex during development in the thymus), peripheral T cells utilize another binary system to define whether a high-​affinity interaction should lead to activation or in- activation. This binary system uses additional cell-​surface molecules (called costimulatory molecules) to denote context. Thus, when per- ipheral T cells recognize an MHC–​peptide complex with high af- finity in the absence of costimulation (through ligation of CD28 by surface CD80 or CD86 on the antigen-​presenting cell), T cells are inactivated or tolerized. This is known as ‘peripheral tolerance’. In contrast, when peripheral T cells recognize an MHC–​peptide com- plex with high affinity in the presence of costimulation, these T cells are activated. In addition to T-​cell tolerance, B-​cell tolerance to self-​components is also actively maintained. Thus, if B cells encounter either soluble or membrane-​bound antigen during development in the bone marrow, these cells are either deleted (tolerance) or inactivated such that they become refractory to specific stimulation by their antigen (anergy). Mechanisms allowing an immune response to be directed against self-​antigens Although tolerance to self-​molecules is stringently maintained at the T-​ and B-​cell levels, reactivity against self-​molecules may still be possible for several reasons. These include the following. Abnormal immunoregulation There are numerous mechanisms used to establish and maintain T-​ and B-​cell tolerance. There is accumulating evidence that defects in regulation of these pathways may result in the failure to elim- inate autoreactive lymphocytes, or an altered activation threshold for lymphocytes. Examples include defects in the Fas/​Fas-​ligand system, a receptor–​ligand pair which is required for removal of ac- tivated, self-​reactive lymphocytes. Mice or humans with defects in this pathway manifest profound lymphadenopathy and a spectrum of autoimmunity. Similarly, defects in regulatory molecules which normally function to dampen the immune response (e.g. CTLA-​4, the inhibitory T-​cell receptor for the costimulatory molecules B7-​ 1 and B7-​2) may result in profound autoimmune responses. Mice lacking CTLA-​4 develop fatal autoimmunity, with widespread T-​cell infiltrates, and CTLA-​4 polymorphisms are associated with auto- immunity in humans. It should be remembered that the immune system is a highly com- plex system, with interdependent regulation present at numerous levels. It is likely that many of the other susceptibility genes in human autoimmunity impinge on these immunoregulatory pathways. Existence of sites of immune privilege Strict sequestration of tissue-​specific antigens behind anatomical and immunological barriers prevents the development of tolerance to molecules expressed preferentially at these sites. Events (e.g. penetrating trauma) which breach this tight boundary may allow initiation of an immune response to these previously hidden self-​ molecules. Relevant examples include antigens within the eye, testis, and central nervous system. In the eye, for instance, penetrating in- jury to one eye may be followed by development of inflammation in the contralateral eye (sympathetic ophthalmitis) approximately 1 to 2 weeks after injury. Several mechanisms have been proposed to be responsible for maintaining the immune-​privileged status of these tissues. One powerful mechanism appears to involve the con- stitutive expression of Fas ligand in the relevant tissue (e.g. eye). When this molecule binds to and activates its receptor on lympho- cytes, these cells undergo apoptotic death, and are prevented from entering the tissue. Immunodominance and cryptic determinants Not all regions of a molecule are equally immunogenic. Regions of the molecule that are well-​captured by class II MHC molecules during natural processing of self-​antigens are able to tolerize T cells (these determinants have been termed ‘immunodominant’ by Sercarz and colleagues). In contrast, regions of self-​molecules that are not gen- erated in significant amount during natural antigen processing (so-​ called ‘cryptic determinants’) cannot effectively tolerize T cells, since they are never seen by these cells either in the thymus or peripherally. This immunodominance appears to be influenced by the intrinsic affinity of the peptide for MHC class II, as well as by neighbouring structural determinants on the antigen that may influence its binding to the peptide-​binding groove. On self-​molecules, two sets of deter- minants can therefore be functionally defined (Fig. 4.6.1): • those that are easily processed and presented (comprising the dominant self), which readily tolerize developing T cells • those that are not presented in appreciable amounts after natural processing (comprising the cryptic self), which do not tolerize There are unusual circumstances in which processing of self-​ antigens may load different peptides into the groove of MHC class  II than those generated during normal antigen processing. Examples include (i) somatic mutation of the autoantigen (e.g. in cancer), which might result in loading a unique peptide from that autoantigen which has not previously been seen and tolerized; (ii) mutation might also cause modified proteolysis of the autoantigen (creating or destroying a proteolytic cleavage site, which destroys the dominant epitope or generates a new dominant epitope); (iii) unusual proteolysis of an autoantigen prior to entry into the nat- ural processing pathway, allowing emergence of a previously cryptic epitope; (iv) high-​affinity binding of the antigen to specific receptors or antibodies, which can hinder access of the dominant epitope to the antigen-​binding groove of MHC class II molecules, or optimize the loading of a previously cryptic epitope. Since T cells recognizing these cryptic peptides have not previously been tolerized, such ‘autoreactive’ T cells can now be activated (Fig. 4.6.2).

384 SECTION 4  Immunological mechanisms (c) High affinity binding to another protein that alters processing • Antibody binds with high affinity to dominant epitope, and stops it from getting into the class II groove • The protein is cleaved with a different pattern to that normally observed during processing, and the
cryptic epitope is generated in amounts sufficient to load MHC class II • T cells have never seen this epitope before, and have therefore not been tolerized
• Autoreactive T-cell response initiated
MHC II Antibody/high affinity receptor The dominant epitope is preferentially loaded onto class II MHC during normal antigen processing Other regions are trimmed away, and are not presented in significant amount of MHC II processing Dominant Cryptic Ag processing No autoreactive T cells recognizing the dominant self exist in the host, because they have been tolerized MHC II (a) Normal processing of intact Ag self antigen (b) Generation of novel fragments Autoreactive T-cell response initiated • Dominant epitope destroyed
• Cryptic epitope loaded onto class II MHC • T cells specific for the cryptic epitope have never seen this self-antigen before, and are activated MHC II Ag processing Fig. 4.6.1  Dominant and cryptic T-​cell epitopes in autoimmune disease. (a) The default processing pathway for intact antigen results in the preferential and reproducible loading of the ‘dominant’ peptide determinant into the antigen-​binding groove of MHC class II. During establishment of thymic and peripheral tolerance, T cells recognizing this dominant epitope are purged from the repertoire, but T cells recognizing cryptic epitopes do not encounter their antigens, and are not deleted or anergized. (b, c) When the processing of self-​antigens is altered (e.g. by novel proteolysis or through high-​affinity binding to another molecule), a different hierarchy of epitopes is loaded on to class II MHC. If cryptic epitopes are loaded in sufficient amounts, these peptides can stimulate autoreactive T-​cell responses directed against the cryptic self, and drive the autoimmune process.

4.6  Autoimmunity 385 There are several clear demonstrations that autoreactive T cells recognizing cryptic epitopes can be activated in vivo through al- tered processing of self-​molecules to reveal these previously immunocryptic epitopes. For instance, high-​affinity binding of the HIV surface protein gp120 to CD4 alters the processing of CD4, and activates T cells which recognize epitopes of CD4 not generated during normal antigen processing. This mechanism may account for the autoimmune response to CD4 seen during HIV infection. Similarly, although intact mouse cytochrome c is not immunogenic in mice, cleavage of the molecule into smaller peptides induces a robust T-​cell response to cryptic areas of cytochrome c, which were never previously presented by the natural processing pathway, and therefore did not induce tolerance. The revelation of cryptic epitopes in self-​antigens is likely to be a highly relevant mechanism in many human autoimmune dis- eases, but the studies to demonstrate the importance of this mech- anism have only recently begun in earnest. Since the structure of autoantigens influences the hierarchy of dominant and cryptic and determinants generated when the molecule is processed, unique pro- cesses which alter the structure of molecules may play critical roles in initiation of autoimmune diseases. These unique events likely do not occur during normal homeostasis, but may occur preferentially during infectious or other proimmune events occurring at the host-​ environment interface. Relevant examples include: • Somatic mutation may create novel epitopes that bind with high affinity to the patient’s MHC class II alleles and effectively stimulate a CD4 T-​cell response. Autoimmunity with damage to self-​tissue can be driven when this immune response against the ‘mutanome’ also cross-​reacts with the unmutated wild type autoantigens present in the patient’s tissues. Recent studies have demonstrated that this mechanism is relevant in patients with scleroderma, where mutations in a major scleroderma autoantigen (RNA polymerase 3) in patients’ cancers appear to initiate the immune response against that antigen. • Activation of unique proteolytic pathways that specifically alter the structure of autoantigens during immune effector pathways. It has recently been observed that most autoantigens targeted across the spectrum of human autoimmune diseases are specific- ally cleaved by granzyme B during killing of infected target cells by cytotoxic lymphocytes. This cleavage generates unique molecular fragments never generated in the organism during development or homeostasis. Interestingly, this cleavage is a unique feature of autoantigens, and does not affect non​autoantigens. Although it (a) Ag processing B T ‘Help’ Strong TH epitope T-cell epitope = Antibody = MHC II = TCR Processing TH X-reactive Self • Self-antigen release in setting of crossreactive activated B cell
• Revelation of cryptic self TH epitope Foreign Cryptic Foreign B Self B X-reactive Ab T Vs foreign ‘Help’ B T Autoreactive against cryptic epitope in self- antigen ‘Help’ Crossreactive Ab driven by self antigen B cell (b) Fig. 4.6.2  Molecular mimicry. (a) Foreign antigens, which clearly differ from their homologous self-​antigens in some areas, may nevertheless bear significant structural similarity to self-​antigens in other regions. Initiation of an immune response to the foreign antigen may generate a cross-​reactive antibody response that also recognizes the self-​protein. When the self-​antigen is a cell-​surface molecule, antibody-​mediated effector pathways can lead to host tissue damage. Although the antibody response is cross-​reactive with self-​molecules, the T cells that drive this response are directed exclusively at the foreign antigen. (b) Under highly novel conditions, the simultaneous liberation of significant amounts of self-​antigen in the setting of a cross-​reactive antibody response may allow effective presentation of cryptic epitopes in the self-​antigen to autoreactive T cells by activated cross-​reactive B cells. These autoreactive T cells can now continue to drive an autoantibody response to the self-​antigen. If continued release of self-​antigen occurs as part of this process, a specific, adaptive immune response to self will be sustained.

386 SECTION 4  Immunological mechanisms has been proposed that these cleavage events allow the efficient presentation of previously cryptic epitopes, this remains to be for- mally demonstrated. • Additional post-​translational modifications that alter conform- ation of antigens, and modify their subsequent processing. It is noteworthy that numerous post-​translational modifications of autoantigens occur, and that in some cases initiation of the auto- immune response is strictly dependent on the occurrence of these modifications. One of the most prominent examples is the post-​ translational deimination of arginine to citrulline in rheumatoid arthritis. Other examples include phosphorylation, acetylation, and isoaspartyl formation, among others. • Formation of high-​affinity complexes between autoantigens and other viral or self-​proteins. In all these examples, it should be remembered that the initiating event in autoimmunity requires that, on the background of appro- priate susceptibility genes, several stringent criteria needed to ini- tiate a primary immune response must be simultaneously satisfied. These include the generation of suprathreshold concentrations of self-​molecules that have a structure not previously tolerized by the immune system, and the presentation of these unique molecular forms to T lymphocytes in the presence of costimulation (i.e. in a proimmune context). Molecular mimicry Foreign antigens, which clearly differ from their homologous self-​ antigens in some areas, may nevertheless bear significant structural similarity to self-​antigens in other regions. Initiation of an immune response to the foreign antigen may generate a cross-​reactive anti- body response that also recognizes the self-​protein (molecular mim- icry). When the antigen is a cell-​surface molecule, antibody-​mediated effector pathways can lead to host tissue damage. Although the anti- body response is cross-​reactive with self-​molecules, the T cells that drive this response are directed at the foreign antigen (see following paragraphs). Diseases involving this sort of antigen mimicry there- fore tend to be self-​limited. It is important to realize that molecular mimicry alone cannot explain self-​sustaining autoimmune diseases, which are driven by self-​antigens and autoreactive T cells. In these cases, there is a requirement for overcoming T-​cell tolerance to the self-​protein. The simultaneous liberation of self-​antigen in the pres- ence of the cross-​reactive antibody response likely play critical roles in this regard (see next). Mechanistic insights into molecular mimicry Although several microbial and viral antigens have regions of high homology with various human autoantigens, a causal link between exposure to these foreign antigens and the onset or exacerbation of autoimmune disease has been extremely difficult to establish. There are, however, clear examples that suggest the existence of ‘one-​shot’ autoimmune processes, in which cross-​reactive antibodies directed against surface self-​antigens are generated following infection, and result in tissue damage. This persists until infection is cleared, and the immune response wanes. Although the mechanistic details of this scheme are difficult to prove in vivo, several pertinent examples exist. One of these is a seasonal epidemic form of Guillain–​Barré syndrome seen in northern China, which follows Campylobacter jejuni infection. Affected patients make antibodies recognizing gangliosides, and the disease has a self-​limited course, which rarely recurs. The antiganglioside antibodies generated are likely respon- sible for the pathological findings of acute motor axonal neur- opathy. Another plausible example of this mechanism (although with meagre in vivo evidence) is immune thrombocytopenia (ITP) in children. This process characteristically (1) follows an infectious process; (2) demonstrates antiplatelet antibodies, and (3) frequently shows durable remissions. The mechanistic details of this process have been difficult to prove in vivo, and cross-​reactive epitopes on potentially initiating pathogens have not yet been defined. The single episodes of tissue damage in the setting of a cross-​ reactive immune response following infection must be contrasted to the sustained, autoamplifying disease frequently seen in other auto- immune syndromes. The central issues in this regard are (1) how T-​ cell tolerance to self-​antigens might initially be broken, and (2) once this has occurred, why these antigens continue to drive the immune response to self. Examination of tolerance to cytochrome c, a ubi- quitous protein that has regions of homology and divergence across different species, has been very useful in understanding molecular mimicry of cross-​reactive epitopes. Mouse cytochrome c shares sig- nificant homology with human cytochrome c, although the proteins are entirely different in other areas. When Mamula and colleagues used mouse cytochrome c to immunize mice, no T-​cell or anti- body response to the murine protein was observed. When human cytochrome c was similarly used to immunize mice, strong T-​cell epitopes on the foreign cytochrome c were able to induce a strong antibody response to the foreign protein. The antibodies induced recognized both the murine and the human forms of cytochrome c (i.e. cross-​reactive antibodies that recognize the self-​protein were produced). The T-​cell response to cytochrome c was, however, dir- ected entirely against the foreign (human) form of the protein, and no T cells against the murine protein could be found. These cross-​ reactive antibodies disappear as the immune response to the foreign protein wanes. Interestingly, when mouse cytochrome c was included with human cytochrome c during the immunization, a T-​cell response to human cytochrome c, and a humoral response to the human pro- tein that cross-​reacts with the murine protein, was induced. Within a few days, a strong helper T-​cell response specific for murine cyto- chrome c was detected. This breaking of T-​cell tolerance to murine cytochrome c was dependent on activated B cells specific for cyto- chrome c, which likely exert their effect through altering the pro- cessing of mouse cytochrome c, potentially uncovering previously cryptic epitopes in the self-​protein (see Fig. 4.6.2). In the presence of continued release of self-​antigen, this response may become self-​ sustaining—​self-​antigen driving autoreactive T cells, providing help to autoantibody-​producing B cells (Fig. 4.6.2). Molecular mimicry may therefore induce the production of cross-​reactive antibodies, which, in the absence of liberation of sig- nificant amounts of self-​antigen, should disappear when the for- eign pathogen is cleared. The form of epidemic motor axonopathy described here is likely representative of this scenario. Under highly novel conditions, the simultaneous liberation of significant amounts of self-​antigen in the setting of a cross-​reactive antibody response may allow effective presentation of cryptic epitopes in the self-​antigen to autoreactive T cells by activated cross-​reactive B cells. If continued release of self-​antigen occurs, a specific, adaptive immune response to self will be sustained. Antigen release from

4.6  Autoimmunity 387 tissues likely plays a critical role in driving this autoimmune pro- cess. Understanding the mechanisms of ongoing antigen release at sites of tissue damage in autoimmune disease (e.g. unique pathways of cell injury and death) is a high priority for future work, as it pro- vides a novel target for therapy (see next). It is clear from the previous discussion that extraordinary com- plexity is operative in initiation of the human autoimmune diseases. The patient population is genetically heterogeneous, the human im- mune system is complex and extremely plastic, and it interacts with a plethora of environmental stimuli and stochastic events. The sim- ultaneous confluence of susceptibility factors and initiation forces to set off the self-​sustained and autoamplifying process is there- fore an extremely rare occurrence. In contrast, once activation of autoreactive T cells has occurred, the ability of the immune system to vigorously respond to vanishingly low concentrations of antigen, to amplify the specific effector response to those antigens, and to spread the response to additional antigens in that tissue, greatly re- duces the stringency that must be met to keep the process going (Fig. 4.6.3). Effector mechanisms in autoimmune diseases The initiation phase of autoimmunity requires cooperation be- tween many different cell types, including antigen-​presenting cells, T cells and B cells, as well as numerous soluble mediators including antibodies, chemokines, and cytokines. The effector phase of autoimmunity uses the same immune and inflammatory effector mechanisms that the immune system has evolved for removing and destroying pathogens. These include activation of the com- plement cascade, which generates signals that effect inflammatory cell recruitment and activation. Similarly, ligation of activating Fc receptors on inflammatory cells by immune complexes activates macrophage and neutrophil effector function. Autoantibodies dir- ected against cell-​surface antigens initiate antigen-​dependent cel- lular cytotoxicity, likely mediated by macrophages and natural killer cells. Cytokines and chemokines play a central role in inflammatory cell recruitment and activation in the target tissue. Tissue damage can also be effected by cytolytic lymphocytes. The pathology char- acteristic of each autoimmune disease reflects both the particular antigens targeted, as well as the predominant effector mechanisms activated. One principle of central importance in the effector phase of auto- immunity is autoamplification, which appears to play a central role in the self-​sustaining nature of the autoimmune process. Thus, im- mune effector pathways cause damage of cells in the target tissue, liberating antigen which further stimulates the immune response and effector pathways, thus liberating more antigen. Although this is likely an oversimplification, the view that the immune system plays a role in generating an ongoing supply of autoantigen is useful Environmental insult (e.g. viral infection) Antiviral immune response inflammation Clearance of infection repair of damage Normal Infected tissue Normal host Antiviral immune response Clearance of infection Generation of novel autoantigen structure Antiself immune response Infected tissue Autoimmune host Tissue damage Environment Susceptibility genes Initiation Propagation Fig. 4.6.3  Model of initiation and propagation of autoimmune disease. Autoimmune diseases are highly complex disorders, which require the simultaneous cooperation of multiple factors for their development. Numerous susceptibility genes (some of which regulate the immune response) appear to determine the threshold for disease initiation. In many diseases, a discrete, proimmune trigger (environmental or somatic mutation as occurs in cancer) likely plays a role in disease initiation, but is infrequently recognized. A critical requirement for disease initiation is the generation of suprathreshold concentrations of self-​antigen with novel structure. Development of a recognizable disease phenotype generally requires marked antigen-​driven amplification of the autoimmune response, in which immune effector pathways play a role in generating the ongoing supply of antigen to sustain the process.

388 SECTION 4  Immunological mechanisms therapeutically, since it focuses attention on controlling both the supply of antigen as well as immune effector pathways (see next). Principles of amplification One of the central features of human autoimmunity is the tendency of the process to amplify progressively with the accumulation of significant immune-​mediated tissue damage. Furthermore, in the vast majority of cases, once such amplification begins, the process is very unlikely to resolve spontaneously. Properties of autoantigens themselves may be very important in this phase, in terms both of acquisition of adjuvant properties, and of regulation of expression. The essential features of amplification are a substrate cycle, in which antigen expression and adjuvant properties induce an immune re- sponse, which induces increased antigen expression and tissue damage—​and further drive the immune response. The importance of tissue-​specific autoantigen expression in focusing such immune responses is only beginning to be recognized. Acquisition of adjuvant properties by disease-​specific autoantigens In recent years there have been dramatic advances in the under- standing of the mechanisms whereby specific molecules are selected as antigens in the various autoimmune syndromes. In spite of the fact that tens of thousands of molecules could be targeted by the immune system in autoimmunity, the number of molecules that are frequently targeted in the different phenotypes are markedly restricted—​limited perhaps to a few hundred molecules at most. This has led to the proposal that frequently targeted autoantigens may themselves have properties that make them proimmune. This was first suggested by Plotz and colleagues. who observed that the autoantigenic histidyl aminoacyl-​tRNA synthetase which is targeted in autoimmune myositis (but not non-​auto-​antigenic lysyl-​ and aspartyl-​aminoacyl-​tRNA synthetases) is chemoattractant to im- mature dendritic cells and other leucocytes. The authors suggested that the selection of a self-​molecule as a target for an autoantibody response may be a consequence of proinflammatory properties of the molecule itself. They further suggested that modification of autoantigen structure during processes of cell damage or death may be critical in recruiting these additional functions of autoantigens. Toll-​like receptors (TLRs) and other nucleic acid sensors One of the most likely receptor systems to sense and transduce the proinflammatory properties of autoantigens is the TLR family, which is the primary innate immune system transducer of pathogen-​ associated molecular patterns. Ligands for TLRs include both micro- bial and endogenous molecules, the latter group being particularly relevant to autoimmunity (see next). Microbial ligands include com- ponents of Gram-​positive bacteria, Gram-​negative bacteria, yeast, and protozoans. Although viral and bacterial nucleic acids are the most likely ligands for TLRs, accumulating data demonstrates that complexes containing endogenous nucleic acids are also able to signal through TLRs. Although the exact nature and source of endogenous ligands for TLRs in vivo remains unclear, recent studies have demon- strated that components from stressed, injured, and dying cells may play critical roles. Working in several models, numerous investigators have now provided evidence that the targeting of frequently targeted nucleoprotein autoantigens (which contain DNA or RNA) results from the ability of these nucleic acid components to ligate TLRs both in vitro and in vivo. For example, when TLR9-​deficiency is bred on to MRL-​lpr mice—​which are an excellent model of SLE—​animals no longer get autoantibody responses to chromatin. Similarly, when mice are rendered TLR7-​deficient, the autoantibody response to Sm is markedly inhibited, and severity of the SLE phenotype is improved. These data confirm that autoantigens frequently selected in different autoimmune phenotypes likely have the dual property of being able to simultaneously activate the innate and adaptive immune systems, and that the ability to colligate TLRs plays a critical role. The like- lihood that the TLR-​autoantigen interface will be therapeutically relevant in autoimmune processes is very high. Recent work has also underscored the importance of families of cytoplasmic nucleic acid sensors as targets of the immune response in autoimmunity, and as potential amplification hubs in tissue. These include MDA5, IFI-16 and alter autoantigens. One of the major pathways downstream of TLR ligation in auto- immunity appears to centre on a relatively rare class of immature den- dritic cells (plasmacytoid dendritic cells or pDCs), which can secrete large amounts of type I interferons upon TLR ligation, and which ex- press TLR7 and TLR9 at high levels. Ronnblom and colleagues have demonstrated that, when added to material from apoptotic or necrotic cells, autoantibodies from SLE and Sjögren’s syndrome patients with specificity for DNA or RNA autoantigens induce striking interferon secretion. Type I interferons have a broad set of functions which likely contribute to the feed forward, propagation phase of systemic auto- immune diseases. For example, they (1) promote the differentiation of monocytes into mature DCs, which drive autoreactive T-​ and B-​ cell responses; (2) increase target cell sensitivity to killing pathways; (3) upregulate cytotoxic effector pathways; and (4) upregulate expres- sion of autoantigens. Targeting interferon pathways in systemic auto- immunity is currently a major focus for therapeutic intervention. Autoantigen expression in the target tissue in autoimmunity Another important component of the amplification cycle is the target tissue itself, and particularly the amounts and forms of autoantigens expressed at these sites. Unfortunately, very little is currently known about such parameters in vivo in relevant target tis- sues, in either normal or pathological circumstances. Insights from studies on human autoimmune myopathies and rheumatoid arthritis have begun to provide important insights into this problem. Thus, myositis-​specific autoantigens are expressed at very low levels in normal muscle, but at high levels in myositis tissue, where antigen expression is at highest levels in regenerating muscle cells. Similarly, cells in synovial fluids from rheumatoid arthritis (RA) patients have very high levels of citrullinated protein antigens, apparently gener- ated by immune-​mediated membranolytic pathways. These data sug- gest that enhanced autoantigen expression in the target tissue may be a feature of disease propagation, and that antigen expression during tissue damage or repair may provide an ongoing antigen source to sustain and amplify tissue damage. In this regard, the regulation of antigen expression (rather than exclusively pathways of immune-​ mediated damage) may have important therapeutic potential. Clinical features The clinical features of the different autoimmune diseases are ex- tremely diverse, and reflect the specific tissue dysfunction which

4.6  Autoimmunity 389 results from activity of immune effector pathways. Almost all tissues may be affected, including prominent involvement of endocrine or- gans, nervous system, eye, bone marrow elements, kidney, muscle, skin, liver, and gastrointestinal tract, blood vessels, lung, and joints. For tissue-​specific autoimmune processes (e.g. type 1 diabetes, ITP, autoimmune haemolytic anaemia (AIHA)—​see Table 4.6.1), symp- toms may relate to tissue hypofunction resulting from (1)  target cell destruction (for type 1 diabetes, destruction of the β cells of the pancreatic islets; for ITP and AIHA, destruction and phagocytosis of platelets and erythrocytes); (2) antibody-​mediated interference with function or downregulation of autoantigen expression (e.g. myasthenia gravis, bullous pemphigoid). In other cases, symptoms may arise from tissue hyperfunction (e.g. Graves’ disease) due to activating effects of antibody binding (where antibodies to the TSH receptor induce non​physiological secretion of thyroid hormone). In the case of systemic autoimmune processes (Table 4.6.2), symptoms frequently result both from localized target tissue de- struction (e.g. skeletal muscle in polymyositis, skin disease in SLE) as well as from the more general activities of inflammatory effector pathways. The latter result from (1) immune complex deposition Table 4.6.1  Autoantigens targeted in several tissue-​specific autoimmune diseases Disease Tissue target Prominent autoantigen(s) Proposed disease mechanisms Clinical features Autoimmune haemolytic anaemia Erythrocyte surface Components of the Rh antigen, band 3.1, glycophorin, and several unidentified molecules Antibody-​mediated destruction and clearance of erythrocytes Anaemia Autoimmune hepatitis Hepatocytes Smooth muscle cell cytoskeletal components Cytocshrome P450-​2D6 ASGP-​receptor Multiple Mild to severe chronic hepatic dysfunction in young women Epidemic Guillain–​ Barré syndrome
(N. China) Motor axons Axonal gangliosides Infection with Campylobacter jejuni induces cross-​reactive antibody, which mediates axonal damage Acute autoimmune axonopathy Flaccid paralysis with areflexia Elevated cerebrospinal fluid protein Grave’s disease Thyroid gland TSH receptor Antibody-​mediated stimulation of TSH receptor, leading to excessive thyroid hormone secretion Hyperthyroidism Goitre Grave’s ophthalmopathy Localized dermopathy Diabetes (type 1) β cells of the islets of Langerhans Glutamic acid decarboxylase (65-​kDa form) Insulin Carboxypeptidase Cytotoxic lymphocyte-​mediated destruction of islet cells Insulin deficiency and diabetes Idiopathic thrombocytopenia Platelet surface Platelet integrins Antibody-​mediated platelet destruction and phagocytosis Thrombocytopenia, bleeding Inflammatory bowel disease Gastrointestinal tract Atypical p-​ANCA ASCA Cytokine and lymphocyte-​mediated epithelial damage and dysfunction Chronic intestinal inflammation marked by remission and relapse Multiple sclerosis Myelinated nerve fibres Myelin basic protein PLP MOG Transaldolase Activated cytokine pathways Activated effector lymphocytes Autoantibodies Demyelinating disorder primarily affecting young adults: protean clinical manifestations depending on location and size of classic plaques Myasthenia gravis Neuromuscular junction Nicotinic AChR Antibody-​induced blockade and downregulation of AChR Striated muscle fatigue and weakness Myocarditis Myocardium Cardiac myosin Adenine nucleotide transporter Branched-​chain ketodehydrogenase Infection with coxsackievirus induces myocardial damage and immunization with cardiac autoantigens Subacute congestive heart failure Pemphigus vulgaris Hemidesmosome junctions Desmoglein-​3 Antibody-​mediated disruption of epithelial cell junctions, with epidermal cell detachment Blistering skin lesions Psoriasis ADAMTSL5 Non​cytotoxic CTL effects on melanocytes and keratinocytes Among the most frequent of T-​cell-​mediated
disorders with chronic, relapsing, hyperproliferative skin inflammation Rasmussen’s encephalitis Inhibitory neurons Type 3 glutamate receptor Antibody-​mediated blockade of inhibitory neurotransmitter signalling Severe epileptic seizures, progressive degeneration of a single cerebral hemisphere Stiff man syndrome GABA-​ergic neurons modulating spinal cord reflexes GAD67 Amphiphysin Blockade of inhibitory neurotransmitter signalling, possible autoantibody-​mediated Rare disease characterized by severe, progressive stiffness with superimposed episodic muscle spasms, may be associated with autoimmune disease or malignancy Vitiligo Melanocytes Tyrosinase, TRP-​1 Cytotoxic lymphocyte-​mediated damage of melanocytes Skin depigmentation AChR acetylcholine receptor; ASCA, anti-​Saccharomyces cerevisiae antibodies; MOG, myelin/​oligodendrocyte glycoprotein; PLP, proteolipid protein.

390 SECTION 4  Immunological mechanisms at multiple sensitive sites (e.g. joints, kidney, skin, and blood vessel walls) with activation of the complement cascade and recruit- ment and activation of myelomonocytic cells; (2) ongoing secre- tion of proinflammatory cytokines. In this regard, the profoundly positive clinical effects of inhibitors of multiple cytokine pathways (including tumour necrosis factor (TNF), IL-​6, IL12/​23, IL-​17) on the inflammatory symptoms and tissue destruction in rheumatoid arthritis and psoriasis underscore the central role of these general inflammatory mediators in generation and maintenance of the dis- ease phenotype in systemic autoimmune diseases. Prognosis Although the barriers that need to be overcome in terms of initiating an autoimmune disease are stringent and difficult to satisfy even in the setting of appropriate susceptibility genes, the immune system is equipped with a powerful memory. The mechanisms of this memory are still incompletely defined but include the generation of a population of memory cells specific for the antigen that initiated the response, which respond vigorously (both in terms of clonal ex- pansion, as well as effector function) to very low concentrations of antigen if they encounter it again. Since the autoimmune diseases are disorders driven by the ongoing release of self-​antigen, this immunological memory con- stitutes a major barrier to complete cure. Autoimmune diseases therefore tend to be self-​sustaining over long periods, and are often punctuated by clinical exacerbations (flares), which are likely due to re-​exposure of the primed immune system to antigen (e.g. SLE, autoimmune myositis, rheumatoid arthritis). The possibility of dis- ease recurring, even after long clinical remission, remains present in most of the autoimmune diseases. It is notable that early recognition of disease and early intervention may avoid the significant amplifica- tion of disease that renders the process so resistant to therapy. Early recognition is therefore a major goal for effective management. Tissue-​specific autoimmune diseases may result in the complete destruction of the target tissue over time, with loss of function of that tissue accompanied by a waning immune response (e.g. type 1 dia- betes). Interestingly, in cases where immune-​mediated tissue path- ology results from effector pathways being driven by a cross-​reactive T-​cell response to a foreign antigen (e.g. epidemic Guillain–​Barré syndrome), disease has a finite duration, and generally does not recur. Therapy It is not possible to discuss the therapy of this broad group of dis- orders in any detail in this chapter, but a few principles that underlie current approaches to therapy are discussed. Autoimmune diseases cause significant tissue dysfunction through (1)  inflammation, (2) tissue destruction with loss of functional units, (3) the conse- quences of healing, and (4) functional disturbances (e.g. interference with acetylcholine signalling by autoantibody to the acetylcholine receptor and inducing receptor downmodulation in myasthenia Table 4.6.2  Systemic autoimmune diseases Disease Prominent tissue target Prominent autoantigen(s) Proposed disease mechanisms Clinical features PM/​DM Skeletal muscle Mi-​2 helicase Aminoacyl-​tRNA synthetases DNA repair machinery Complement activation (DM) Activated effector lymphocytes (PM) Proximal muscle weakness (PM/​DM) Heliotrope/​skin rash (DM) Interstitial lung disease Rheumatoid arthritis Synovial joints IgG Fc Citrullinated peptides (CCP) Citrullinated vimentin, fibrin, calpastatin Peptidyl arginine-​deiminase (PAD)-​4 Activated cytokine pathways (TNF) Activated effector lymphocytes Immune complex deposition Symmetric, erosive polyarthritis Scleroderma Skin, lung, GI, kidney, heart Topoisomerase-​1 (diffuse form) RNA polymerases (diffuse) Centromere proteins (CREST form) Blood vessel damage by activated effector lymphocytes and autoantibodies Progressive fibrosis of skin, and multiple internal organs (including GI, lung, kidney, and heart) Raynaud’s phenomenon Vasculopathy Sjögren’s syndrome Exocrine glandular epithelial tissue Ro/​SS-​A; La/​SS-​B Epithelial cell death induced by cytotoxic lymphocytes and other immune effector pathways Keratoconjunctivitis sicca Systemic lupus erythematosus Numerous, including skin, kidney, joints, haematologic elements, nervous system dsDNA/​nucleosomes Splicing ribonucleoproteins (e.g. Sm, U1-​RNP) Ro/​SS-​A; La/​SS-​B Ribosomal P proteins Phospholipid-​protein complexes Cell death/​abnormal clearance of apoptotic cells Nucleoprotein complex ligation of TLRs inducing prominent interferon secretion Autoantibody-​mediated pathology Immune complex deposition Multisystem inflammatory disease Skin lesions Arthritis Renal disease Anaemia, thrombocytopenia Granulomatosis with polyangiitis (GPA, formerly Wegener’s granulomatosis) Numerous, including upper airways, lungs, kidneys, and skin Neutrophil proteinase-​3 (c-​ANCA) c-​ANCA binding to neutrophil surface induces degranulation in the vessel wall with consequent damage Multisystem inflammatory vascular disease with predominance of sinuses, middle ear, lung, and renal involvement DM, dermatomyositis; GI, gastrointestinal; PM, polymyositis.

4.6  Autoimmunity 391 gravis). Therapeutic interventions in autoimmune diseases are gen- erally focused on controlling immune and inflammatory pathways, and at replacing or accommodating lost function. Control of immune and inflammatory pathways responsible for ongoing damage Since in most instances the critical autoantigens and effector path- ways responsible for disease have not been fully defined, this goal is frequently extremely challenging. Thus, frequent use is made of anti-​inflammatory and immunosuppressive therapies which broadly target many aspects of the immune response (e.g. steroids, azathioprine, cyclophosphamide, methotrexate, mycophenolate). Since a robust immune response is required to protect the host from a myriad of infectious threats, this non​targeted suppression of the immune system can have deleterious consequences in terms of in- creased susceptibility to infection, with its attendant high morbidity and mortality. The hazards of non​targeted immune suppression makes thera- peutic targeting of specific inflammatory pathways extremely at- tractive, and there are recent examples in which this approach has been highly successful. In many patients with rheumatoid arthritis, the maintenance of chronic inflammatory joint pathology appears to be dependent on the activity of several cytokines. Specific inhib- ition of TNF, IL-6, IL-17 through the use of either soluble receptors or humanized monoclonal antibodies has led to an astonishing ef- fect on disease activity in various inflammatory arthritides, with abolition of systemic symptoms and a striking decrease in the rate of joint destruction. These positive effects were associated with only a minimal increase in susceptibility to infection, although this risk is certainly present. These therapies also served as a model demonstrating that the use of injectable forms of biological ther- apies (monoclonal antibodies or receptors) as therapeutic agents in the general population was feasible. The potential that early diagnosis and specific intervention may have a higher chance of reversing the process if this occurs before the immune response amplifies and spreads to multiple antigens remains tantalizing, and early observations in RA and cancer-​ associated autoimmunity are hopeful in this regard. As noted here, modulation of additional immune effector pathways, TLR or cytoplasmic nucleic acid sensor signalling, or autoantigen expression in the target tissue are also important targets for novel therapy in the autoimmune diseases. Another example of specific targeting of proinflammatory path- ways is that of intravenous immunoglobulin (IVIG). This is prepared from pooled serum and its major component is immunoglobulin G (IgG). IVIG therapy has been used as a treatment of several auto- immune diseases, including ITP, autoimmune myositis, and acute demyelinating polyneuropathy, but is only available at prohibitive cost. Recent data from mice has demonstrated that IVIG induces surface expression of the inhibitory Fcγ receptor (Fcγ RIIB) on macrophages, and shifts the balance of signalling through Fc re- ceptors towards inhibition, down regulating the proinflammatory response to immune complexes. It is likely that continued iden- tification of additional agents that precisely modulate specific inflammatory pathways will have a major therapeutic impact on this group of diseases. Interventions aimed at replacing or accommodating lost function Most autoimmune diseases are associated with loss of function of organs and tissues, many of which perform essential physiological functions. Indeed, recognition of the autoimmune phenotype in many instances requires that tissue damage is sufficiently severe to have led to characteristic loss of function. For example, loss of insulin-​secreting β cells of the pancreatic islets results in type 1 diabetes, and blockade and downregulation of the nicotinic acetyl- choline receptor causes striated muscle weakness and fatigue in my- asthenia gravis. Similarly, chronic immune complex deposition in glomeruli causes renal inflammation and scarring in SLE. Where significant functional reserve is still present in a particular dis- ease, a strong argument can be made for preventing further damage through specific or general immunosuppressive strategies described here. This is particularly relevant where the ‘supply’ of tissue that could be damaged is essentially inexhaustible (e.g. most instances of systemic autoimmune disease). Where functional impairment is already established, interventions aimed at replacing or accommo- dating lost function are indicated. For example, insulin replacement is required for type 1 diabetes, and treatment for hyperthyroidism is indicated in Graves’ disease. FURTHER READING Banchereau J, Pascual V (2006). Type I interferon in systemic lupus erythematosus and other autoimmune diseases. Immunity, 25, 383–​92. Cho JH, Feldman M (2015). Heterogeneity of autoimmune diseases: pathophysiologic insights from genetics and implications for new therapies. Nat Med, 21, 730–​8. Feldmann M, Maini RN (2001). Anti-​TNF alpha therapy of rheuma- toid arthritis: what have we learned? Annu Rev Immunol, 19, 163–​96. Gammon G, Sercarz EE, Benichou G (1991). The dominant self and the cryptic self: shaping the autoreactive T-​cell repertoire. Immunol Today, 12, 193–​5. Joseph C, et al. (2014). Association of the autoimmune disease sclero- derma with an immunologic response to cancer. Science, 34, 152–​7. Marshak-​Rothstein A, Rifkin IR (2007). Immunologically active autoantigens: the role of toll-​like receptors in the development of chronic inflammatory disease. Annu Rev Immunol, 25, 419–​41. Miossec P, Kolls JK (2012). Targeting IL-​17 and TH17 cells in chronic inflammation. Nat Rev Drug Discov, 11, 763–​76. Pincetic A, et al. (2014). Type I and type II Fc receptors regulate innate and adaptive immunity. Nat Immunol, 15, 707–​16. Radic MZ, Weigert M (1994). Genetic and structural evidence for antigen selection of anti-​DNA antibodies. Annu Rev Immunol, 12, 487–​520. Rosen A, Casciola-​Rosen L (2016). Autoantigens as partners in initi- ation and propagation of autoimmune rheumatic diseases. Annu Rev Immunol, 34, 395–420.

4.7 Principles of transplantation immunology 392

4.7 Principles of transplantation immunology 392

ESSENTIALS The survival of transplanted organs remains limited by the body’s immune responses, which are designed to discriminate between ‘self’ and ‘non-​self’ or ‘altered-​self’, and many of the complications of transplantation result from the crude nature of our attempts to suppress these. The immune response to transplanted tissue The immunological response that follows transplantation of tissue between genetically non​identical individuals is complex. (1) Inflammatory signals are generated at the site of transplantation and activate cells of the innate immune system, promoting the pres- entation of alloantigens—​particularly molecules of the major histo- compatibility complex—​to recipient T cells. (2) Activation and clonal expansion of alloreactive recipient T cells results in the production of populations of effector lymphocytes. (3)  Activated lympho- cytes, along with macrophages and neutrophils, migrate to the graft. (4) Many effector mechanisms contribute to graft destruction, including the delayed-​type hypersensitivity response, direct cytotox- icity, and B-​cell alloantibody production. Clinical features of allograft rejection In clinical practice allograft rejection is categorized by the timing in relation to the transplantation procedure and the dominant arm of the immune system involved, cellular or humoral. (1) Hyperacute re- jection—​caused by preformed complement-​fixing antibodies against allogeneic major histocompatibility complex molecules or ABO antigens (modern cross-​match techniques have made this extremely rare). (2) Acute rejection—​may be predominantly due to acute cel- lular or acute antibody-​mediated rejection; leads to a sudden de- terioration in graft function over days to weeks. (3) Chronic graft dysfunction (‘chronic rejection’)—​may be partly due to chronic ac- tivation of the immune system; causes gradual deterioration in graft function occurring over weeks to month. Immunosuppressive therapy Many different immunosuppressive regimens for solid organ
transplantation are in clinical use. The agents employed in- clude glucocorticoids, antiproliferative agents (e.g. azathioprine, mycophenolate mofetil), calcineurin inhibitors (e.g. ciclosporin, tacrolimus), mammalian target of rapamycin (mTOR) inhibitors (e.g. sirolimus, everolimus), depleting antibodies (e.g. antithymocyte globulin, alemtuzumab), and other biological agents (e.g. daclizumab and basiliximab, both of which are directed against CD25, the high-​ affinity IL-​2 receptor α-​chain, and belatacept, a fusion protein linked to CTLA-​4 (CD152) that blocks T-​cell costimulation). Clinical perspective and future prospects Modern immunosuppressive therapy has improved 1-​year graft survival to above 90% for kidney and liver recipients, and above 80% for most other solid organ transplants, but late graft loss and the adverse effects of chronic immunosuppression remain a signifi- cant problem. Challenges include improving donor organs prior to transplantation and the development of better assays to monitor the immune response following transplantation and make it easier to individualize immunosuppressive therapy. Ongoing trials of novel therapies provide the exciting possibility of donor-​specific hyporesponsiveness or even operational transplantation tolerance. Introduction Since the first successful transplant of a kidney between identical twins in 1954, transplantation has progressed from being an experi- mental procedure to a routine clinical therapy offering immense benefits for patients with organ failure. However, the survival of transplanted organs remains limited by the body’s immune re- sponses, and many of the complications of transplantation result from the crude nature of our attempts to suppress these. The immune system has evolved to protect the individual from invasion by pathogenic microorganisms as well as malignant or premalignant mutation of the individual’s own cells, hence strin- gent discrimination of ‘self’ from ‘non-​self’ or ‘altered-​self’ is cru- cial to ensure immunological responses are directed correctly and to limit damage to self tissues that might otherwise occur during the response to a foreign pathogen. To complicate matters, the immune system is continually exposed to new antigens, and since many of these are harmless or even beneficial (e.g. bacteria composing the in- testinal flora), the immune response generated must be proportional to the threat that each new antigen indicates. 4.7 Principles of transplantation
immunology Elizabeth Wallin and Kathryn J. Wood

4.7  Principles of transplantation immunology 393 The character of an immune response is determined partly by the particular antigens involved and partly by the context in which these antigens are encountered. In particular, priming of T cells with antigen from sites of inflammation is much more likely to produce an aggressive immune response aimed at clearing the antigen than priming with antigen in the absence of inflammatory signals. In the context of the highly inflammatory microenvironment sur- rounding transplantation, the introduction of tissue from a genet- ically disparate (allogeneic) individual (see Table 4.7.1) containing a multitude of non-​self peptides (alloantigens) triggers a vigorous immune response that almost invariably results in destruction of the transplanted tissue unless active steps are taken to suppress the im- mune system. Acute allograft rejection is a complex T-​cell-​dependent process, summarized in Fig. 4.7.1. Inflammation triggered by the retrieval of the donor organ or tissue and the transplant procedure itself generates so-​called ‘danger’ signals that activate both the innate and adaptive immune systems. Triggering of the innate immune system creates an environment that promotes activation of the adaptive response, which includes T cells and B cells. T-​cell in- tegration of signals delivered through recognition of alloantigen, costimulation, and inflammation leads to activation, clonal ex- pansion, and differentiation of donor-​reactive lymphocytes into effector cells. While these events begin in the transplant itself, activation of the adaptive immune system occurs predominately in the lymphoid tissue draining the transplant site. Innate and in- flammatory changes in the graft then provide homing signals for the migration of mature effector lymphocytes to orchestrate the destruction of the transplant. While T cells, both cytotoxic and helper, are absolutely required for transplant rejection, recent evidence has shown that the roles played by B cells and cells of the innate immune system, including monocytes and natural killer (NK) cells, are more important than previously thought, and can increase the speed and magnitude of the response to the graft, as well as initiating long-​term immunological memory directed against the donor antigens. In clinical practice allograft rejection is frequently categorized depending on the timing in relation to the transplantation procedure or the dominant arm of the immune system involved (Table 4.7.2). Internationally accepted scoring systems have been developed for most solid organ transplant biopsies to characterize rejection and fa- cilitate the decision-​making process regarding the implementation of antirejection therapy. The best known of these is the Banff scoring system for renal allografts, but similar systems are in place for liver, heart, lung, and vascularized composite allografts. This chapter will outline the cellular and molecular events in- volved in alloantigen recognition and subsequent allograft rejection, as well as the mechanism of action of the current pharmacological agents available in clinical practice to suppress graft rejection. Initiation of response to graft The initiation of a response to transplanted tissue is complex and multilayered, involving all aspects of the immune system—​ inflammation, innate immune cells, and an adaptive response. Multiple factors, both donor and recipient, interact and affect the magnitude of this response. Broadly these can be divided into innate and adaptive responses, with the innate consisting of both inflam- mation and innate immune cell responses. Innate Both the physical process of removing and reimplanting tissue for transplantation, also the state of the donor organ, can have a pro- found impact on the initiation of responses against the graft. The type of donation (cadaveric heart beating, cadaveric non-​heart Table 4.7.1  Transplantation terminology Autograft Tissue transplanted from one part of an individual’s body to another (e.g. skin grafts in patients with burns; vascular grafts for coronary or peripheral vascular disease) Isograft Tissue transplanted between genetically identical (syngeneic) members of the same species (e.g. grafts between monozygotic twins; grafts between members of the same inbred strain of mouse or rat) Allograft Tissue transplanted between genetically disparate (allogeneic) members of the same species (e.g. grafts between unrelated humans; grafts between different inbred strains of mice or rats) Xenograft Tissue transplanted between individuals of different species (e.g. pig to human, rat to mouse) Generation of DAMPs, ROS, HSPs, cytokines, complement products Ischaemia/ reperfusion injury Surgical injury (retrieval/ implantation) Brain death, donor factors, e.g. infection Invasion of innate cells increasing inflammation Migration of APCs to SLOs, setting up adaptive immune response Macrophages, neutrophils, NK cells, complement CD8 cytotoxic T-cell damage CD4 T cell help B-cell antibody responses GRAFT REJECTION Fig. 4.7.1  Overview of allograft rejection. Multiple cell types and effector mechanisms contribute to graft destruction, including both innate and adaptive arms of the immune system. DAMPS, damage associated molecular patterns; ROS, reactive oxygen species; HSPs, heat shock proteins; APCs, antigen-​presenting cells; SLOs, secondary lymphoid organs; NK cells, natural killer cells.

394 SECTION 4  Immunological mechanisms beating or living donor) as well as the age and physical health of the donor can all affect the degree of preimplantation damage that oc- curs within the graft. Ischaemia from hypoxia and hypoperfusion, as well as the cytokine storm associated with brainstem death, initiates a sequence of changes in gene expression within the donor tissue that have a profound influence on the immunological response of the recipient. Adding to this the injury from direct surgical trauma, and further injury from reactive oxygen species released when the ischaemic tissue is reperfused, and the graft environment becomes profoundly proinflammatory. The initial responders to this inflammatory milieu are innate im- mune cells, which express pattern-​recognition receptors. These en- able them to recognize markers of tissue injury and to detect the presence of pathogens. The best characterized of these are the Toll-​ like receptors (TLRs) which bind to phylogenetically conserved mo- lecular features unique to microorganisms, as well as endogenous molecules that are produced as a consequence of tissue injury, such as reactive oxygen species, activated complement components, and heat shock proteins. In the context of transplantation, damage associated molecular patterns (DAMPs) lead to graft infiltration with innate immune cells and the local production of inflammatory mediators, chemokines (chemoattractant cytokines), and P-​selectin (CD62P), an adhesion molecule necessary for leucocyte transmigration into tissue. This identifies the transplanted tissue as a site of inflammation and trig- gers the migration of donor-​derived antigen-​presenting cells (APCs) to recipient lymphoid tissue, as well as the recruitment of recipient immune cells into the graft. Recently, particular interest has fallen on the role of monocytes, which are one of the earliest invaders (Fig. 4.7.1), and their role in initiating an alloimmune response in the case of a genetically non​identical donor. Many of the events initiated by the transplant operation are a non​specific response to tissue damage, and the invasion of recipient cells occurs regardless of whether the transplantation procedure occurs between genetically identical or genetically disparate indi- viduals. However, recent work has shown that recipient monocytes invading the graft respond differently to genetically disparate in- dividuals and are capable of differentiating self from non-​self. The proinflammatory milieu is sensed by TLRs, but in the case of gen- etically identical individuals, the stimulation is insufficient to fully activate these monocytes and the response subsides, leading to reso- lution of inflammation and healing. In allogeneic transplants, the monocytes differentiate into inflammatory dendritic cells, capable of presenting antigen and providing both costimulation and inflam- matory cytokine production. Adaptive In addition to the innate immune response, an adaptive response is initiated in the context of allogeneic transplants. Although the innate immune reaction occurs in auto-​, iso-​ and allo-​grafts, only in the case of genetically non​identical individuals is an adaptive re- sponse both initiated and sustained by the alterations in the innate response. There are several components to this (see Fig. 4.7.1) al- though T cells remain the key players. T-​cell allorecognition Animals that lack T cells do not reject fully major histocompatibility complex (MHC)-​mismatched allografts or xenografts, but adop- tive transfer of wild-​type T cells to these animals is able to restore allograft rejection. In contrast, B-​cell deficient mice reject cardiac allografts at control rates. In clinical transplantation, therapies that deplete peripheral T cells are highly effective at reversing episodes of acute rejection. These observations highlight the role of T cells in transplant rejection. Several different approaches have been used to evaluate the rela- tive contribution of CD8+ and CD4+ T cells to allograft rejection. The available animal data indicate that, in general, CD4+ cells are essential for allograft rejection. However, CD8+ cells contribute to rejection and in certain circumstances are capable of rejecting MHC class I mismatched allografts in the absence of CD4+ cell help. There are many cell surface and intracellular molecules that are variable, or polymorphic, between different members of the same species, and recognition of this genetic variation by T cells is a cru- cial step in initiating transplant rejection. One of the reasons that transplantation induces such a dynamic immune response is the high precursor frequency of T cells able to respond to mismatched Table 4.7.2  Characterizing the immune response following transplantation Time after transplant Cellular mechanisms Humoral mechanisms Innate mechanisms Hyperacute—​ minutes to hours If preformed complement-​fixing antibodies against allogeneic MHC molecules or ABO antigens are present at the time of transplantation, catastrophic rejection occurs. In solid organ transplantation this is characterized by rapid widespread vascular thrombosis leading to infarction of the graft Complement Phagocytes Acute—​days to weeks Infiltration of the graft by activated allospecific CD8 T cells, causing acute TCMR Pre-​existing or de-​novo generated antibodies against allogeneic MHC molecules causes acute ABMR Infection may trigger TLRs via DAMPs or PAMPs, creating a proinflammatory environment and triggering adaptive responses Chronic graft dysfunction—​ weeks to months Repeated episodes of acute TCMR may contribute to fibrosis and scarring, although CD8 responses are no longer thought to be prominent in chronic graft dysfunction Now thought to be largely due to chronic antibody-​ mediated damage, histologically this is typically associated with fibrointimal proliferation in intragraft arteries and interstitial fibrosis NK cells may be involved in the response to antibodies. Maladaptive macrophage responses to injury can switch healing and resolution of inflammation to fibrosis and scarring ABMR, antibody-​mediated rejection; DAMP, damage-​associated molecular pattern; MHC, major histocompatibility complex; PAMP, pathogen-​associated molecular pattern; TLR, Toll-​like receptors; TCMR, T-​cell mediated rejection.

4.7  Principles of transplantation immunology 395 MHC molecules, which can be as high as 1 in 10, orders of mag- nitude higher than the proportion of T cells that are able to react to a nominal peptide antigen–​self-​MHC complex (1 in 20 000 to 1 in 100 000). However, although MHC molecules are the most im- portant alloantigens, transplants between siblings with identical MHC molecules are still vulnerable to rejection, albeit at a slower tempo than MHC-​mismatched transplants. Rejection in this set- ting is a result of T-​cell recognition of other antigens. These include polymorphic non-​MHC molecules called minor histocompati- bility antigens (miH) that are derived from a wide variety of pro- teins and not necessarily expressed by cells of the immune system. Additionally, responses to endothelial antigens and other non-​MHC molecules have been described. Pathways of allorecognition Transplantation is a unique immunological situation in which priming of recipient T cells with antigen can occur via three dis- tinct pathways. Direct allorecognition is the interaction of recipient T cells with intact allogeneic MHC–​peptide complexes on the sur- face of donor-​derived APCs. Indirect allorecognition occurs when peptides derived from donor antigens are degraded and presented by recipient APCs. The final pathway, semi-​direct allorecognition, involves the exchange of intact MHC–​peptide complexes between recipient and donor APCs (Fig. 4.7.2). Direct allorecognition Transplanted tissue contains bone marrow-​derived haematopoi- etic cells of donor origin that have the characteristics of immature dendritic cells. In response to the inflammatory milieu that follows transplantation, the donor-​derived passenger leucocytes rapidly leave the graft and migrate to the secondary lymphoid tissues of the recipient. During migration the passenger leucocytes acquire the phenotype and functional characteristics of mature dendritic cells, expressing high levels of MHC class I and II molecules, as well as other cell-​surface costimulatory molecules necessary to fully acti- vate naive CD4+ and CD8+ T cells. Once in the secondary lymphoid tissues they act as professional antigen-​presenting cells, presenting donor-​derived MHC molecules in the transplanted tissue to re- cipient T cells, which recognize them as non-​self. Antigen presentation via the direct pathway was originally thought to play a dominant role in initiating the response to a trans- plant as T cells that recognize alloantigen in the context of donor MHC constitute 90% of the total alloreactive T cell repertoire. Evidence for the dominance of this pathway is variable: some animal models have suggested that depletion of donor APCs from the tissue prior to transplantation leads to long-​term graft survival without immunosuppression, but other studies show that donor APCs are rapidly killed within secondary lymphoid tissue by recipient NK cells, before they are able to directly activate T cells. Models showing that the expanded T-​cell repertoire post-​transplant is comprised of T cells stimulated by indirect allorecognition have supported this observation. It is therefore likely that direct allorecognition plays a much smaller role that originally thought. Semi-​direct allorecognition With new evidence suggesting a much more limited role for direct allorecognition in the transplant response, but older evidence sup- porting the role of donor derived APCs, it has been suggested that, rather than acting as direct APCs, the main role of donor-​derived DCs is to transport antigen from the graft to lymphoid tissue. It has been shown that MHC–​peptide complexes can be directly passed from one DC to another on membrane fragments, in this case permitting donor MHC-​peptide to be passed to recipient DCs, allowing semi-​direct allorecognition of non-​self by alloreactive T cells. There is increasing evidence to support this pathway of allorecognition as being the pathway that dominates early in the Recipient APC Direct pathway Semi-direct pathway Donor MHC Recipient MHC TCR Recipient T cell Donor APC Donor MHC TCR Recipient T cell Recipient APC Recipient T cell Indirect pathway Fig. 4.7.2  Pathways of allorecognition. In the direct pathway donor, APCs present donor peptide to recipient T cells in the context of donor MHC. In the semi-​direct pathway, intact donor MHC–​peptide complexes are passed from donor APCs to recipient APCs, and presented to recipient T cells. In the indirect pathway, which dominates over time, donor MHC is taken up by recipient APCs, processed and presented on recipient MHC to recipient T cells in the classical pathway of antigen presentation.

396 SECTION 4  Immunological mechanisms response to transplanted tissue, but detailed mechanisms are yet to be elucidated. Indirect allorecognition Whatever the precise mechanism of early allorecognition, the long-​ term alloresponse is dominated by antigen presented via the indirect pathway. This is the standard pathway for recognition of non-​self, where donor peptides are taken up by recipient APCs, processed and presented on self-​MHC to T cells. While antigen capture can take place both in the graft and transplant-​draining lymphoid tissue, presentation of donor antigen-​self-​MHC occurs in the secondary lymphoid organs (SLOs). As in direct and semi-​direct pathways, the dominant antigenic peptides presented by the indirect pathway are the hypervariable peptide-​binding regions of MHC molecules, but unlike direct and semi-​direct pathway allorecognition, the in- direct pathway is available for antigen presentation for as long as the graft remains in situ and therefore becomes the dominant mode of allorecognition in the long term. T-​cell activation Regardless of the method of antigen presentation, T cells cannot be activated by recognition of peptide-​MHC complex alone. This signal, delivered via the TCR-​CD3 complex, is known as ‘signal 1’ and, in isolation, suggests recognition of self-​antigen, which will lead to deletion or anergy rather than activation of the naive T cell. T cells therefore require additional signalling—​‘signal 2’—​in the form of costimulation, and ‘signal 3’ in the form of cytokines. Signal 2 is pro- vided by the interaction of pairs of cell-​surface molecules present on T cells and antigen-​presenting cells called costimulatory molecules. Many of these molecules are homologous and in general they can be divided into two families: the CD28/​B7 family, best characterized by the T-​cell costimulatory molecules CD28 and CD152 (CTLA-​4), which both interact with the APC molecules CD80 and CD86; and the tumour necrosis factor (TNF)/​tumour necrosis factor receptor (TNFR) family of which the prototype receptor–​ligand pair are CD40 and CD154 (CD40L) (Table 4.7.3). T-​cell costimulatory molecules CD28 is constitutively expressed by T cells and binds to the B7 family molecules CD80 and CD86 on antigen-​presenting cells. Signalling via CD28 lowers the threshold for T-​cell activation, increases expression of IL-​2, and promotes T-​cell proliferation and resistance to apoptosis. During an immune response, activated T cells upregulate expres- sion of CD152 (CTLA-​4), a molecule that has close homology to CD28. CD152 also binds to CD80 and CD86, but it has an inhibitory effect on T-​cell activation and is able to attenuate immune responses by competing with CD28 for ligation of these molecules. The im- portance of CD152 as a negative regulator of immune responses was demonstrated by the generation of CD152 knockout mice, which develop a fatal disorder characterized by massive proliferation of lymphocytes. Another effect of CD28 signalling during T-​cell activation is to upregulate expression of other costimulatory molecules such as CD154 (CD40L). CD154 is the ligand for CD40 expressed by antigen-​presenting cells, and as well as delivering a positive signal to the T cell, CD40–​CD154 ligation activates APCs, leading to in- creased expression of B7 family molecules and therefore an en- hanced ability to activate further T cells. As more novel costimulatory molecules are identified, it is becoming clear that the outcome of interaction between T cells and antigen-​presenting cells is determined both by the avidity of the cognate TCR–​MHC–​peptide interaction and the balance of positive and negative signals delivered by the costimulatory molecules present on the surface of the participating cells (see Table 4.7.3). Transduction of costimulatory signals occurs in parallel to TCR–​CD3 complex signalling and can be blocked independently, and the development of pharmaceutical agents able to interrupt costimulation has provided evidence of the importance of these pathways in transplant rejection. Administration of a fusion protein constructed from the extracellular domains of CD152 (CTLA-​4Ig or CTLA-​4Fc; see ‘Immunosuppression in trans- plantation’, to follow), or monoclonal antibodies directed against CD80 and CD86, are able to block costimulation via CD28, and this is sufficient to prevent allograft rejection in animal models. Monoclonal antibodies directed against CD154 are also able to prevent acute allograft rejection. Both of these examples of costimulation blockade lead to long-​term rejection-​free survival of vascularized and non​vascularized allografts in experimental rodent models, but only CD152 fusion proteins have made it into clinical practice. Table 4.7.3  T-​cell costimulation molecules and their ligands T cell Antigen-​presenting cell Effect on T cells CD28 CD80 (B7–​1) CD86 (B7–​2) Activatory CD27 CD70 CD154 (CD40-​L) CD40 ICOS (inducible costimulator) ICOS-​L (ICOS ligand) OX40 (CD134) OX40-​L (CD252) 4–​1BB (CD137) 4–​1BB-​L (CD137L) CD152 (CTLA-​4—​cytotoxic T lymphocyte antigen-​4,) CD80 (B7–​1) CD86 (B7–​2) Inhibitory PD-​1 (programmed death-​1, CD279) PD-​L1 (programmed death ligand 1, CD274, B7-​H1) PD-​L2 (programmed death ligand 2, CD273, B7-​DC)

4.7  Principles of transplantation immunology 397 TCR signal transduction and ‘signal 3’ The intracellular signalling pathway downstream of the TCR is complex. Briefly, TCR–​MHC–​peptide engagement results in the recruitment and phosphorylation of several signalling mol- ecules. These phosphorylation events initiate several intracel- lular biochemical processes, resulting in activation of the Ras-​ and Rac-​mitogen-​activated protein (MAP) kinase pathways and hy- drolysis of membrane phosphatidylinositol 4,5-​biphosphate to generate the secondary messengers, inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 leads to the release of stored calcium from the endoplasmic reticulum and activation of the phosphatase calcineurin, which in turn dephosphorylates the transcription factor NFAT (nuclear factor of activated T cells), allowing it to translocate to the nucleus. Generation of DAG results in the activation of an- other transcription factor, NF-​κB, and a third transcription factor, AP-​1, is generated by the MAP kinase cascades. The action of these transcription factors alters expression of many genes, in particular leading to upregulation of the T-​cell growth factor IL-​2 and the high-​affinity IL-​2 receptor α-​chain (CD25). Soon after activation the generation of large amounts of IL-​2 and other proproliferative cytokines act in an autocrine and para- crine fashion to provide what has been described as ‘signal 3’ (see Fig. 4.7.3). Transduction of signals delivered by IL-​2 promotes cell-​cycle progression and initiates the clonal expansion and dif- ferentiation of activated T cells. B-​cell activation For many years, transplantation immunology has focused on T cells as the key cells in both initiation and maintenance of a re- sponse to transplanted tissue. However, interest has grown in the role of B cells, both in terms of rejection and, more recently, poten- tial protection of the graft. B cells have long been defined by their main antibody-​producing role, but they are also known to function as APCs, providing costimulation and activation signals to T cells. Several groups have described B cells with the potential to regulate immune responses in a similar way to regulatory T cells, although not as powerfully. The role of B cells in transplantation is clearly multifaceted, and they are by no means bystanders in the early re- sponses to the graft. B-​cell activation also occurs in secondary lymphoid organs. Mature naive B cells continuously sample antigen, either that held on follicular dendritic cells within the B-​cell zone or if presented on subcapsular sinus macrophages. When the B cells recognize antigen via their B-​cell receptor (BCR) they can take it up, process it, and then combine peptide fragments into the binding site of class  II MHC. The antigen-​MHC complex is then displayed on the B-​cell surface and presented to CD4 T cells. B cells therefore recognize antigen when intact, whereas the peptide they present will be a much smaller fragment, introducing additional layers of complexity into the recognition and attack of non-​self by T cells. For full activation of B cells, they then require help and recip- rocal signals from the CD4 T cells (see Fig. 4.7.4), a process which usually occurs at the border between the T-​cell zone and the B-​cell zone in the SLOs. Following antigen recognition there are several fates for B cells: those recognizing non-​self can either go on to form extrafollicular responses, leading to the rapid but short-​lived pro- duction of low affinity antibody, or they can receive signals that allow them to migrate further into the B-​cell zone and form clusters called germinal centres where they undergo recombination of their BCR, with each round of somatic hypermutation an attempt to im- prove receptor specificity for the antigen. Those B cells that increase their affinity in this process compete with specialized CD4 T cells, which have also entered the B-​cell follicle, to receive survival and Upregulation of gene transcription TCR-CD3 complex Calcineurin Antigen presenting cell NFAT NF-κB MAP kinases AP-1 Protein kinase C Nucleus mTOR Signal 1 Signal 2 Signal 3 IL-2 production IL-2 CD4 Costimulatory molecules IL-2 receptor T cell Cell cycle progression MHC molecule + peptide Upregulation of gene transcription Fig. 4.7.3  Three-​signal model of T-​cell activation. Signalling through the TCR is not sufficient for activation of T cells, and in isolation leads to anergy or deletion of cells. For activation of cells and development of a pathogenic response, T cells also require signal 2 in the form of costimulation. Signal 3 is required to drive the differentiation of the activated cell into a subset (e.g. Th1, Th2), and thus the downstream response. B B T T Tfh Tfr GC APC APC Memory B cell Plasma cell B-cell follicle T-cell zone Fig. 4.7.4  The germinal centre reaction. B cells are activated by APCs and migrate to the T-​B border. Here they interact with recently activated CD4 T cells, reciprocal signals allow the B cells to move deeper into the B-​cell follicle and form germinal centres, and induce CXCR5 expression in CD4 T cells, allowing them to enter the germinal centre as T follicular helper (Tfh) cells and support B-​cell differentiation into plasma and memory cells. T follicular regulatory (Tfr) cells control the size of this reaction.

398 SECTION 4  Immunological mechanisms differentiation signals allowing them to differentiate into antibody secreting plasma cells, or memory B cells. This interaction between cells of the innate immune system (macrophages, dendritic cells) and adaptive immune system (T cells, B cells) leads to a complex and multilayered response to the graft, providing both a rapid and less specific response but setting up im- munological memory and refining responses to be highly targeted against non-​self-​antigens. This long-​lived response is the biggest hurdle in terms of transplantation. Ongoing responses and immunological memory While the early response to pathogens is predominately innate, the high frequency of alloantigen-​specific T cells means the early re- sponse to transplanted tissue is more aggressive and more specific than that to pathogens. These initial responses also set in motion the development of a long-​lived response to alloantigens which, if left unchecked, will rapidly destroy the graft. The actions of recipient and donor-​derived innate cells with allospecific T cells play key roles in the development of the ongoing response. Additional factors modify the character of the immune response to an allograft, including the site of transplantation, the organ or tissue transplanted, the antigen-​ presenting cells involved in T-​cell priming, and the immune status of the recipient at the time of transplantation. Innate immune system in transplant rejection The innate immune system comprises a group of cells and mol- ecules (Table 4.7.4) that provide a first line of defence against pathogens and which also play an important role in allograft re- jection. Primary adaptive immune system responses, which rely on the activation and expansion of antigen-​specific lymphocytes, take several days to reach maturity. In contrast, the innate im- mune system represents a ‘preformed’ defence that is immediately available to defend the host until either the dangerous stimulus is cleared or the adaptive immune system is able to mount an antigen-​specific response. As just described, the physical process of graft retrieval and im- plantation generates ‘danger signals’ in the form of heat shock pro- teins, complement breakdown products, and reactive oxygen species that activate cells of the innate immune system via TLR ligation. Macrophages and other phagocytic cells ingest necrotic tissue and, when activated, release cytokines such as tumour necrosis factor α (TNFα), interleukin 1 (IL-​1), and interleukin 6 (IL-​6) that con- tribute to the local inflammatory environment. Complement Activated complement components constitute a proteolytic cas- cade that generates a range of effector molecules (see Fig. 4.7.5). The anaphylatoxins C5a and C3a are chemoattractant molecules that as- sist leucocytes to home to the graft, while other soluble mediators are able to opsonize cells, targeting them for destruction by phago- cytes. Recognition of C3b, C4b, or their fragments covalently bound to target cells by complement receptors on the surface of leucocytes, facilitates antigen presentation and T-​cell activation. Generation of the terminal components of the complement cascade (C5b-​9) re- sults in formation of the membrane attack complex (MAC) within the target cell membrane and initiation of target cell lysis. The al- ternative pathway, mediated by C3, has been shown to play a key role in ischaemia-​reperfusion injury, with C3 knockout mice being resistant to the damage associated with this insult. The production of the MAC also rises following reperfusion and components of the terminal complement pathway, particularly C5a and C5a receptor, have been shown to be upregulated in donors with brainstem death. The proinflammatory effects of complement can play a signifi- cant role in activating the adaptive immune system, particularly in deceased donors, and despite strategies to reduce ischaemia-​ reperfusion injury (see later paragraphs on therapeutics), this re- mains a significant hurdle in improving outcomes for deceased donors compared to live donors. Innate lymphoid cells NK cells are large granular lymphocytes, comprising 5–​20% of lymphocytes in spleen and blood, which have natural cytotoxic ac- tivity against virus-​infected or mutated host cells. They also produce several cytokines and mediate antibody-​dependent cellular cytotox- icity (ADCC), so have multiple roles in the response to allograft. NK cells express invariant cell-​surface receptors, including activating receptors that bind to widely expressed carbohydrate residues on self cells and inhibitory receptors that bind self-​MHC class I mol- ecules. Some malignant or virally infected cells down-​regulate MHC class I expression or express altered class I molecules as a strategy to evade CD8+ T-​cell cytotoxicity. As a result, they are unable to stimu- late inhibitory receptors and are vulnerable to NK cell killing, in the same way as transplanted tissue expressing non-​self-​MHC class I. In addition, some cells express both a TCR and a NK phenotype, and are known as NKT cells. These cells recognize glycolipids pre- sented by the MHC class I-​like molecule CD1d, and are divided into type I  (iNKT), expressing an invariant TCR reactive to α-​ galactosylceramide, and type II, with diverse TCR specificity. Interest has grown in the role of both NK and NKT cells in the past few years. It has long been known that NK cells are respon- sible for the early killing of donor-​derived APCs, which would otherwise present antigen through the direct pathway, and therefore they may play a role in tolerance induction by limiting the presen- tation of donor antigen. However, in solid organ transplantation, ischaemia-​reperfusion injury has been shown to upregulate ligands to NK activating receptors, and NK cells have been shown to mi- grate into grafts prior to CD8 T cells, producing IFNγ and thereby inducing upregulation of MHC I and MHC II on graft endothelial cells. This makes grafts more susceptible to cytotoxic T-​cell attack, Table 4.7.4  Components of the innate immune system Component Primary function Macrophage/​neutrophil Phagocytosis; secretion of cytokines, enzymes, and inflammatory mediators Dendritic cell Antigen uptake and presentation to lymphocytes Natural killer (NK) cell Cytotoxic to virally infected or mutated cells Complement Opsonization, target cell lysis, and chemoattraction Eosinophil Killing of antibody-​coated parasites

4.7  Principles of transplantation immunology 399 and the proinflammatory cytokines secreted by NK cells can both drive DC maturation and promote the activation of allospecific T cells, enhancing the response against donor tissue. It is therefore not clear whether NK cell responses are positive or negative in the con- text of transplantation. Other innate cells The role of DCs and other APCs has been discussed earlier in terms of leucocyte activation, but many other components of the innate immune system contribute to inflammation (neutrophils, macro- phages), activation of the adaptive immune system (macrophages, NK cells, NKT cells, complement) and tissue damage (NK cells, macrophages). In practice, the response to the graft represents a con- tinuum between early non​specific responses, and later allo-​specific and subsequently donor-​specific responses, with both innate and adaptive components involved at all time points. Adaptive responses to the graft CD8 T-​cell responses Following activation in SLOs, activated CD8 T cells migrate to the graft site, recruited by chemotactic cytokines called chemokines produced by the inflammatory process ongoing in the graft. Inflammatory signals also affect blood vessels in the vicinity of the transplant, causing vasodilation and endothelial activation. This causes upregulation of the adhesion molecule P-​selectin, as well as endothelial surface expression of chemokines released from the graft. Leucocytes are usually conveyed within the fast laminar flow at the centre of blood vessels, but when activated leucocytes reach postcapillary venules close to the graft they are able to leave this rapid flow and move towards the edge of the vessel. Here they bind to P-​selectin and begin the process of extravasation into the graft it- self, moving along the chemokine gradient. Once in the graft, activated CD8 T cells recognize allogenic class I MHC on donor cells and form an immunological synapse with their intended target. The CD8 T-​cell releases granules con- taining cytotoxic molecules such as perforin and granzyme B, as well as up-​regulating cell-​surface expression of Fas ligand (FasL) and secreting soluble mediators such as TNF. Perforins polymerize and insert into the target cell membrane, forming a pore that facili- tates the entry of granzyme B and other compounds into the cell. Granzyme B is a protease that is able to initiate apoptosis by several mechanisms, including activation of caspase cascades. These mech- anisms are designed to lead to cell death by apoptosis. Binding of FasL to Fas on the target cell surface is also able to trigger apoptosis by activating caspases. CD4 T-​cell responses CD4 T cells have multiple and very diverse roles in transplant- ation. Alloantigen-​specific CD4+ T cells (typically T helper 1 cells) can contribute to inflammation and the effector phase of allograft C3a C3b C5 convertases Classical pathway Lectin pathway Alternative pathway C1q, C1r, C1s C4 C2 MBL/ficolin, MASPs C4 C2 C3i B D C3 C3 convertases C5a C5b C5b C6 C7 C8 C9 (n) Terminal pathway Lysis Sublytic effects C5 C3b Membrane-attack complex Antigen-antibody complexes pathogen surfaces Apoptotic cells—infected cells Carbohydrate residues Activating surfaces (e.g. pathogen surfaces, tumour cells) Fig. 4.7.5  A simplified overview of the complement cascade. For more detail see Chapter 4.2. In the context of organ transplantation, all three pathways have been implicated in damage to organs, with the classical pathway being most relevant in ABMR, but both lectin and alternative pathways implicated in ischaemia-​reperfusion injury. The use of drugs that interfere with the complement cascade represents a promising new therapeutic avenue in transplantation.

400 SECTION 4  Immunological mechanisms rejection via a non​specific effector mechanism referred to as the delayed-​type hypersensitivity (DTH) response. DTH reactions are characterized by the release of multiple soluble mediators, including the proinflammatory cytokines IL-​1, interferon-​γ, and TNFα. These induce infiltration of the graft by of activated leucocytes, including monocytes, macrophages and eosinophils, and the production of non​specific mediators such as nitric oxide, reactive oxygen spe- cies, and inflammatory arachidonic acid derivatives (prostaglandin E2, thromboxane, and leukotrienes). Although this activity is trig- gered in an antigen-​specific manner by the T helper cell, the effector ­mechanisms that lead to the destruction of the graft are non​specific. DTH reactions have been shown to directly affect graft physiology by altering cell permeability and vascular smooth muscle tone and play a role in both acute and chronic allograft rejection. CD4 T cells also play a key role in the humoral response to alloantigen (Fig. 4.7.4) by providing survival signals to activated B cells and receiving reciprocal signals, allowing them to migrate into the B-​cell follicle of SLOs and participate in the germinal centre (GC) response. These reciprocal interactions maintain the GC and allow the formation of long-​lived memory B cells and plasma cells. CD4 T cells can also have a regulatory role, suppressing immune responses to antigen in order to control the size of the response. The best studied regulatory or suppressor T cells (Treg) are a popula- tion of naturally occurring CD4+ cells that constitutively express the IL-​2 receptor α-​chain (CD25). These cells develop within the thymus under the direction of the transcription factor Foxp3 and have a critical role in limiting immune responses, particularly to self-​antigens. Mutation of Foxp3 in humans is responsible for the IPEX syndrome (immune dysregulation, polyendocrinopathy, en- teropathy, X-​linked), which causes severe disease and death by aged two in most cases. Mouse models of Treg deficiency also lead to overwhelming autoimmune disease. Tregs are known to play key roles in downregulating immune responses to all antigens, as well as prevention of autoimmunity, but can also prevent responses to malignant cells. B-​cell responses The B-​cell response to alloantigen following transplantation results in the generation of alloantigen-​specific antibodies (alloantibodies—​ also referred to as donor-​specific alloantibodies (DSA)), which play an important role in allograft rejection. B cells use surface immuno- globulin as their antigen receptor (B-​cell receptor or BCR) and are able to internalize antigens, which are then degraded and presented in conjunction with class  II MHC molecules. Early antibody re- sponses to the graft do not require significant interaction with T cells, but long-​lived antibody responses require B cells to go through the GC reaction and are absolutely dependent on interactions with CD4 T cells (Fig. 4.7.4). When B cells recognize their antigen in SLOs they migrate to the edge of the B-​cell follicle, where they interact with recently primed CD4 T cells. If two cells recognize the same antigen, the T cell pro- vides the B-​cell with survival and activation signals, allowing it to migrate further into the B-​cell follicle. The B-​cell provides recip- rocal signals to the CD4 T cell, causing upregulation of CXCR5 via the transcription factor Bcl6, a chemokine receptor that allows the CD4 T cell to follow the B-​cell into the follicle and become a follicular helper T cell, or Tfh. Here the Tfh and B cells interact to form a germinal centre. This process is absolutely required for the formation of memory B cells and plasma cells, and involves random mutation of the variable region of their BCR. With each mutation, B cells alter the affinity of their BCR for the antigen and compete with each other for survival and maturation signals from the Tfh. There are also regulatory cells within the GC (T follicular regulatory cells or Tfr) that control the size of the GC response by limiting the interactions between B cells and Tfh and ensure that only the cells with the highest affinity BCR for antigen go on to become plasma or memory cells (Fig. 4.7.4). As in the T-​cell response to alloantigen, the predominant anti- genic targets of DSA are mismatched MHC molecules, but anti- bodies that recognize other antigens such as miH or blood group antigens also contribute to rejection. The development of de-​novo DSA after transplantation in kidney transplant recipients is strongly associated with graft decline and failure. The mechanism of antibody-​mediated rejection is primarily via complement fixation and membrane attack complex formation leading to target cell lysis. NK cells and macrophages express recep- tors that bind to the non-​antigen-​specific (Fc) portion of antibodies, stimulating them to kill target cells that have antibody bound on the surface (a process called antibody-​dependent cell-​mediated cyto- toxicity or ADCC), and this provides a second mechanism by which alloantibodies can induce donor cell death. Antibody-​mediated rejection Antibody-​mediated rejection (ABMR) can occur immediately in patients with preformed antibodies in bouts of acute rejection, or in a more chronic and insidious manner. Hyperacute rejection, now exceedingly rare, occurs if patients have preformed DSA at the time of transplantation and results in graft destruction within minutes of organ reperfusion. In this situation the antibodies (which are usually directed at allogeneic MHC molecules or ABO blood group antigens expressed on graft endothelium) cause local activation of the coagu- lation and complement cascades, resulting in extensive thrombosis within the vascular supply to the graft and culminating in infarction. Modern cross-​matching techniques screen for preformed DSA and in deceased donor transplants this is considered an absolute contra- indication to transplantation. Some living donor kidney transplant recipients can undergo desensitization protocols where preformed ABO or HLA antibodies are removed prior to the operation, which only goes ahead if antibody levels are within acceptable limits: these procedures require careful planning. Humoral responses to transplanted tissue are more commonly seen in both acute and chronic rejection of grafts. Acute antibody-​ mediated rejection is uncommon with modern cross-​matching techniques: it is very rare in liver transplantation, very uncommon in lung and renal transplantation, and moderately uncommon in cardiac transplantation, although rates vary as it is rarely screened for outside of renal transplantation. The presence of DSA does not in itself indicate antibody-​mediated rejection, and many scoring systems have been used to define histological indicators of ABMR. In the Banff renal histology scoring system, positive staining for complement component 4d (C4d) in biopsies allows indirect iden- tification of antibody deposition and complement fixation, and peritubular C4d staining is strongly associated with early as well as late graft failure. C4d staining is also used in lung and cardiac transplantation as an indicator of ABMR, but there is no clear con- sensus on its use.

4.7  Principles of transplantation immunology 401 More recently, evidence has emerged that the presence of DSAs, even without histological evidence of rejection, is associated with late graft loss, and it is becoming clear that antibodies may play a more complex role than originally thought, with suggestion that the subclass of and ability of antibodies to fix complement may indicate how pathological they are, but this is an emerging field and much needs to be elucidated. As with T cells, evidence has emerged that suggests B cells can also have a regulatory role. These cells have been identified in both murine models and human peripheral blood and shown to have the capacity to suppress immune responses. However, regulatory B cells are less well characterized than regulatory T cells (see Table 4.7.5) and the mechanisms underlying their suppressive action are yet to be fully elucidated. Immunological memory in transplantation As just described, the generation of memory is a key step in the re- sponse to non-​self-​antigen. Following primary antigen exposure, long-​lived antigen-​specific memory T and B cells are generated which are able to deliver a larger and more rapid immune response if the same antigen is encountered on a subsequent occasion. Memory lymphocytes have a reduced activation threshold and are less de- pendent on costimulation. As a result they are able to upregulate effector function and cytokine secretion more rapidly than naïve lymphocytes. With increasing age, the proportion of memory T cells within an individual’s peripheral T-​cell pool increases, reflecting cu- mulative antigen exposure, and can be as high as 50% in adult hu- mans. While the generation of immunological memory is beneficial for protection against infectious pathogens, in transplantation the presence of allospecific memory produces an accelerated or ‘second-​ set’ rejection response. In clinical transplantation, evidence of prior sensitization to donor antigens is associated with increased risk of acute rejection episodes and premature graft failure. Memory-​type responses towards alloantigens are frequently a result of exposure to alloantigens at the time of a previous blood transfusion, pregnancy, or transplantation. However, it is now rec- ognized that memory-​type responses may also be generated as a consequence of antigen receptor cross-​reactivity (heterologous im- munity) or by homeostatic proliferation of lymphocytes following an episode of lymphopenia. Sensitization Patients on the transplant waiting list have their HLA genotype charac- terized and are regularly monitored for the development of anti-​HLA antibodies as a marker of sensitization to potential donor antigens. This was previously carried out using a ‘panel’ of random donors with known HLA types and testing the serum of potential recipients to see what proportion of donor lymphocytes was lysed. This would give the percentage of panel reactive antibodies, or PRA. In practice, this was a crude measure of reactivity as it depended on large numbers of donors or immortalized cell lines, was work intensive, and only de- tected complement-​fixing antibodies. In most centres it has been re- placed with solid phase assays, which use purified HLA antigens to detect antibodies, whether complement fixing or not, in the serum of recipients. These assays are extremely sensitive, but also give an idea of antibody levels, allowing thresholds to be set for low, medium, and high-​risk transplants, depending on the amount of antibody present, thus allowing patients with extremely high (i.e. broad) sensitization to receive transplants, where previous PRA methodology would limit their options. Sensitization is now given as either a calculated PRA or a calculated reaction frequency, both indicating the proportion of the population to which a patient is likely to react. Many events can lead to sensitization. In most cases antibodies develop in response to previous transplants, paternal antigens in multiparous women, and in patients who have received blood product transfusions. The risk of sensitization from blood product transfusion is highly variable, although overall sensitization rates are low. No obvious sensitizing event can be identified in some cases and it has been proposed that heterologous immunity or homeostatic proliferation after lymphopenia may contribute to memory cell for- mation and hence antibody production. Heterologous immunity Experiments in mice have found that sequential viral infections can generate populations of alloreactive memory-​phenotype T cells, possibly through epitope sharing or TCR cross-​reactivity, and the presence of these cells prevented transplant tolerance induction by protocols that usually result in reliable long-​term graft acceptance. It is therefore possible that memory lymphocytes generated by ante- cedent viral infections in humans may be able to cross-​react with epitopes presented following transplantation, resulting in memory-​ phenotype responses towards the graft without prior sensitization to donor alloantigen. Homeostatic proliferation The size of the peripheral T-​cell pool, as well as the relative ratios of CD4+ to CD8+ and naive to memory cells, are tightly regulated by homeostatic mechanisms. A consequence of this is that reduction of the overall T-​cell population, either during illness or following med- ical intervention, strongly induces the residual T cells to proliferate, whether or not cognate antigen is present. A proportion of T cells that have undergone homeostatic proliferation exhibit memory T-​ cell-​like phenotype and behaviour. Homeostatic proliferation there- fore presents a particular concern in transplantation both in terms of Table 4.7.5  Characteristics of regulatory T and B cells (Tregs and Bregs) Tregs Bregs Surface markers (not exhaustive) CD4+, CD25hi, IL-​7Rlo, ICOS+, CD152+ (CTLA-​4) CD19+, CD24hi, CD38hi, CD5+, CD1d+, CD27+, IgD+, IgM+ Transcription factor Foxp3 Unknown Cytokines produced TGF​β, IL-​10 IL-​10 Mechanism of action Contact dependent inhibition of T-​cell activation via inhibitory receptors and cytokine dependent suppression Unknown—​possibly via IL-​10, possibly via induction of Tregs

402 SECTION 4  Immunological mechanisms pretransplant sensitization, but also as many patients receive mono- clonal or polyclonal antibody therapy designed to deplete leucocytes and/​or T cells either as part of induction immunosuppression or as therapy for an acute rejection episode. Since as many as 10% of naive T cells are able to respond to alloantigen, it is likely that homeo- static expansion in this setting could generate alloreactive memory-​ phenotype T cells. Cross-​matching In all except liver transplants, a ‘cross-​match’ of recipient reactivity towards donor is carried out to exclude the presence of complement-​ fixing and binding antibodies directed against donor HLA. This is normally done immediately prior to the transplant, with labora- tory tests including the complement-​dependent cytotoxicity (CDC) assay and/​or flow cytometric cross-​matching. This combination al- lows detection of complement-​fixing alloantibodies which would cause hyperacute rejection (where the CDC is positive) as well as non-​complement fixing or lower level antibodies that may cause problems in future with acute or chronic rejection (CDC negative, flow positive cross-​match). In patients with no antibodies, or long-​standing and well-​ characterized HLA-​specific antibodies, current guidelines suggest a ‘virtual’ cross-​match is possible. In the virtual cross-​match the donor HLA genotype is compared with the potential recipient’s HLA genotype and any known antibodies to ensure that the donor genotype is suitably matched and avoids any known antibodies. In practice, most transplants proceed after a pre-​emptive laboratory cross-​match, and in those where a virtual cross-​match is performed, a laboratory cross-​match or solid phase assay will be carried out within 24–​48 h to confirm the lack of reactivity (see Fig. 4.7.6). Patients who have become highly sensitized to HLA antigens have a reduced chance of transplantation and may remain on the transplant waiting list for a long time. Patients with renal failure who have a living kidney donor can sometimes be successfully transplanted following a programme of ‘desensitization’. Typically this involves a strategy of removing existing alloantibodies by plasma exchange or immunoadsorption. However, in isolation this is insufficient to prevent antibody rebound, and additional strategies include the use of IVIg, and reducing alloantibody pro- duction by the administration of B-​cell depleting agents (such as rituximab) in combination with other immunosuppressive agents (see ‘Immunosuppression in transplantation’, next). There is no clear consensus on the best therapeutic strategy at present. Similar strategies have also been employed to allow transplantation be- tween ABO-​incompatible donor–​recipient pairs (Table 4.7.6). However, desensitization requires profound immunosuppression, which cannot be maintained for long periods without deleterious effects on the recipient. As deceased donor transplants are unpre- dictable in terms of timing, these approaches are currently only applicable to live-​donor recipients. Immunosuppression in transplantation The dramatic improvements in allograft survival that have occurred since the first successful transplants were performed in the 1950s have been achieved by the development of potent immunosuppres- sant medications able to inhibit the aggressive immune response to the transplant. These agents work by depleting lymphocytes, interfering with lymphocyte signal transduction pathways, and/​or altering lymphocyte trafficking. The risk of acute graft rejection is greatest during the initial 3 months after transplantation and therefore most solid organ trans- plant recipients initially receive strong immunosuppression, often consisting of an induction agent alongside triple therapy with gluco- corticoids, a calcineurin inhibitor (CNI), and an antiproliferative agent. Provided there are no episodes of acute rejection, the doses of these agents are gradually reduced and then maintenance immuno- suppression is continued indefinitely. Liver transplant patients tend to have a much lower risk of rejection compared to other solid organ transplants and therefore generally receive low-​dose CNI with an antiproliferative agent. Kidney-​pancreas patients tend to be given a steroid-​sparing regimen, and—​given their plethora of side effects—​ there is a move towards reduction or cessation of glucocorticoids in most patients where feasible. Chronic immunosuppression is associated with several undesir- able sequelae, in particular an increased relative risk of infections and malignancy. Recognized side effects of particular immunosup- pressive agents are listed in Table 4.7.7. Many different immunosuppressive regimens for solid organ transplantation are in clinical use, and a detailed discussion of these is beyond the scope of this chapter. What follows is an introduction to the immunosuppressive agents in widespread clinical use cur- rently (Fig. 4.7.7). Glucocorticoids Corticosteroids were developed in the 1950s and have complex immunosuppressive as well as anti-​inflammatory effects. They act principally by binding to cytoplasmic glucocorticoid receptors, although at higher doses they can exhibit receptor-​independent effects as well. The steroid–​receptor complex translocates to the nucleus where it is able to alter the expression of multiple cyto- kines through DNA-​binding and by targeting transcription factors such as AP-​1 and NF-​κB. Corticosteroids reduce the expression of many molecules important in the immune response, including interleukins 1, 2, 3, and 6, TNFα, interferon-​γ and chemokines. By inhibiting cyclooxygenase, corticosteroids are also able to reduce the production of inflammatory mediators such as leukotrienes and prostaglandins. Glucocorticoids are used as induction and main- tenance immunosuppression, as well as the first line of therapy for rejection episodes. Antiproliferative agents Azathioprine and mycophenolate mofetil interfere with DNA syn- thesis and prevent cell-​cycle progression. In the context of allograft rejection this impairs the clonal expansion of alloreactive T cells. The introduction of azathioprine into clinical practice in the 1960s and its use in conjunction with corticosteroids allowed transplantation to progress from an experimental procedure into a practical therapy for patients with organ failure. It is widely metabolized (mostly in erythrocytes and the liver) to the purine analogue 6-​mercaptopurine and incorporated into DNA. By inhibiting purine nucleotide synthesis (and therefore DNA and RNA synthesis), azathioprine reduces gene transcription and

Donor cells Donor cells Recipient serum with donor-specific antibodies Antihuman globulin (AHG) Complement Complement binding and cell death Recipient serum with donor-specific antibodies Fluorescently tagged AHG Stained with T- and B-cell markers Negative CDC Positive CDC Flow cytometry- positive peak in red (a) (b) (c) Luminex: Serum is added to a cocktail of microbeads, each coated with a purified HLA antigen and with a different fluorescence ELISA: Serum is added to a plate of purified HLA antigens, then a secondary antibody is added to allow measurement of levels Solid assays phase Fig. 4.7.6  Methods used in cross-​matching. (a) CDC cross-​match. Recipient serum is added to donor leucocytes: if there are donor-​specific antibodies (DSA) present, these will bind to the cells. An antihuman globulin is then added to enhance the effects of complement. Complement is added and, if antibodies are bound, the classical pathway is activated and the cells are lysed. (b) Flow cross-​match. This is similar to the CDC except the antihuman globulin is labelled with a fluorescent tag, which can be detected by a flow cytometry machine. Additional fluorescently labelled antibodies are added to divide the leucocytes into B cells (anti-​CD19) and T cells (anti-​CD3). Positive fluorescence is seen on the flow cytometer. (c) Solid phase assays. These are not routinely used in the immediate pretransplant cross-​match but are used regularly to assess sensitization to HLA antigens. The antigens are purified and can be coated on a plate where individual wells contain different antigens, and antibody binding is then measured using enzyme-​linked immunosorbent assay (ELISA). Alternatively, and more sensitively, the HLA antigens can be used to coat microbeads, each with a slightly different fluorescence, and antibody binding of these can then be measured using a Luminex machine. Both give read outs of the antibodies in a recipient’s serum, but they cannot say whether it is complement fixing or not, and hence cannot accurately predict whether an antibody is likely to cause rejection. In general, the higher the level of antibody, the more likely it is to cause a problem, and each laboratory will have a level that they consider to be high risk, intermediate risk, and low risk. Luminex results reproduced with the kind permission of the Transplant Immunology Laboratory, Churchill Hospital, Oxford.

404 SECTION 4  Immunological mechanisms prevents cell-​cycle progression. The effects of azathioprine are not lymphocyte-​specific and patients must be monitored closely for bone marrow suppression. Mycophenolate mofetil is metabolized in the liver to mycophenolic acid, which is a non​competitive, reversible inhibitor of inosine monophosphate dehydrogenase (IMPDH). Cells are able to gen- erate purines either de novo by converting inosine monophosphate to guanosine monophosphate (catalysed by IMPDH), or from guanine via the salvage pathway. The salvage pathway is less active in lymphocytes and therefore they are relatively dependent on the de-​ novo pathway of purine synthesis compared to other cell types. As a result, the effects of mycophenolate are more lymphocyte-​specific than azathioprine, and it is less myelosuppressive although it is com- monly associated with diarrhoea, which can be severe. Calcineurin inhibitors The introduction of ciclosporin in the early 1980s was a great step forward in transplant immunosuppression as this was the first drug able to selectively block T-​cell activation. Subsequently a second calcineurin inhibitor, the macrolide antibiotic tacrolimus, was de- veloped. Both drugs bind cytoplasmic immunophilins (cyclophilin in the case of ciclosporin and FK506-​binding protein 12 in the case of tacrolimus) to form complexes that can inhibit the calcium-​ dependent phosphatase calcineurin, a rate-​limiting enzyme in the T-​cell receptor signal transduction pathway. By preventing trans- location of the transcription factor NFAT to the nucleus, calcineurin inhibition impairs upregulation of many molecules important for T-​cell proliferation and the generation of an effective immune re- sponse, including the cytokines IL-​2, IL-​4, TNFα, and interferon-​γ, and costimulatory molecules such as CD154 (CD40L). In practice, ciclosporin is gradually being phased out in favour of tacrolimus, which is more immunosuppressive with fewer side effects. mTOR inhibitors Sirolimus and everolimus bind to the same immunophilin as tacrolimus (FKBP12), although the complexes they form are un- able to interact with calcineurin. Instead they bind to the regulatory kinase mammalian target of rapamycin (mTOR), which has a critical role in cytokine receptor signal transduction. The usual actions of mTOR are to activate the ribosomal enzyme p70 S6 kinase and block an inhibitory protein 4E-​BP1, both of which are required for trans- lation of proteins necessary for progression from the G1 (growth) phase to the S (DNA synthesis) phase of the cell cycle. Inhibition of this pathway (‘signal 3’) in T cells therefore blocks the action of cyto- kines such as IL-​2, IL-​4, and IL-​15, preventing cell-​cycle progression and clonal expansion. Depleting antibodies Polyclonal antithymocyte globulin (ATG) is produced by im- munizing either rabbits or horses with human lymphocytes. Immunoglobulins directed against lymphocyte epitopes can then be harvested from the animal’s serum for clinical use. ATG causes profound lymphocyte depletion that takes many months to recover, and is highly effective as an induction agent and in the treatment of steroid-​resistant rejection. CAMPATH-​1H or alemtuzumab is a humanized monoclonal anti- body directed against human CD52, a surface molecule that is ex- pressed by most nucleated bone-​marrow-​derived cells, including T and B cells, monocytes, macrophages, and eosinophils. Administration of alemtuzumab causes profound lymphopenia that recovers over months to years, although patients can remain CD4 lymphopenic for many years. It is used routinely in induction for simultaneous kidney-​ pancreas transplant recipients as part of a steroid-​sparing regimen, and widely used for high immunological risk transplants as an alter- native induction agent to basiliximab (discussed next). It can also be used for the treatment of steroid-​resistant rejection Administration of either ATG or alemtuzumab can result in the massive release of cytokines caused by the initial activation of lympho- cytes prior to their depletion. Clinical manifestations of this range from fever and flu-​like symptoms to a potentially fatal severe systemic inflammatory response syndrome characterized by hypotension, rigors, and pulmonary oedema. This is more common with ATG. Rituximab is a monoclonal antibody licensed for use in B-​cell lymphoma and rheumatoid arthritis. It is directed against CD20, which Table 4.7.6  ABO compatibility for transplantation Blood group Can receive transplant from: Can donate transplant to: O O O, A, B, AB A O, A A, AB B O, B B, AB AB O, A, B, AB AB Table 4.7.7  Principal side effects of immunosuppressive agents Corticosteroids Glucose intolerance, osteoporosis, weight gain, hyperlipidaemia, cosmetic changes Ciclosporin Nephrotoxicity, hypertension, glucose intolerance, gingival hyperplasia, hyperlipidaemia, hirsutism, neurological sequelae (including tremor) Tacrolimus Nephrotoxicity, hypertension, new-​onset diabetes after transplantation (NODAT), alopecia, neurological sequelae (including tremor) Azathioprine Bone marrow suppression, macrocytosis, hepatotoxicity Mycophenolate Diarrhoea, bone marrow suppression Sirolimus Hyperlipidaemia, thrombocytopenia, mouth ulcers, interstitial lung disease Anti-​CD25 (IL-​2 receptor α-​chain) antibodies Occasional hypersensitivity reactions ATG Cytokine release syndrome (fever, influenza-​like symptoms, hypotension), leukopenia, thrombocytopenia, serum sickness Alemtuzumab Cytokine release syndrome, cytopenias, infections

4.7  Principles of transplantation immunology 405 is expressed on most B cells (except plasma cells). In transplantation practice rituximab is rarely used outside of desensitization protocols and occasionally in ABMR. Attempts to use it for induction therapy had to be abandoned due to a high incidence of acute cellular rejection. Other biological agents Basiliximab is a monoclonal antibody directed against CD25 (the high-​ affinity IL-​2 receptor α-​chain). The use of basiliximab as induction therapy was shown to reduce 6-​month acute rejection rates by one-​ third, with minimal side effects, and it is now in widespread use as part of induction immunosuppression for many solid organ transplants. Another biological agent now in routine clinical use is belatacept (LEA29Y). This is a fusion protein consisting of the extracellular domain of CD152 (CTLA-​4, cytotoxic-​T-​lymphocyte-​associated antigen 4)  combined with the Fc (non-​antigen-​binding) portion of IgG. As discussed earlier, CTLA-​4 has a higher affinity for the costimulatory molecules CD80 and CD86 than does CD28, and belatacept is therefore able to block CD28:CD80/​86 costimulation, thus increasing the T-​cell activation threshold. Belatacept is used as maintenance therapy in cases where patients are intolerant of CNIs and are not able to take mTOR inhibitors, and has shown non​inferiority to ciclosporin when combined with mycophenolate mofetil and steroids, although is yet to be directly compared to tacrolimus. Ongoing clinical trials and future possibilities in transplantation Advances in the management of transplant recipients have been accompanied by both improved 1-​year graft survival and reduced acute rejection rates, offering many patients with organ failure great improvements in both morbidity and mortality. However, these ad- vances still rely on the continuous administration of potent non-​ specific immunosuppressive medication, which is associated with increased risk of infection and malignancy as well as drug toxicity. The improvement in short-​ to medium-​term outcomes has un- fortunately not been followed by comparable improvement in long-​ term graft survival, with considerable late attrition of grafts resulting from the complex interaction of drug toxicity and chronic immune activation. A further major problem facing patients on transplant waiting lists is organ availability, with average waiting list time stable or increasing for most organs in the United Kingdom at present, and rates of cadaveric donation remain insufficient to meet demand. There is therefore considerable interest in strategies that might allow reduced exposure to immunosuppression medication with improved long-​term outcomes (which in itself would decrease the pressure on scarce organ resources), as well as improvement in organ quality, allowing more offered organs to be utilized. While there are many therapies that are not currently in routine use, the past few years have seen a plethora of new drugs and ther- apies enter into clinical trials. Some therapies that were considered purely experimental are now in early trials, and the targets for therapy are being extended. Treatments aimed at immunosuppres- sion reduction (e.g. regulatory cell therapy), new drugs targeting the innate immune system (e.g. complement and TLRs) and ther- apies aimed at improving the quality of donor organs are all under- going trials. There are still many hurdles to overcome, which will be discussed next. Adjunctive cell therapies Perhaps the most exciting development in recent years is the moving of regulatory cell therapy from an experimental technique into early clinical trials. Trials have begun in several centres across the United States and Europe using regulatory T cells as well as regulatory macrophages and dendritic cells. Currently these phase I/​IIa trials in living donor kidney transplants are aimed at establishing safety, not efficacy, but it is hoped that future studies will allow assessment of efficacy and reduction of immunosuppression. The central concept behind the use of regulatory cell therapy is the fact that regulatory cells are able to switch immune responses off and convert the response to an antigen from immunogenic to tol- erant. The hope with infusion of regulatory cells is that the immune system can be ‘persuaded’ to see the transplant as non​threatening by inhibiting the proliferation of T cells in SLOs (Treg) or by rendering alloantigen-​specific cells anergic (Mreg or tolerogenic DCs). Interest has also grown in the idea of using regulatory B cells, a relatively newly described subset of B cells, which may work by dir- ectly suppressing responses, or by inducing Tregs. Present work is entirely experimental, but cell therapy appears to be a promising av- enue towards the ultimate goal, which is transplant tolerance. Transplantation tolerance In healthy individuals, autoimmunity is avoided by specific mech- anisms that maintain immunological tolerance to self-​antigens (Table 4.7.8). The optimal outcome for patients after transplant- ation would be to harness these mechanisms to induce specific tol- erance to the graft. Transplantation tolerance can be defined as the Upregulation of gene transcription Calcineurin Antigen presenting cell NFAT NF -κB MAP kinases AP-1 Protein kinase C Nucleus mTOR Signal 1 Signal 2 Signal 3 IL-2 production IL-2 CD4 Costimulatory molecules IL-2 receptor T cell Cell cycle progression MHC molecule + peptide Upregulation of gene transcription Basiliximab Sirolimus Belatacept CD52 Alemtuzumab CD52 CNI Azathioprine Mycophenolate Corticosteroids Fig. 4.7.7  Molecular targets of common immunosuppressive drugs.

406 SECTION 4  Immunological mechanisms lack of a destructive immune response towards the graft without the requirement for indefinite non​specific immunosuppressive therapy, while preserving immune responses to pathogens. The science of immunological tolerance and the technology of transplantation have evolved hand in hand. The first description of acquired tolerance to foreign antigen in mice by Billingham, Brent, and Medawar in 1953 (for which Medawar was awarded the Nobel Prize for medicine in 1960)  actually preceded the first successful renal transplant (between identical twins) by Murray and colleagues in 1955. Billingham, Brent, and Medawar demonstrated that it was possible to acquire immunological tolerance to foreign antigen, and much effort has gone into defining strategies that would lead to toler- ance to alloantigens in transplantation. Many successful experimental techniques can produce durable hyporesponsiveness to mismatched allografts in rodent models, but so far few of these have been success- fully translated into large animal models or clinical trials. Progress in the field of transplantation tolerance is also hampered by the lack of definitive laboratory parameters able to give a clear indication of whether a particular recipient is tolerant of their graft. Of the proposed strategies for inducing transplant tolerance, few have been adopted into clinical practice. Regulatory cell therapy re- mains the most promising at present, although other strategies have been trialled on a smaller scale. Peripheral depletion Therapies that deplete circulating leucocytes, and in particular T cells, have been used successfully both as induction immunosup- pression and in the treatment of acute allograft rejection episodes for many years. The efficacy of this strategy in preventing allograft rejec- tion led to the hypothesis that profound lymphocyte depletion at the time of transplantation may allow the development of donor-​specific immunological hyporesponsiveness. Trials have been undertaken using peripheral T-​cell depletion with several poly and monoclonal antibodies, the most widely studied of which is alemtuzumab. Initial reports are excellent and show efficacy of alemtuzumab with low rates of early rejection. However, there is counter evidence that sug- gests the homeostatic proliferation of cells following therapeutic lymphopenia may paradoxically increase the response to the trans- plant. Patients who were given alemtuzumab with no or little main- tenance therapy have a higher incidence of late rejection, and there is some evidence to suggest that those given alemtuzumab induction are at higher risk of DSA development in the longer term, even when given maintenance triple therapy, hence this strategy does not seem likely to induce transplant tolerance. Macro-​ and mixed chimerism The most robust experimental strategies for the induction of toler- ance to foreign antigen utilize the mechanisms of central deletion to eliminate T-​cell clones with specificity for the foreign antigens in question, thereby preventing them from entering the periphery. This can be reliably achieved by the establishment of haematopoietic chimerism through bone marrow transplantation. Stable engraft- ment of donor haemopoietic stem cells results in repopulation of the recipient thymus with donor-​type thymic dendritic cells, with the result that developing T cells with antidonor specificity are deleted by negative selection. In experimental models, full donor chimerism induced by myeloablative therapy (frequently a combination of total body irradiation and cytotoxic medication) followed by donor bone marrow transplantation produces tolerance to a subsequent allo- graft from an identical donor. There are several patients who have undergone successful bone marrow transplantation for haem- atological indications and have subsequently been successfully transplanted with a kidney from the same donor, without the re- quirement for increased immunosuppression. However, the es- tablishment of full donor chimerism is not acceptable for most recipients on the transplant waiting list as the risk profile (i.e. the toxicity and mortality associated with myeloablative condi- tioning regimens and the high incidence of graft-​vs.-​host disease in patients), is not acceptable when compared to the alternative (i.e. lifelong immunosuppression with the agents just described). However, this may be an option for young patients who are highly sensitized and who may not otherwise be suitable for transplant- ation, for whom the risks of long-​term dialysis may be more sig- nificant than those associated with myeloablative conditioning. Data from North America in 12 patients treated with this combin- ation therapy has shown that 7 of these are immunosuppression free by 4 years post-​transplant, so this seems a promising option for those with limited alternatives. Mixed chimerism may be a less toxic alternative, where recipi- ents receive donor stem cell infusion aimed at transient mixed chimerism, rather than full donor chimerism, just around the trans- plant period. The aim is to transiently fool the immune system, al- lowing a tolerogenic rather than pathogenic response to be initiated towards the graft, which is then maintained indefinitely. Mixed chimerism regimens are less toxic than full chimerism regimens and generally involve costimulatory blockade or non​myeloablative treat- ment such as cyclophosphamide with high dose of donor stem cells. This approach is still experimental in nature. Targeting of the innate immune system As interest grows in the role of the innate immune system in trans- plantation, new therapeutics are being trialled. As previously dis- cussed, there is renewed interest in the role of complement in transplantation, in particular with regards to the damage caused by ischaemia-​reperfusion injury. Eculizumab, a C5a inhibitor, is Table 4.7.8  Mechanisms of immunological tolerance Deletion Deletion of autoreactive T cells occurs in the thymus during T-​cell development (central deletion). Peripheral deletion of T cells can also occur if antigen is encountered under suboptimal condition (such as the absence or blockade of costimulatory signals) Anergy Anergy is the functional inactivation of T cells following antigen encounter and another possible outcome of antigen encounter under suboptimal conditions Immunoregulation Several populations of lymphocytes are able to suppress or regulate the ability of other lymphocytes to respond to a particular antigen Ignorance T cells do not have access to antigens present at certain sites of immune privilege (such as the anterior chamber of the eye)

4.7  Principles of transplantation immunology 407 used with success in kidney transplantation in patients with atyp- ical haemolytic uraemic syndrome (aHUS) to prevent immediate disease recurrence, and it has also been used in cases of severe antibody-​mediated rejection, particularly in patients who have undergone desensitization. There is potential for using eculizumab in deceased donor transplants, where complement components are known to be upregulated. Studies are in progress to investigate the possibility of blocking Toll-​like receptors, and a phase II study of a monoclonal antibody to TLR2 in extended-​donor criteria organs. The hope is that blocking receptors to DAMPs may prevent activation of phagocytes and hence reduce responses to inflammatory stimuli and it is likely that other therapeutics will be introduced in the coming years. Strategies to improve donor organs While many trials of therapeutics are limited to recipients of living kidney donors, particularly regulatory cell therapies which must be prepared in advance, most kidney donors are deceased, and outcomes for deceased donors are worse than for living donors. Furthermore, some solid organs have no possibility of being do- nated by living donors. Strategies are therefore required to increase the donor pool, both by sociological intervention and encouraging donation, but also by extending the criteria for accepting organs and improving organs retrieved from deceased donors. For many years, the gold standard of retrieval was perfusion of the organ with ice cold fluid at the time of organ harvesting. However, it is known that prolonged ischaemia leads to anaerobic metabolism and production of damaging and proinflammatory molecules on reperfusion. Trials into the use of ex-​vivo, hypothermic machine perfusion in kidney transplantation, and ex-​vivo normothermic machine perfusion in liver transplantation are ongoing, with prom- ising early results. In addition to potentially improving outcomes by reducing ischaemic time, the ex-​vivo perfusion allows surgical teams to assess function of the potential transplant, looking at bile and urine production, which may allow assessment of the potential for delayed graft function and guide decisions about which organs are suitable for implantation (see Fig. 4.7.8). Xenotransplantation Since the 1960s attempts have been made to use animals as an alter- native source of organs for transplantation. Initial attempts to trans- plant kidneys from non​human primates to humans resulted in renal function for weeks to months, although non​human primates are too scarce to be a realistic long-​term source of organs. Instead, most re- search has focused on other mammals, and in particular pigs, which can be raised in sufficient numbers in pathogen-​free facilities, can be genetically engineered, and have organs that are a suitable size. However, there remain significant practical as well as ethical hur- dles to overcome before clinical xenotransplantation becomes a possibility. The practical considerations include the robust immune re- sponse to xenografts. Although genetic engineering has been employed to remove Galα1, 3Gal, a carbohydrate determinant ex- pressed by cells of pigs and other lower mammals that commonly causes hyperacute rejection, other molecules have emerged to cause problems with acute rejection and further genetic manipu- lation is under investigation. There also remains a risk of viral transmission, which is theoretical but could present a significant public health problem. Xenografts are used for heart valve replacement, but no other xenografts are in routine clinical use or trials outside of non​human primates. Bioengineering At present a very theoretical possibility, the interest in bioengineered grafts is very considerable. The theory is that organs could be grown using the intended recipient’s own stem cells, seeded onto an organ scaffold (usually a decellularized donated organ, harvested up to 6 hours after death) and then implanted. This would allow transplant- ation of an entirely self-​derived organ, with no risk of rejection and thus no requirement for immunosuppression. A few case studies have given proof of concept, but they are currently limited to simple, avascular tissues, such as tracheas, bladders, and blood vessels, which rely on simple diffusion for oxy- genation and nutrients. Bioengineering of more complex grafts has been attempted, and some success has been achieved in producing animal liver, kidney, pancreas, and intestinal grafts, but implant- ation of these has had limited success. One major concern is the malignant potential of the seeder cells, as the reprogramming of stem cells can cause genetic alterations with the potential to be- come malignant. It is likely to be many years before bioengineered organs become a reality for human transplantation. However, with the shortage of donor organs, the increased lifespan of transplant recipients leading to problems with immunosuppressive burden and the limited pro- gress in the field of xenotransplantation, the possibility of bioengin- eering remains attractive. Fig. 4.7.8  Ex-​vivo liver perfusion. The OrganOx machine was designed by transplant surgeon Professor Peter Friend and biomedical engineer Professor Constantin Coussios as a way of supporting the retrieved liver prior to implantation. The liver is perfused with oxygenated blood at 37oC and supported by infusions of nutrients, bile salts, prostacyclin, heparin, and insulin. Sensors allow assessment of haemodynamic, metabolic, and synthetic liver performance during preservation. The machine is intended to allow assessment of liver function, longer preservation times, and use of extended criteria donors. Image and information reproduced with the kind permission of Professor Peter Friend.

408 SECTION 4  Immunological mechanisms FURTHER READING Abecassis M, Kaplan B (2015). Transplantation:  biomarkers in transplantation—​the devil is in the detail. Nat Rev Nephrol, 11, 204–​5. Batista FD, Harwood NE (2009). The who, how and where of antigen presentation to B cells. Nat Rev Immunol, 9, 15–​27. Benichou G, et al. (1992). Donor major histocompatibility complex (MHC) peptides are presented by recipient MHC molecules during graft rejection. J Exp Med, 175, 305–​8. Billingham RE, Brent L, Medawar PB (1953). Actively acquired toler- ance of foreign cells. Nature, 172, 603–​6. Carney EF (2015). Transplantation: induction of chimerism and im- mune tolerance using belatacept. Nat Rev Nephrol, 11, 66. Clatworthy MR (2013). B cell responses to allograft—​more common than we thought? Am J Transplant, 13, 1629–​30. Clatworthy MR (2014). B-​cell regulation and its application to trans- plantation. Transpl Int, 27, 117–​28. Crotty S (2015). A brief history of T cell help to B cells. Nat Rev Immunol, 15, 185–​9. Edwards JK (2015). Immunology: clonal deletion contributes to allo- graft tolerance. Nat Rev Nephrol, 11, 196. Feng G, et al. (2009). Donor reactive regulatory T cells. Curr Opin Organ Transplant, 14, 432–​8. Gandhi AM, et al. (2008). Costimulation targeting therapies in organ transplantation. Curr Opin Organ Transplant, 13, 622–​6. Gasteiger G, Rudensky AY (2014). Interactions between innate and adaptive lymphocytes. Nat Rev Immunol, 14, 631–​9. Girmanova E, Hruba P, Viklicky O (2015). Circulating biomarkers of tolerance. Transplant Rev (Orlando), 29, 68–​72. Gosset C, Lefaucheur C, Glotz D (2014). New insights in antibody-​ mediated rejection. Curr Opin Nephrol Hypertens, 23, 597–​604. Grakoui A, et al. (1999). The immunological synapse: a molecular ma- chine controlling T cell activation. Science, 285, 221–​7. Hu Q, Liu Z, Zhu H (2014). Pig islets for islet xenotransplantation: cur- rent status and future perspectives. Chin Med J (Engl), 127, 370–​7. Juvet SC, et al. (2014). Harnessing regulatory T cells for clinical use in transplantation: the end of the beginning. Am J Transplant, 14, 750–​63. Kawai T, et al. (2018). Summary of the third international workforce on clinical tolerance. Am J Transplant, doi: 10.1111/​ajt.15086. Kinnear G, Jones ND, Wood KJ (2013). Costimulation blockade: current perspectives and implications for therapy. Transplantation, 95, 527–​35. Kolev M, Le Friec G, Kemper C (2014). Complement—​tapping into new sites and effector systems. Nat Rev Immunol, 14, 811–​20. Kugelberg E (2014). Neutrophils:  bugging transplantation. Nat Rev Immunol, 14, 430–​1. Land WG (2013). Chronic allograft dysfunction: a model disorder of innate immunity. Biomed J, 36, 209–​28. McDonald-​Hyman C, Turka LA, Blazar BR (2015). Advances and challenges in immunotherapy for solid organ and hematopoietic stem cell transplantation. Sci Transl Med, 7, 280rv282. Meier-​Kriesche HU, et al. (2004). Lack of improvement in renal allo- graft survival despite a marked decrease in acute rejection rates over the most recent era. Am J Transplant, 4, 378–​83. Mella A, et al. (2014). Complement cascade and kidney transplant- ation: the rediscovery of an ancient enemy. World J Transplant, 4, 168–​75. Messner F, Etra JW, Dodd-O JM, Brandacher G (2019). Chimerism, transplant tolerance and beyond. Transplantation, doi: 10.1097/ TP.0000000000002711. Michel SG, et al. (2015). Current progress in xenotransplantation and organ bioengineering. Int J Surg, 13, 239–​44. Montgomery RA, et  al. (2018). HLA in transplantation. Nat Rev Nephrol, 14, 558–​70. Nasralla D, et al. (2018). A randomized trial of normothermic preserva- tion in liver transplantation. Nature, 557(7703), 50–6. Nayak DK, Mohanakumar T (2014). Transplantation:  recognizing self versus non-​self: new territory for monocytes. Nat Rev Nephrol, 10, 548–​9. Oberbarnscheidt MH, Lakkis FG (2014). Innate allorecognition. Immunol Rev, 258, 145–​9. Ochando J, Ordikhani F, Boros P, Jordan S (2019). The innate immune response to allotransplants: mechanisms and therapeutic potentials. Cell Mol Immunol, 16(4), 350–6. Pratschke J, Stauch D, Kotsch K (2009). Role of NK and NKT cells in solid organ transplantation. Transpl Int, 22, 859–​68. Quezada SA, et al. (2004). CD40/​CD154 interactions at the interface of tolerance and immunity. Annu Rev Immunol, 22, 307–​28. Reddy UG, Danovitch GM (2014). Transplantation. T-​cell depletion—​ balancing acute rejection and cancer risk. Nat Rev Nephrol, 10, 301–​2. Resch T, et al. (2015). The role of natural killer cells in humoral rejec- tion. Transplantation, 99, 1335–​40. Rickert CG, Markmann JF (2019). Current state of organ trans- plant tolerance. Curr Opin Organ Transplant, doi: 10.1097/MOT. 0000000000000670. Sachs DH, Kawai T, Sykes M (2014). Induction of tolerance through mixed chimerism. Cold Spring Harb Perspect Med, 4, a015529. Saidi RF, Hejazii Kenari SK (2014). Clinical transplantation and toler- ance: are we there yet? Int J Organ Transplant Med, 5, 137–​45. Schinstock CA, Stegall M, Cosio F (2014). New insights regarding chronic antibody-​mediated rejection and its progression to trans- plant glomerulopathy. Curr Opin Nephrol Hypertens, 23, 611–​8. Schroder PM, et al. (2019). The past, present, and future of costimulation blockade in organ transplantation. Curr Opin Organ Transplant, doi: 10.1097/MOT.0000000000000656. Stolp J, Turka LA, Wood KJ (2014). B cells with immune-​regulating function in transplantation. Nat Rev Nephrol, 10, 389–​97. Thomson AW (2018). A view of the future or regulatory immune cell therapy in organ transplantation. Curr Opin Organ Transplant, 23, 507–​8. Turka LA, Lechler RI (2009). Towards the identification of biomarkers of transplantation tolerance. Nat Rev Immunol, 9, 521–​6. Willis JC, Lord GM (2015). Immune biomarkers: the promises and pit- falls of personalized medicine. Nat Rev Immunol, 15, 323–​9. Wood KJ, et al. (2014). Bridging innate with adaptive immunity in transplantation: triggers, sensors, and modulators. Transplantation, 98, 1021–​4. Yolcu ES, Leventhal JR, Ildstad ST (2015). Facilitating cells in tolerance induction for kidney transplantation. Curr Opin Organ Transplant, 20, 57–​63. Zachary AA, Leffell MS (2014). Desensitization for solid organ and hematopoietic stem cell transplantation. Immunol Rev, 258, 183–​207. Zhuang Q, Lakkis FG (2015). Dendritic cells and innate immunity in kidney transplantation. Kidney Int, 87, 712–​8. Suggested websites British Transplantation Society. http://​www.bts.org.uk ESOT. http://​www.esot.org Immune Tolerance Network. http://​www.immunetolerance.org NHS Organ Donation. http://​www.organdonation.nhs.uk Project HLA Sequence Database. http://​www.ebi.ac.uk/​imgt/​hla The ONE Study. http://​www.onestudy.org The Transplantation Society. http://​www.tts.org

SECTION 5 Principles of clinical oncology Section editors:  John D. Firth, Christopher P. Conlon, and Timothy M. Cox 5.1 Epidemiology of cancer  411 Anthony Swerdlow and Richard Peto 5.2 The nature and development of cancer:
Cancer mutations and their implications  445 James D. Brenton and Tim Eisen 5.3 The genetics of inherited cancers  456 Rosalind A. Eeles 5.4 Cancer immunity and immunotherapy  471 Charles G. Drake 5.5 Clinical features and management  487 Tim Eisen and Martin Gore 5.6 Systemic treatment and radiotherapy  497 Rajesh Jena and Peter Harper 5.7 Medical management of breast cancer  505 Tim Crook, Su Li, and Peter Harper

ESSENTIALS The epidemiology of cancer is the investigation of the incidence and causes of the disease in people under different conditions of life. Such investigations have generally been the way in which reliable evidence about causal agents for cancer, and the magni- tude of the risks from these agents, have been found. They have shown that any type of cancer that is common in one population is rare in some other, and that the differences between popu- lations are mostly not genetic, but rather the consequences of behaviours and circumstances of life. In principle, cancers are therefore largely preventable. The range of incidence rates between geographical and ethnic groups is more than 10-​fold for each of the common cancers, and for some cancers is more than 100-​fold. Large changes in rates of many tumours can occur in migrants compared with rates in their homeland, and large changes have occurred in rates within popula- tions over time, indicating the scope for prevention. The causes of cancer These can be divided into nature (biological factors), nurture (envir- onment and behaviours), and chance. Biological factors—​important biological factors are genetic suscep- tibility, age, and sex. Tobacco smoking—​this is the most important extrinsic factor causing cancer in developed countries, and is a major cause of can- cers of the mouth, pharynx (other than nasopharynx), oesophagus, larynx, lung, pancreas, renal pelvis, and bladder (and it also causes a proportion of several other types of cancer). In 2010, smoking is esti- mated to have caused 27% of all fatal cancers in the United Kingdom. Other extrinsic causes—​include (1) alcohol—​a cause of at least six types of cancer, including liver, various upper aerodigestive sites, and breast; (2) ionizing radiation—​which can cause cancer in most tis- sues; in the United Kingdom the main sources of exposure are natural sources including radon, and medical uses; (3) ultraviolet radiation—​ causes skin cancer; (4) infection, principally viral, but also bacterial and parasitic—​a major cause of cancer of several sites, especially in developing countries; (5)  immunosuppression—​patients with per- sistent immunosuppression from therapeutic, infective, or genetic causes have raised risks of certain cancers, notably non-​Hodgkin’s lymphoma; (6) chemotherapeutic agents—​about 20, used for treat- ment of specific diseases and including several that are used to treat cancer, have been shown themselves to cause cancer, of dif- ferent anatomical sites according to the agent; (7)  other drugs—​ hormone replacement therapy and oral contraceptives, both widely used in the general population, affect the risk of certain female reproductive-​related malignancies, increasing risk for some cancers but (for combined steroid contraceptives) decreasing it for others; (8) occupation—​numerous occupational groups have been found to be at increased risk of cancer, mainly of the respiratory tract, and es- pecially the lung; (9) air, water, and food pollution—​these are probably responsible for a small percentage of cancers in Western countries; (10) diet—​this may well have an effect on the aetiology of a substantial proportion of cancers, but there is considerable uncertainty on the figure and specific dietary associations are largely unknown. Other factors of particular note are (1) menstrual and reproductive history—​these affect the risks of breast, endometrial, and ovarian cancers in women; (2) obesity—​relates to increased risks of cancers of the breast (postmenopausally), endometrium, colon, kidney, and gallbladder, adenocarcinoma of the oesophagus, and possibly other cancers; (3)  physical inactivity—​relates to increased risk of breast, endometrial, colonic, and possibly other cancers. Epidemiology and aetiology of particular cancers The most common cancers worldwide are those of the lung, breast, and colorectum, and the most common causes of cancer death are lung, liver, and stomach cancers. Descriptive and aetiological epidemiological information is given about 33 types of cancer in this chapter. Lung cancer—​the major cause is smoking tobacco, particu- larly cigarettes. Lung cancer became epidemic in men in Western countries during the mid-​20th century, with rates rising later in women, and in Western countries there have been consider- able decreases in men in recent years. In developing countries, however, the epidemic has arrived later, with rising rates to be expected in future years as a consequence of current smoking levels. Occupational causes of lung cancer include exposures to asbestos, polycyclic hydrocarbons, and radon. Air pollution in towns may have also been a factor, largely in smokers, and radon in indoor air contributes to a small percentage of cases. 5.1 Epidemiology of cancer Anthony Swerdlow and Richard Peto

412 SECTION 5  Principles of clinical oncology Breast cancer—​the most common cancer worldwide in women. It has greatest incidence in Western countries, where rates have tended to increase slowly over decades; rates have generally been much lower in Asia and Africa. Hormonal and reproductive factors are important to risk: early menopause, late menarche, nulliparity, and older age at first full-​term pregnancy all increase risk, as do postmenopausal hormone replacement therapy and combined
oral contraceptives, while tamoxifen treatment of unilateral breast cancer decreases risk in the unaffected breast. There is also raised risk of breast cancer in relation to a history of benign breast disease, alcohol consumption, lack of physical exercise, postmenopausal obesity, taller height, and ionizing radiation exposure at young ages, as well as genetic predisposition. Introduction Cancer is a major cause of death and an increasing one: in England and Wales (Fig. 5.1.1) the proportion of deaths attributed to cancer has risen inexorably for over a century. All cancers have certain pathological and clinical characteristics in common, but those arising in different organs often have very different causes. The epidemiology of cancer, by which is meant the study of the incidence and causes of the disease in people under different conditions of life, is, therefore, the epidemiology of spe- cific types of cancer, usually—​but not always—​defined as cancers of specific organs. In this sense, the subject has a history dating back nearly 300 years to Ramazzini’s observation that cancer of the breast occurred more often in nuns than in other women of similar age and to Pott’s observation, over 200 years ago, that scrotal cancer in young men occurred characteristically in chimney sweeps. The high risk in nuns (which largely reflected the protective effect of multiple pregnancies in the general population) helped the real- ization that hormonal factors can substantially affect the incidence of several types of cancer, while the latter led to the recognition that the combustion products of coal to which sweeps had been exposed could cause cancer on any part of the skin with which they came into repeated contact and to the isolation of the first specific chemical carcinogen. Many other similar observations were made over the next 150 years, mostly as a result of the acumen of individual doctors who noticed clusters of cases of a particular type of cancer occurring in patients with a similar occupational or cultural background. Lip and tongue cancers were found in pipe smokers; bladder cancer in certain aniline dye workers; buccal cancer in those who habitually chewed mixtures of tobacco and betel in India; lung cancer in miners of particular ores (who, it was subsequently realized, were heavily exposed to radon and its daughter products); and skin cancer in the early radiologists and radiographers who were heavily exposed to X-​rays and in farmers and seamen heavily exposed to sunlight. Gradually, however, clinical anecdotes were replaced by statistics as epidemiological methods began to be applied to the study of cancer and other non​infectious diseases. As a result, many other causes were identified with sufficient certainty to justify preventive action and data were obtained to suggest hypotheses that could be tested in the laboratory. Preventability of cancer Perhaps the most important result of such observations has been the realization that any type of cancer that is common in one population is rare in some other, and that the differences between populations are mostly not genetic. Hence, where they are common these can- cers occur, in large part, as a result of the way people behave and the circumstances in which they live and they are, therefore, at least in principle, preventable. This does not mean that we can at present en- visage a society in which any of the common cancers are completely eliminated (although this may prove to be possible when we under- stand more clearly the mechanisms by which they are produced). What it does mean is that we can envisage a society in which the age-​specific risk of developing any type of cancer is low. Differences in incidence between communities Variation in incidence of cancer between different ethnic and geo- graphical groups around the world can be ascertained from data provided by population-​based cancer registries. Table 5.1.1 shows, for selected types of cancer, the range of variation recorded by cancer registries that have produced data sufficiently reliable for the purpose of international comparison, or, in one instance, the range determined by special surveys. Types of cancer have been included if they are common enough somewhere to have a cumu- lative incidence among men or women of at least 2% by 75 years of age. The range of variation is never less than 14-​fold and is sometimes more than 100-​fold. Despite the selection of reason- ably reliable registries, some of this tabulated variation may still be an artefact due to differing standards of medical service, case registration, and population enumeration. In many cases, how- ever, the true ranges will be greater, because much of the world is not covered by reliable cancer registries, and because the data generally refer to cancers of whole organs and do not distinguish between different histological types or different locations within an organ, for which greater variation may apply. 40 30 20 10 0 1910 1920 1930 1940 1950 1960 Year Females Males Percentage of all deaths due to neoplasms 1970 1980 1990 2000 2010 2020 Fig. 5.1.1  Percentage of all deaths due to neoplasms, England and Wales, 1911–​2014.

5.1  Epidemiology of cancer 413 The variation in incidence is not limited to the common cancers. Burkitt’s lymphoma, for example, never affects more than 1 in 1000 of the population, but it is at least 100 times as common among chil- dren in parts of Uganda as it is in Europe and North America; while Kaposi’s sarcoma, which was extremely rare in most of the world until the advent of AIDS, was so common in children and young adults in parts of Central Africa, even before 1970, that it accounted for 10% of all tumours seen in one of the African hospitals surveyed by Cook and Burkitt. Some few cancers occur with approximately the same frequency in all communities, but all are relatively un- common. Acute myeloid leukaemia at 15–​25 years of age is an ex- ample; nephroblastoma is another, except that it appears to be only half as common in Japan as elsewhere. The aforementioned figures refer to cancer incidence in commu- nities defined by geography, but substantial differences are found be- tween communities defined in other ways such as by ethnic origin, religion, or socioeconomic status. Jewish women, for example, have a low incidence of cervical cancer irrespective of the country in which they live, and Mormons and Seventh Day Adventists living in the United States of America have low incidence of cancers of the respiratory, gastrointestinal, and genital systems. Few of the large differences between communities can be ex- plained by genetic factors, apart from some of the differences in the incidence of cancer of the skin, the risk of which is much greater for whites than for blacks, and possibly also for some of those in the incidence of testis cancer, which rarely affects black popula- tions, and in chronic lymphocytic leukaemia, which rarely affects people of Chinese or Japanese descent. Genetic factors cannot ex- plain the differences observed on migration or with the passage of time, which are discussed in the next sections of this chapter, nor can they explain the correlations observed between the national rates for particular types of cancer and aspects of the lifestyle in different countries. Changes in incidence in migrant groups That changes in the incidence of cancer occur on migration is cer- tain. Numerous groups have been studied, particularly migrants from many countries to Australia, Israel, and the United States. These show, for example, that Afro-​Americans experience in- cidence rates for internal cancers that are generally much more like those of white Americans than those of the black populations in West Africa from which most of their ancestors came, while Japanese in Hawaii have experienced rates that are much more like those of the white residents of Hawaii than those of Japanese living in Japan (Table 5.1.2). The ancestors of black Americans and Hawaiian Japanese will have come from many different parts of Africa and Japan, some of which are likely to have cancer rates somewhat different from those cited in Table 5.1.2. Nevertheless, the contrasts are so great that there can be no serious doubt that new factors were introduced with migration. Table 5.1.1  Range of incidence rates of common cancers Site of origin of cancera High-​incidence areab,c Cumulative incidence (%) 
in high-​incidence aread Low-​incidence area,c same sex Ratio of cumulative rates in high-​ and
low-​incidence arease Non​melanoma skin Australia (Queensland)f

20 Several non​white populations 200 Prostate US (Delaware, black) 28 China (Yancheng) 250 Oesophagus China (Cixian) 24 Kuwait, non-​Kuwaitis 480 Stomach China (Yangcheng) 22 Kuwait, non-​Kuwaitis 92 Breastg Hawaii, Hawaiian 13 China (Yanting) 14 Lung US (Wisconsin, black) 12 India (Dindigul, Ambilikkai) 25 Cervix uterig Zimbabwe (Harare, black) 10 Egypt (Gharbiah) 58 Liver China (Qidong) 8 Brazil (Forteleza) 64 Kaposi’s sarcoma Malawi (Blantyre) 8 Several 500 Colong US (Alaska, American Indian) 6 India (Dindigul, Ambilikkai) 200 Melanoma of skin Australia (Queensland) 6 Several 500 Bladder Italy (Syracuse) 5 India (Sikkim, and Dindigul, Amilikkai) 32 Corpus uterig New Zealand, Pacific Islander 4 Libya (Benghazi) 43 Rectum Czech Republic 3 India (Dindigul Ambilikkai) 20 Kidney Czech Republic 3 China (Yanting) 135 Nasopharynx China (Zhongshan) 3 Several 280 Non-​Hodgkin’s lymphoma US (Washington, Seattle) 2 India (Sikkim) 68 a Sites of cancer are shown if somewhere they reach a cumulative incidence by age 75 of at least 2% in either sex. b The geographic area of highest recorded incidence in either sex by age 75. Since most are males, footnotes to indicate sex are only given where the rate is in females. c Excluding very small cancer registry populations, with unstable numbers. d By age 75 years, in the absence of other causes of death. e By age 65 years, in the absence of other causes of death. f Special survey. g Women.

414 SECTION 5  Principles of clinical oncology Changes in incidence over time Within one population there may be substantial changes in the in- cidence of a particular type of cancer over a period of a few decades that provide conclusive evidence of the existence of preventable fac- tors. Changes in incidence over time may, however, be difficult to assess reliably. This is chiefly because it is difficult to compare the thoroughness of the diagnosis and registration of particular types of cancer at different periods and partly because few incidence data have been collected for long enough, so we often have to fall back on changes in mortality rates even though these may be influenced by changes in treatment, as well as changes in incidence. There are no simple rules for deciding which changes in recorded cancer incidence and mortality rates are reliable indicators of real changes in incidence. Each set of data has to be assessed individu- ally. It is relatively easy to be sure about changes in the incidence of cancer of the oesophagus, as the disease can be diagnosed without complex investigations and its occurrence is nearly always recorded, at least in middle age, because it is nearly always fatal. By contrast, it is much more difficult to be sure about changes in the incidence of basal cell carcinomas of the skin, which, although easy to diagnose, seldom cause death and can be treated effectively outside hospital, and so often escape registration. What appears to be a change in in- cidence may therefore be a change only in the completeness of regis- tration. Cancers of the pancreas, liver, and brain, and myelomatosis, in contrast, usually cause death, but even when they do they may be misdiagnosed as another disease, so that an increased incidence or mortality rate may be wholly or partly due to improvements in diag- nosis or in the availability of medical services. Such changes are par- ticularly likely to affect the rates recorded for people over 65 years of age, as many older people who were terminally ill used not to be intensively investigated. Despite these difficulties, some of the decreases and increases in the recorded rates of particular types of cancer have been so gross that there must have been real changes in their incidence. Examples include the increase in lung cancer throughout most of the world (and its recent large decrease in men in the United Kingdom), the in- crease in mesothelioma of the pleura in men in industrialized coun- tries, the decrease in cancer of the tongue in the United Kingdom, and the decrease in cancers of the uterine cervix and stomach throughout Western Europe, North America, and Australasia. For a fuller account, see Doll et al., 1994 and Swerdlow et al., 2001. Identification of causes More-specific evidence of the preventability of cancer, and of meas- ures to enable this, has come from identification of agents and cir- cumstances that cause the disease. In general, reliable evidence of causality (and particularly of the magnitude of any risks) has come from epidemiology and not from laboratory experiments, although the latter can often provide reinforcement of epidemiological find- ings and understanding of the mechanisms of cancer causation. Reliable epidemiological evidence does not require randomized trials within particular populations, but it does require the study of different individuals within populations and not just the com- parison of incidence rates between populations. Non​randomized epidemiological studies of individuals have often yielded proof of causation beyond reasonable doubt, and have provided the decisive evidence of aetiology for almost all proven carcinogens. Action based on such evidence has, moreover, often been followed by the desired result; for example, a reduction in the incidence of bladder cancer in the chemical industry on stopping the manufacture and Table 5.1.2  Comparisons of cancer incidence rates in migrantsa and residents in homelands and adopted countries (men, unless otherwise specified), mid-​1990s. Cumulative rate to age 65, per 1000 persons Japanb Hawaii West Africab United States Japanese Whites Blacks Whites Oesophagus 6.5 3.0 1.7 1.9 7.4 2.6 Stomach 32.9 7.4 3.6 7.3 6.6 3.1 Colon 14.8 17.2 14.0 0.8 16.3 11.0 Rectum 10.0 11.6 6.6 0.9 6.8 6.4 Liver 17.3 3.5 2.6 32.1 4.9 1.9 Pancreas 4.4 4.0 3.9 1.3 7.0 3.4 Larynx 1.7 1.8 5.2 0.4 6.6 3.2 Lung 15.2 13.4 21.7 1.9 49.5 25.8 Breastc 24.2 61.7 72.2 10.5 59.8 65.1 Uterine cervixc 4.7 3.4 5.7 27.5 7.5 5.3 Corpus uteric 3.0 13.1 9.9 1.9 7.6 12.4 Ovaryc 4.7 7.4 10.2 1.4 5.5 9.1 Prostate 2.0 17.6 40.6 2.2 91.2 47.4 Testis 1.0 1.7 5.1 0.2 0.8 4.1 Non-​Hodgkin’s lymphoma 3.4 4.3 10.4 1.4 11.0 10.1 a ‘Migrant’ rates are based on ethnicity, and hence include all generations of migrant. b Average of rates in two regions. c Women.

5.1  Epidemiology of cancer 415 use of 2-​naphthylamine and, on a national scale, the reduction in the incidence of lung cancer in men in the United Kingdom following the decrease in smoking since the mid-​20th century. The causes of cancer can, briefly, be divided into nature (biological factors), nurture (behaviour and environment), and chance. Biological factors Genetic susceptibility Many common types of cancer tend to cluster in families to some extent. Differences of this sort do not in themselves imply that the familial clusters are genetic in origin; they could be due to familial similarities of behaviour or environment. Nevertheless, various genetic factors are known that affect cancer risks to different ex- tents. Very large risks are seen in patients with certain rare cancer-​ associated genetic syndromes in which bearers of one gene (if the condition is dominant) or two (if recessive) almost invariably de- velop a particular type of cancer. Examples include the dominant genes for polyposis coli that lead to cancer of the large bowel, and the recessive gene for retinoblastoma, and that for xeroderma pigmentosum, which leads to squamous carcinoma and (less com- monly) melanoma of the skin. Similar evidence has shown that other genetic syndromes frequently, but not invariably, lead to cancer, such as von Recklinghausen’s neurofibromatosis leading to fibrosarcoma, the Peutz–​Jeghers syndrome leading to carcinoma of the small bowel, the Wiskott–​Aldrich syndrome leading to non-​ Hodgkin’s lymphoma, and ataxia telangiectasia, Bloom’s syndrome, and Fanconi’s anaemia leading to leukaemia. Very high cancer risks are also present in individuals with various cancer susceptibility genes; for example, raised risks of breast and ovarian cancers in pa- tients with mutations in BRCA1 and BRCA2 genes. The recognition of these genes is important to the individual, as it may provide an opportunity for prophylactic surgery, or enable the diagnosis of ma- lignancy to be made at an early stage when treatment is more likely to be effective, or (rarely) enable precautions to be taken to prevent exposure to the relevant carcinogens, as in the case of sufferers from xeroderma pigmentosum or albinism, who can be protected against sunshine. The proportion of all cancers that occur in people who are highly susceptible to cancer in this way is, however, very small, al- though substantially greater at young than at older ages. Genes conveying less-​raised risks of cancer are far more wide- spread in the population, and are involved in the causation of a much larger proportion of cancers. Large numbers of relatively common susceptibility variants influencing cancer risks have been discovered in the last few years. Discovery of genetic factors that affect particular types of cancer is unlikely to explain much of the social and geographical differences in the distribution of cancer other than skin cancer, but it can help to elucidate mechanisms and to focus health education and early diag- nosis on the sections of the populations that are most at risk. Age Some risk of cancer occurs at every age, but the risk of developing any particular type varies with age. The most common relationship with age is a progressive increase in incidence from near zero in childhood and adolescence to a high rate in old age. This type of relationship is shown by carcinomas of the skin, lung, and gastro- intestinal and urinary tracts, and by myelomatosis and chronic lymphatic leukaemia. The rate of increase is rapid, being typically proportional to the fourth, fifth, or sixth power of age in years, so that the annual incidence may be 100 or 1000 times greater above age 75 than before age 25. It is probable that this reflects the cu- mulative effect of processes that operate steadily throughout life, starting at around the time of birth or at young ages (e.g. for lung cancer, in adolescence). With most of these cancers, the recorded incidence may stabilize, or even decrease, in the oldest age groups, but this is partly or wholly an artefact due to incomplete investiga- tion of the terminal illnesses of old people. A less common pattern is a peak incidence early in life, which may be followed either by a decline virtually to zero or by a slow rise in middle and old age. Retinoblastomas and nephroblastomas occur only in childhood, with peak incidences (respectively) in the first and second years of life. Teratomas and seminomas of the testis have peak incidence rates at about 25 and 35 years of age, respectively, and later almost cease to occur, while osteogenic sarcomas have a peak incidence in adolescence and then show a slow increase with age from a lower rate in young adult life. The remaining cancers show a variety of patterns. Carcinomas of the breast and uterine cervix of women, for example, begin to ap- pear in young adulthood and become rapidly more common up to the menopause. After the menopause the incidence of carcinoma of the breast may remain approximately constant, or may even be- come slightly reduced for a few years, before increasing again with age, though at a slower rate. Carcinoma of the cervix continues to increase fairly steeply for a few years after the menopause, before showing a stable or declining rate. Hodgkin’s disease, on the other hand, appears in childhood but thereafter continues relatively evenly throughout life with only modest peaks in young adult life and at older ages, while connective tissue sarcomas become progressively more common from childhood onwards, but with a much slower rate of increase than is shown by the common carcinomas. Some of these relationships with age, like that for retinoblastoma in early childhood, seem to be invariant everywhere and, as far as is known, at all times. Others vary from community to community, or from time to time. In postmenopausal women, for example, cancer of the breast becomes progressively less common with increasing age in parts of Asia, but more common in Europe, while carcinoma of the lung used to show a peak incidence at about 60 years of age in the United Kingdom, which gradually moved to older ages, as a generation that had not smoked substantial numbers of cigarettes throughout adult life was replaced by one that had, and the same process is now being repeated in many developing countries. These various patterns provide information, either about the period of activity of the stem cells from which the cancers derive, or about the period when the main exposure to causative agents occurs and the duration of that exposure. Some of this variation has already helped to explain some of the causes of cancer, as was the case with the shift in the peak incidence of bronchial carcinoma, but much still awaits elucidation. Sex Cancer used to be more common in women than in men in many countries due to the great frequency of carcinoma of the breast and of the uterine cervix, and to the rarity of bronchial carcinoma. However, now the reverse is generally true except in a few popu- lations in Africa and Asia for which similar conditions to those

416 SECTION 5  Principles of clinical oncology formerly persist. This overall male preponderance hides, however, a variety of sex ratios for cancer of different organs. Aside from sites of cancer that are peculiar to one sex, the sex ratio varies (in Britain) from a male excess of 6 to 1 for pleural mesothelioma and carcinoma of the larynx, through many types of cancer with only a small male preponderance, to carcinomas of the thyroid and gallbladder, which are about twice as common in women, and breast cancer which is over 100 times more common in women. For many types of cancer the sex ratio is much the same in different countries and at different times. For some, however, and particularly for cancers of the mouth, oesophagus, larynx, and bronchus, the sex ratio is extremely variable—​not only between countries and at dif- ferent times, but sometimes also between different ages at the same time and in the same country. The most marked variation is shown by cancer of the oesophagus, which may affect both sexes equally or be 20 times more common in men than in women. As with the pat- terns of incidence with age, these sex ratios and their variation can provide useful clues to the causation of particular types of cancer, not all of which have yet been successfully followed up. Delay between cause and effect One reason why it has been difficult to recognize causes of cancer in humans is the long delay that characteristically occurs between the start of exposure to a carcinogen and the appearance of the clin- ical disease. This ‘latent period’, as it is commonly, but rather mis- leadingly, called is often several decades, although it may be as short as 1 year or as long as 60. The exact relation between the date of exposure and the date of the appearance of different cancers is still uncertain, partly because the interval is subject to random factors, partly because few cancers are induced by a single, brief exposure, and partly because there are still relatively few sets of quantitative data with detailed information about the dates when exposure began and ended. When cancer is induced by short but intensive exposure to ion- izing radiation, as following the explosions of the atomic bombs in Hiroshima and Nagasaki or in patients treated by radiotherapy, the excess incidence of solid tumours rises for 15–​20 years and then may continue to rise, level off, or decline. In the case of acute leukaemia, however, peak incidence occurs much earlier (c.5 years after irradi- ation) and relatively few cases appear after more than 30 years. Short, intensive exposure to a carcinogen is, however, exceptional. The more usual situation is for sporadic or continuous exposure to a carcinogen to be prolonged for years—​a decade or two in the case of occupational exposure, several decades in the case of tobacco smoking, and a lifetime in the case of ultraviolet radiation. In this situation, the incidence of cancer increases progressively with the length of exposure. In the last two cases cited, the incidence appears to increase approximately in proportion to the fourth power of the duration of exposure, so that the effect after (say) 40 years is more than 10 times as great as that after 20 years, and more than 100 times as great as that after 10 years. Whether the same holds for occupa- tional exposure is not known, but it has been shown to hold in some experiments in which chemicals were repeatedly applied to the skin of genetically similar mice and it may prove to be a general biological rule for many types of carcinoma and many carcinogens. There is still less quantitative information about what usu- ally happens when exposure ceases, but in the case of cigarette smoking the rapidly rising annual risk among those who continue to smoke stabilizes for one or two decades after smoking ceases be- fore increasing again slowly. The ex-​smoker consequently avoids the enormous progressive increase in risk suffered by the continuing smoker. These delayed effects accord with the idea that the appearance of clinical cancer is the end result of a multistage process in which sev- eral mutations have to be produced in a single stem cell to turn it into the seed of a growing cancer. From a practical point of view, the important conclusions are that cancer may be very much more likely to occur after prolonged exposure to a carcinogen than after short exposure; that it is seldom likely to appear within a decade after first exposure (except in the case of leukaemia, certain hormone-​related and immune suppression-​related cancers, and the specific cancers of childhood); that it commonly occurs several decades after first exposure; and that some excess risk may continue to occur for dec- ades after exposure has ceased. The exact relationship may, how- ever, differ for different carcinogens and different types of tumour. Bladder tumours, for example, began to appear within 5 years of intensive exposure to 2-​naphthylamine in the dye industry, while mesotheliomas of the pleura have seldom, if ever, appeared within 10 years of exposure to asbestos, but they continue to increase in incidence for up to 50 years after first exposure, even if the exposure was relatively brief. Chance There remains the influence of chance, which is important for the individual as it is the reason why two animals of identical genetic constitution that have been treated in the same way do not, in gen- eral, develop cancer in the same place at precisely the same age. It reflects the element of chance that determines whether a particular series of events all occur in one particular stem cell out of the many thousands of stem cells that exist that do not give rise to a malignant clone. For any one individual, the role of chance in determining the occurrence of cancer may be large, but in a large population it has little net effect on the incidence of cancer and only nature and nur- ture are important. Avoidable factors Tobacco Tobacco is by far the most important single cause of cancer in de- veloped countries. Chewed, it can cause cancers of the mouth and oesophagus; smoked, it is a major cause of cancers of the mouth, pharynx (other than nasopharynx), oesophagus, larynx, lung, pan- creas, renal pelvis, and bladder. For these eight cancers, epidemio- logical evidence indicates that prolonged smoking of average numbers of cigarettes per day increases the risk 3–​20 times. It is, however, now clear that cigarette smoking also causes a propor- tion of several other types of cancer, increasing the incidence up to thrice that in non​smokers: namely, cancers of the lip, nose, naso- pharynx, stomach, colorectum, cervix, liver, and renal body, mu- cinous ovarian tumours, and also myeloid leukaemia. Although the proportional increases are not large, the consistency of the findings in different countries, the evidence of dose–​response re- lationships, the lower mortality in ex-​smokers than in continuing smokers, the lack of evidence for important confounding, and the presence in the smoke of many different carcinogens, provide strong grounds for believing that most or all of these observed as- sociations are causal.

5.1  Epidemiology of cancer 417 In sum, smoking is estimated to have caused 27% of all fatal can- cers in the United Kingdom in 2010, down from 34% 35 years earlier. The reduction was substantial in men (down from 52% in 1975 to 39% in 1995 and 31% in 2010) but it was largely counteracted by the increase in women (from 12% in 1975 to 20% in 1995 and 23% in 2010). Comparable figures from the United States and from some other developed countries are shown in Table 5.1.3. In men, there have in the past decade been decreases in many developed countries, but in each the proportions remain substantial. In women, the pro- portion of cancer deaths attributed to smoking was generally low in 1975, but has subsequently increased in all developed countries and must be expected to increase further. It was, however, still small in countries such as France or Spain, where many young women now smoke but few middle-​aged or elderly women have been smoking for long enough for any material effect to be produced. Fig. 5.1.2 shows the overall trends in UK cancer mortality at ages 35–​69 since 1950, divided by whether or not they were attribut- able (in a statistical sense) to smoking; it can be seen how greatly the trends in men especially have been influenced by changes in smoking-​attributed mortality. In developing countries, the effects of smoking have only recently begun to be studied systematically and much remains unclear. Large, nationally representative studies of smoking and death have, however, been conducted in China and India. In general, women in developing countries do not smoke (although there are particular regions in China and India where they do so). In men, however, Table 5.1.3  Percentage of cancer deaths attributed to smoking, 1955, 1975, 1995, and c.2010, by sex: various countries Country Male Female 1955 1975 1995 c.2010a 1955 1975 1995 c.2010a Australia 20 40 33 26 0 4 14 15 Finland 38 46 37 30 1 1 5 10 France 17 34 37 34 0 0 3 9 Hungary 21 36 51 50 2 5 13 21 Spain 13 28 39 36 0 0 0 4 UK 41 52 39 31 3 12 20 23 USA 23 42 42 37 0 10 25 27 a Most recent year available from WHO (Australia 2006; United States 2008; France, Spain, Hungary 2009; others 2010). 3 2.5 2 1.5 1 0.5 0 1940 1960 Rate per 100 000 age-standardized 1980 Year 2000 2020 (a) Rate per 100000 age-standardized (b) 3 2.5 2 1.5 1 0.5 0 Year 1940 1960 1980 2000 2020 Not attributed to smoking Attributed to smoking Not attributed to smoking Attributed to smoking Fig. 5.1.2  United Kingdom cancer mortality at ages 35–​69, 1950–​2010, attributable to smoking and not attributable to smoking: (a) males, (b) females.

418 SECTION 5  Principles of clinical oncology there has been a very large increase in worldwide cigarette con- sumption over the past few decades, the full effects of which have yet to materialize. China, with 19% of the world’s population, smokes 37% of the world’s cigarettes. By 1987 smoking was responsible for about 20% of male cancer deaths in China, and now accounts for 25%. In India, where many men have smoked ‘bidis’ (small, home-​ manufactured cigarettes) for decades, smoking caused 32% of all cancer deaths in men and 5% in women in the early 2000s (and 20% of all male deaths in India at ages 35–​69 in 2010), partly because smoking can act as a cofactor for the production of cancers of the mouth, oesophagus, or stomach in those who habitually chew quids containing betel and tobacco. In some parts of South America and China the male lung cancer rates from smoking are already as high as in developed countries. Overall, tobacco may be causing about as many cancer deaths in developing as in developed countries, in which case it would be responsible for about 20% of cancer deaths throughout the world. Alcohol At least six types of cancer are caused in part by the consumption of alcohol. One, liver cancer, is produced mainly by the produc- tion of liver cirrhosis and is, consequently, caused mainly by heavy and prolonged consumption. Four are causally related to smoking as well as to alcohol: namely, cancers of the mouth, pharynx (other than nasopharynx), oesophagus, and larynx. The two agents act synergistically, increasing each other’s effect, so that the risk from alcohol in non​smokers or long-​term ex-​smokers is very small, while that in heavy smokers is disproportionately large. The re- maining type, cancer of the breast, has been shown to be related to alcohol more recently. Epidemiological cohort studies show that the risk increases progressively with the amount drunk (at least up to moderately high levels) and laboratory studies that show that al- cohol increases the level of oestrogen in the blood suggest a plaus- ible mechanism. Cancers of the large bowel have also been associated with alcohol in many studies, but the relationship is modest and its nature uncer- tain: it could be due to confounding by diet. Ionizing radiation Ionizing radiations, of whatever type, and whether from external sources or from inhaled or ingested radionuclides, share the char- acteristic of having sufficient energy to damage DNA through ion- ization when they pass through the tissues of the body. It is not surprising, therefore, that they have been found to increase the in- cidence of cancer in practically every organ. The radiosensitivity of different organs varies greatly, however, and particularly large risks, relative to background rates, occur for thyroid cancer in people exposed as children, for myeloid leukaemia, and for can- cers of the breast and bladder. In contrast, there is no good evi- dence that exposure to ionizing radiation can increase the risk of chronic lymphocytic leukaemia, Hodgkin’s disease, or testis cancer. Many exposures to ionizing radiation are specific to certain parts of the body or to certain organs or tissues, and this determines the site of the induced cancer. For example, the sites of cancers after radiotherapy depend on the sites exposed to radiation as a conse- quence of the treatment; Thorotrast (a radioactive contrast agent used in the mid-​20th century) tends to be incorporated in the liver and bone marrow and hence to cause liver cancer and leukaemia; inhalation and ingestion of iodine-​131 was the principal exposure from the Chernobyl accident to the general population in the sur- rounding areas, which led to an increase in thyroid cancer in chil- dren; and inhalation of the natural radioactive gas radon and its progeny gives rise to lung cancer. Estimates of the carcinogenic effect of X-​rays have been derived by following groups of people with unusual but well-​documented exposures, including patients given radiotherapy, or repeatedly screened radiologically, and the survivors of the atomic bombings of Hiroshima and Nagasaki (in whom exposure was principally to γ-​rays, which are high-​energy X-​rays). Estimates of the risk of lung cancer from the inhalation of radon and its progeny have been de- rived both from studies of uranium miners and from studies of in- door radon exposure carried out in the general population. At low doses (<c.20 mGy) it seems probable that the carcino- genic effect of ionizing radiation is linearly proportional to the dose, while at higher doses the same is true for most cancers other than leukaemia, for which the risk is approximately proportional to the square of the dose. For most sites of cancer the risk is higher in people exposed in childhood than those exposed as adults, and it starts within the first 5 years and lasts for several decades. The International Commission on Radiological Protection (2007) has concluded that the lifetime risk of developing a fatal cancer is ap- proximately 10%/​Gy for X-​rays (or per Sievert for other types of radiation) to the whole body if the radiation dose is moderate and given acutely, and about half that if the dose is low and spread out over time (that is, 5 per 100 000 per mGy (or mSv)), with corres- ponding reductions if only part of the body is exposed. It has not been possible to detect by direct observation the effect of very small exposures, including, for example, the effect of a single chest radiograph given to an adult. However, theoretical consider- ations and the dose–​response relationship observed at larger doses both indicate that there is unlikely to be any threshold below which no effect is produced. This conclusion is reinforced by the observa- tion that children who received doses of 10–​20 mGy in utero (be- cause their mothers were irradiated for diagnostic purposes while pregnant) were subject to an added risk of developing cancer in childhood of approximately 1 in 2000. People are exposed to different amounts of radiation in different countries, depending principally on the concentration of radon in indoor air and the medical use of radiation for diagnosis. In the United Kingdom, the average indoor radon concentration is 20 Bq/​m3 and this is estimated to give rise to about 1000 lung cancer deaths each year. In the United States the average radon concentration is about twice that for the United Kingdom and, within the United Kingdom, it varies from one part of the country to another. Most notably, Devon and Cornwall have average indoor radon concen- trations three or four times greater than the national average and there are a few houses with concentrations that are 10 or even 100 times greater. Other sources of radiation are estimated to give rise to an average annual dose of about 1.4 mSv in the United Kingdom, which, on the basis of the risk estimate recommended by the International Commission on Radiological Protection, would lead to about 4200 deaths per year in the national population of about 60 million. This gives an estimated total of 5200 deaths per year from radiation-​induced cancer, or just over 3% of total cancer deaths. The major contributors are radon (20%), other natural sources (55%), and medical uses (24%). In addition, there is a contribution from

5.1  Epidemiology of cancer 419 radiotherapy for cancer, but less than might at first sight be implied from the collective dose, because a substantial proportion is received by people who will not survive long enough for a radiation-​induced cancer to appear. Less than 1% of all radiation-​induced deaths in the United Kingdom can be attributed to occupational exposure, fallout from past nuclear weapons tests, manufactured products, or radioactive waste. Ultraviolet radiation Photon energies in the ultraviolet (UV) range are sufficient to damage DNA and hence to cause cancer mutations. UV does not penetrate much below the skin, so that it is chiefly within the skin that it is directly carcinogenic. Within the skin, however, it is the principal cause of all types of cancer, other than Kaposi’s sarcoma. Whether it has any indirect carcinogenic effect on other tissues (notably the lymphopoietic tissue) remains uncertain, as does the possibility that the effect of UV in raising vitamin D levels may lead to consequent reduction in risks of certain cancers, especially colorectal. The main source of human exposure to UV radiation is sunshine. Infection Infection, principally viral, but also in some cases bacterial and para- sitic, is a major cause of avoidable cancer, especially in developing countries: in sub-​Saharan Africa about 40% of cancers in women and 30% in men, compared with about 10% in developed countries, are attributable to infections. Viral infection Viruses that are known to cause human cancers, or suspected of doing so, are listed in Table 5.1.4, along with the types of cancer with which they are associated. Not all infected people develop the disease. In some cases the proportion doing so is quite small, un- less other factors are also present. Such cofactors include endemic malaria for Burkitt’s lymphoma, the consumption of a type of salted fish for nasopharyngeal cancer, and the consumption of aflatoxin, a metabolic product of fungal infection with Aspergillus flavus, for liver cancer. What they are for the cancers produced by the human papillomavirus is not known. Quantitatively, chronic infection with hepatitis B virus is one of the most important causes of cancer in many parts of the world. In China, for example, liver cancer accounts for about 15% of all cancer deaths, the large majority of which are due to chronic lifelong infec- tion with the virus. Infant vaccination against the virus is now being introduced and will protect those born in the present century, but will not provide retrospective protection for those born in the 20th century. Bacterial infection Only one specific bacterial infection has been closely linked with the development of cancer: Helicobacter pylori. Persistent H. pylori infection acquired early in life leads to chronic gastritis in the an- trum of the stomach and increases the risk of gastric cancer two-​ to threefold. Non​specific chronic infection in the bladder may increase the risk of bladder cancer. Table 5.1.4  Viral causes of cancer Virus Cancer Hepatitis B Cancer of liver Hepatitis C Cancer of liver, non-​Hodgkin’s lymphoma HPV types 16, 18, and others Cancers of cervix, vulva, vagina, penis, anus; oral cavity, oropharynx, tonsil, some skin cancers HHV type 4 (EBV) Burkitt’s lymphoma Post-​transplant lymphoproliferative disease Nasal T-​cell lymphoma Other non-​Hodgkin’s lymphoma Hodgkin’s diseaseb Nasopharyngeal cancer HHV type 8 (Kaposi-​associated herpesvirus) Kaposi’s sarcoma Primary effusion lymphoma Human T-​cell leukaemia type 1 Adult T-​cell leukaemia/​lymphoma HIVa Kaposi’s sarcoma Non-​Hodgkin’s lymphoma Hodgkin’s disease Conjunctival carcinoma Cancers of the cervix and anus EBV, Epstein–​Barr virus; HHV, human herpesvirus; HPV, human papillomavirus. a In most cases, if not in all, by facilitating the effect of other viruses, probably via immunosuppression. b Causal nature of observed association unproved. Simian virus 40 (SV40) has been suspected in the aetiology of mesothelioma and several other types of cancer, without clear proof.

420 SECTION 5  Principles of clinical oncology Parasitic infection In parts of Africa and Asia, parasitic infection is a major cause of cancer. Infection with Schistosoma haematobium, which excretes its eggs through the bladder wall, causes a high incidence of bladder cancer in Egypt and East Africa while infection with S. japonicum, which excretes its eggs through the wall of the large bowel, is respon- sible for a high incidence of intestinal cancer in parts of China. Liver flukes (Clonorchis sinensis and Opisthorcis viverrini) are similarly responsible for the high incidence of cholangiosarcoma of the bile ducts in parts of Southeast Asia. The parasites may not cause cancer directly, but chronic infection may start a chain of events that leads to cancer in other ways, such as chronic bacterial infection and the local formation of nitrites and nitrosamines. Immunosuppression Patients with persistent immunosuppression, either therapeutic (notably immunosuppressive drug treatment for organ transplant- ation) or as a consequence of infection (e.g. HIV) or genetic (e.g. in ataxia telangiectasia and Wiskott–​Aldrich syndrome) have greatly raised risks of non-​Hodgkin’s lymphoma, and often of other viral infection-​related cancers including Kaposi’s sarcoma, although the pattern of malignancy and scale of risk varies according to the type of immunosuppression—​Kaposi’s sarcoma, for instance, predominates after HIV, whereas non-​Hodgkin’s lymphoma is the most common consequence of genetic immunodeficiency and of transplantation. Medical drugs Apart from ionizing radiation, some 20 agents have been used therapeutically that are known to cause cancer in humans. These are listed in Table 5.1.5. That so many carcinogenic agents should have been prescribed medically is not surprising when it is borne in mind that treatment often requires modification of cellular metab- olism and is sometimes intended to interfere with DNA. The hazard of cancer, however, is not necessarily a bar to the use of a drug if this risk is outweighed by the therapeutic benefits, as is commonly the case with antineoplastic agents, immunosuppressive drugs, and radiotherapy. Some of the chemotherapeutic agents listed in Table 5.1.5 were soon abandoned, while others have continued to be used for treat- ment, and the sum of the cancers that these now produce cannot amount to more than 100 or so a year in the United Kingdom. Two of the listed drugs are, however, used extensively in the general population:  hormonal replacement therapy (HRT) for postmenopausal women, and selected steroids for contraception, which increase the risk of breast cancer. Both also increase the risk of endometrial cancer, but HRT does so substantially only when given in the form of oestrogen alone and steroid contraceptives do so only in the form (now abandoned) in which oestrogen and pro- gestogen are given sequentially. The combined steroid contracep- tives currently in use can also rarely cause liver cancer and they may increase the risk of cervix cancer. Combined steroid contra- ceptives, however, also reduce the incidence of endometrial cancer and halve the risk of ovarian cancer for many years after they have been used. Meanwhile, HRT and combined steroid contraceptives are associated with a reduction of some 20% in the risk of colo- rectal cancer, although whether this is causally related to their use remains unknown. Other drugs that may inhibit cancer rather than cause it are non-​ steroidal analgesics, most notably aspirin, the prolonged use of which may somewhat reduce the risk of colorectal cancer and per- haps cancers of certain other sites. Taken altogether, it seems unlikely that medically prescribed drugs can be responsible for more than 1% of all today’s fatal cancers and may, in total, reduce the risk by somewhat more. Occupation In the years that followed Pott’s observation that chimney sweeps tended to develop cancer of the scrotum, many other groups of workers were found to suffer from specific hazards of cancer; indeed, more substances that are known to be carcinogenic to humans have been unearthed by the search for occupational hazards than by any other means. Most of these occupational cancers are in the respira- tory tract, especially the lung. The hazards are listed in Table 5.1.6. Many of the hazards that have been recognized caused large, or at least relatively large, risks, albeit for limited populations, and it may well be that other occupational causes exist that have not yet been detected, either because the added risk is small in comparison with that due to other causes, or because only a few workers have been persistently exposed, or simply because the hazards have not been suspected and so not looked for. It must also be borne in mind that cancer in humans seldom develops until one or more decades after exposure to the carcinogen first occurs and it is, therefore, too soon to be sure whether agents that have been introduced into industry only during the last 20 years are carcinogenic or not. Many groups of workers not listed in Table 5.1.6 have been sus- pected of having a special risk, but it has not been possible to decide whether the risk is real and attributable to their work. Some of these excesses may have arisen by chance alone, especially if the excess has not been confirmed in other studies, and others may be due to confounding; that is, they may have been produced by social factors or behaviours that are associated with the occupation in question rather than by the occupation itself. Given sufficient details and the ability to repeat the observations, it is usually possible to obtain a fairly clear idea of whether an excess incidence in an occupational group reflects an occupational hazard (e.g. by seeing whether the effect is related to the length of employ- ment, the time after first exposure, and a specific type of work within the industry). Unfortunately these details are not always available and the reasons for many of the reported moderate excesses of occu- pational cancer are still uncertain. In addition to the directly occupational cancers discussed earlier, workers may be indirectly exposed to carcinogens while at work (e.g. tobacco smoke from clients or colleagues). At present it seems likely that occupational hazards account for only a few per cent of all fatal cancers in developed countries such as the United Kingdom. The three principal causes are probably as- bestos dust (lung, pleural, and laryngeal cancers), the combustion products of fossil fuels (skin and lung cancer), and ionizing radi- ation (a wide variety of cancers). Pollution The idea that pollution might be an important cause of cancer has been in the forefront of the minds of cancer research workers since it was realized that the incidence of lung cancer tended to be higher in towns than in the countryside and that the combustion products

5.1  Epidemiology of cancer 421 of coal, which used to produce a pall of smoke over all large cities in Britain, contained carcinogenic hydrocarbons. Subsequently, with the rapid expansion of the chemical industry and the discovery that some of its products are mutagenic in vitro and carcinogenic in laboratory animals, anxiety increased about the possible effects of distributing such products ubiquitously in air, water, and food. The effects of pollution of this sort are, however, peculiarly dif- ficult to assess directly by epidemiological methods, as pollutants are likely to be present in most areas, the absolute risk from each is likely to be small, and there may be little difference in the extent to which individuals are exposed over a wide area. Reliance is, there- fore, often placed mainly on two indirect methods: extrapolation from the effects of chronic exposure to much larger amounts in an occupational setting, and prediction of the effects on humans from laboratory tests. Both, however, (but particularly the latter) involve substantial uncertainties. So far as atmospheric pollution is concerned, the epidemiological picture is complicated by the personal pollution produced by to- bacco smoke and the social distribution of smoking habits. Despite this complication, however, the various methods that have been dis- cussed under lung cancer all lead to the conclusion that the pollu- tion of the past may have contributed to the production of a few per cent of all lung cancers in Western countries, but that the levels over the last three decades (principally from the combustion of fossil fuels, but also from asbestos, dioxins, and various other materials) are unlikely to be responsible for more than a fraction of 1% of fu- ture cancers—​although there may be exceptions awaiting discovery in the neighbourhood of particular factories. The greater effect of Table 5.1.5  Carcinogenic agents used in medical practice (other than ionizing radiations) Agent Type of cancer Antineoplastic agents including:   Busulphan Leukaemiaa   Carmustine (BCNU) Leukaemiaa   Chlorambucil Leukaemiaa   Chlornaphazine Bladder   Cyclophosphamide Bladder, leukaemiaa   Lomustine (CCNU) Leukaemiaa   Etoposide in combination with cisplatin and bleomycin Leukaemiaa   Melphalan Leukaemiaa   MOPPb Leukaemia,a probably lungc   Semustine (methyl-​CCNU) Leukaemiaa   Thiotepa Leukaemiaa   Treosulphan Leukaemiaa Arsenic Skin, liver (angiosarcoma), lung Immunosuppressive drugs:   Azathioprine Non-​Hodgkin’s lymphoma, Kaposi’s sarcomad   Cyclosporine Non-​Hodgkin’s lymphoma, Kaposi’s sarcomad Methoxypsoralen (plus UV radiation) Skin Phenacetin Renal pelvis, ureter Plants containing aristolochic acid Renal pelvis, ureter Polycyclic hydrocarbons (coal-​tar ointment) Skin Sex hormones:   Unopposed oestrogens Endometrium, ovary, breast   Transplacental diethylstilboestrol Vagina and cervix (adenocarcinoma)   Diethylstilboestrol during pregnancy Breast   Oxymetholone (an anabolic steroid) Liver (hepatoma)   Oral contraceptives (combined)e Breast, cervix, liver (hepatoma)   Combined oestrogen-​progestogen menopausal therapy Breast, endometrium (diminishing with more days/​month of progestogen use)   Tamoxifene Endometrium a Acute or non​lymphocytic. b Combination of nitrogen mustard, vincristine, procarbazine, and prednisone. c Lung cancer might also be caused by certain other alkylating agents or regimens. d There have been excesses also of several other cancers in transplant patients treated with immunosuppressive drugs. e Oral contraceptives also reduce the risk of ovarian and endometrial cancers and tamoxifen reduces the risk of contralateral breast cancer and of breast cancer in women at high risk.

422 SECTION 5  Principles of clinical oncology the modern type of pollution with ultra-​fine particles and of the in- tense indoor pollution with smoke that occurs in parts of China is examined later under lung cancer and of erionite in certain Turkish villages under pleural cancer. The effect of polluted drinking water and food is more obscure. Modern analytical techniques permit the detection of chemicals at concentrations of less than one part per billion in both food and water and, in consequence, many have been detected that might ar- guably be carcinogenic, including pesticide residues and a variety of halogenated organic materials produced by the chlorination of water supplies. Relationships have been reported between the con- centrations of some of these compounds in water and the mortality from cancers of the bladder and, possibly, the large intestine, in dif- ferent localities, but it is extremely difficult to know what these rela- tionships mean as there are many potentially confounding factors. Mortality rates from cancers of the gastrointestinal and urinary tracts are, for the most part, stable or decreasing in early middle age, when the effects of new agents might be expected to show themselves Table 5.1.6  Occupational causesa of cancer Agent Type of cancer Occupationb Aromatic amines (several including): Bladder Dye manufacturers   4-​Aminodiphenyl Rubber workers   Benzidine Coal-​gas manufacturers   2-​Naphthylamine Some chemical workers Arsenic Skin, lung, bladder Copper and cobalt smelters Pesticide manufacturers Some gold miners Asbestos (all forms) Lung, pleura, peritoneum, larynx Asbestos miners Asbestos textile manufacturers Carpenters and general builders Insulation workers Shipyard workers Benzene Leukaemia Workers with glues and varnishes Berylliumc Lung Beryllium refiners and machiners Bis-​chloromethyl ether and technical-​grade chloromethyl methyl ether Lung Makers of ion-​exchange resins 1, 3-​butadiene Haematolymphatic system Styrene-​butadiene rubber workers Cadmiumc Lung Cadmium refiners Chromiumc Lungd Manufacturers of chromates from chrome ore; pigment manufacturers Dioxin (2,3,7,8–​TCDD) All cancers combined (not yet clear which sites) Exposed chemical workers Formaldehyde Nasopharynx Exposed industrial workers; embalmers Ionizing radiations Lung Uranium and some other miners Bone Luminizers Leukaemia, skin Radiologists, radiographers Mustard gas Larynx, lung Poison-​gas manufacturers Nickelc Nasal sinuses, lung Nickel refiners Polycyclic hydrocarbons in soot, tar, oil Skin, scrotum, lung, and sometimes bladder Coal-​gas manufacturers, roofers, asphalters, aluminium refiners, and many groups exposed to tars and selected oils Silica, when crystalline as quartz or cristobalite Lung Miners, stone workers, refractory brick workers Sulphuric acid mists (strong acid) Nasal sinuses, larynx Many industries, isopropanol manufacture, ‘steel pickling’ Ultraviolet radiation Skin (melanoma and non​melanoma) Farmers, seamen Vinyl chloride Liver (angiosarcoma, hepatocellular carcinoma) PVC manufacturers ? Nasal sinuses Hardwood furniture manufacturers ? Nasal sinuses Leather workers a Additional to those in the table, there have been raised risks of various cancers, of uncertain causation, found in workers in iron and steel founding; painters; and rubber manufacturers. b Typical occupations with the hazard. c Certain compounds or oxidation states. d And possibly nasal sinuses.

5.1  Epidemiology of cancer 423 first. In the absence of more specific evidence, it seems unlikely that chemical pollution of water and food could have a greater effect than the small effect already estimated for pollution of the air. Diet For many years there has been suggestive evidence that most of the cancers that are currently common could be made less so by modifi- cation of the diet, but, with few exceptions, there is still little reliable evidence as to the modifications that would be of major importance. If we define diet to include all materials that occur in natural foods, are produced during the processes of storage, cooking, and diges- tion, or are added as preservatives or to give food colour, flavour, and consistency, the ways in which diet could influence the development of cancer are legion. Ingestion of preformed carcinogens The most obvious is the ingestion of small amounts of powerful carcinogens or precarcinogens. Several have been identified in foodstuffs but only two have been related at all clearly to the produc- tion of cancer in humans. One is aflatoxin, a metabolic product of Aspergillus flavus, which contaminates stored or oily foods such as grains and peanuts in many countries, and is a major cause of liver cancer in the tropics among those individuals who are also chronic carriers of the hepatitis B (or less commonly hepatitis C) virus. Likewise, the salted fish eaten extensively in South China probably acts synergistically with the Epstein–​Barr virus to cause nasopha- ryngeal cancer. A third possible source is bracken fern, an extract of which is carcinogenic in animals. It is eaten extensively in Japan and has been tentatively linked with the development of oesophageal cancer. The polycyclic hydrocarbons and other mutagens that are produced in food by grilling or smoking have often been suspected of playing a role, and there is considerable evidence that processed meat consumption may increase colorectal cancer risk. It therefore seems likely that any effects of diet on the incidence of cancer in the Western world may be mainly by more indirect means, such as affecting the formation, transport, activation, or deactivation of carcinogens in the body or affecting the secretion of hormones. Overnutrition That overnutrition could affect the incidence of cancer was first sug- gested by Tannenbaum’s experiments on mice during the Second World War. These showed that the incidence of various spontaneous tumours and tumours produced experimentally could be halved by moderately restricting the intake of food without modifying the proportions of the individual constituents. This protective effect has subsequently been demonstrated repeatedly. It is now clear, however, that what is considered normal nutrition in developed countries in- creases the risk of breast cancer (by bringing forward menarche and increasing body size). With greater consumption obesity (i.e. a body mass index >25 kg/​m2) has been estimated to be responsible for 5% of all incident cancers in Europe and 10% of all cancer deaths in non​smokers in the United States: most notably, cancer of the breast in women after the menopause and cancers of the endometrium, large bowel, kidney, and gallbladder, oesophageal adenocarcinoma, and perhaps cancers of the prostate and thyroid. The increases in the two female cancers in postmenopausal women are probably at- tributable to the formation of oestrogen from androstenedione in adipose tissue, but for others the explanation is unclear. Meat and fat Figures for food consumption and cancer incidence and mortality rates in different countries show fairly close correlations between the consumption of fat, and to a lesser extent the consumption of meat, and the incidence of several types of cancer. The correlations are closest for breast cancer and cancer of the large bowel and are less strong for cancers of the endometrium, pancreas, and prostate. When, however, attempts are made to associate the consumption of either type of food with the disease in individuals within a country, the evidence is commonly conflicting. This could be because the international correlations are misleading, indicating only that the risks are correlated with something that is itself correlated with fat and meat consumption (e.g. some other aspect of a high gross na- tional product), but it could be partly because of the inaccuracy of dietary histories and partly because people within developed coun- tries eat such similar diets. The published data suggest that a high consumption of fat, and of red meat, is associated with a high risk of colorectal cancer, but these are not established causes. The claim that a high consumption of fat (or of particular types of fat) is associated with high risks of breast and endometrial cancers after the meno- pause, other than by providing a high-​calorie diet leading to obesity, is controversial. Fibre That fibre may play a part was suggested by Burkitt’s observation that several intestinal diseases, including cancer of the colon, were common in countries in which cereals were processed to remove the fibre and rare in rural Africa and Asia where they were not. The idea was attractive, as ‘fibre’ passes through the small bowel unchanged and serves as pabulum for the colonic bacteria, thus increasing faecal bulk and possibly protecting mechanically against the devel- opment of cancer by diluting any carcinogens present and hastening their transit through the bowel. The idea was too simple, however, and has not been confirmed (using the original definition of fibre) by either epidemiological studies on individuals in developed countries or by experiments aimed at reducing the recurrence of colorectal adenomas. In fact, fibre is difficult to define and the term is better replaced by ‘non​starch polysaccharides’ as there are many that share the characteristics of passing through the small bowel unchanged and being, for the most part, partially or wholly degraded by bac- teria in the large bowel. Some starch, moreover, known as ‘resistant starch’ and found in green bananas and cold potatoes, has similar physiological characteristics. It is uncertain whether ‘fibre’ in any of its manifestations can be considered as having a place in protecting against the development of cancer. Retinoids and carotenoids Experiments on animals and on cell cultures in vitro have suggested that vitamin A (retinol) and its esters and analogues (retinoids) may, in appropriate circumstances, reduce the risk of cancer by reducing the probability that partially transformed cells become fully trans- formed and proliferate into clinically detectable tumours, although in other circumstances they appear to have opposite effects. Human studies, however, did not support the idea that serum levels of ret- inol are related to the risk of any type of cancer, at least in countries in which clinical symptoms of vitamin A deficiency seldom or never occur. Such studies suggested that the risks were inversely related to the serum level of β-​carotene, which acts as an antioxidant and

424 SECTION 5  Principles of clinical oncology is broken down to produce retinol. When β-​carotene was put to the test of clinical trials, however, it provided no benefit and the inverse relationship commonly observed in epidemiological studies is pre- sumably due to confounding with some other protective factors in vegetables. Other components Many other components of the diet, including lycopene in tomatoes, indoles in brassicas (e.g. cabbages and sprouts), phyto-​oestrogens (plant chemicals structurally similar to oestradiol), fresh fruit and vegetables, vitamins C, D, and E, and calcium and selenium have also been proposed as protective agents. Conversely, nitrates, ni- trites, secondary amines, and the preservation of food by salting, have been thought to increase the risk of cancer. For some the evi- dence is strongly suggestive:  notably for vitamin C as protective against gastric cancer and for salt-​preserved foods predisposing to it. In general, however, the evidence of benefit or harm is insufficient to justify any firm conclusion. Conclusion Considerable uncertainties remain about the effect of diet on cancer risks. Practicable modifications of the diet may well provide the means for reducing cancer deaths in developed countries by up to one-​third, but the range of uncertainty about this figure is large. Meanwhile the only major dietary changes that can be recommended with confidence in developed countries are a general increase in the use of fresh fruit and vegetables and a sufficient limitation of calories to avoid obesity. Reproduction, other factors affecting secretion of reproductive hormones, and other hormones Epidemiological observations have shown clear relationships be- tween a woman’s menstrual and reproductive history and the risks of cancers of the breast, endometrium, and ovary, which are gen- erally thought to reflect changes in hormonal secretions. Which hormones are concerned, however, and the mechanisms by which they act are, for the most part, still uncertain. An exception is endo- metrial cancer, the risk of which is directly related to the degree of exposure to oestrogen not followed after an appropriate interval by progestogen. Strong evidence that oestrogenic stimulation of the mammary tissues is a cause of most cases of breast cancer in de- veloped countries has been provided by randomized trials of tam- oxifen, an antioestrogenic drug that blocks the oestrogen receptors in the cells of the normal breast. The effect is large and rapid: five years of tamoxifen approximately halves the incidence of contralat- eral breast cancer in a woman who has had previous breast cancer, not only while the drug is being taken but also for some years afterwards. Exogenous oestrogen also increases the risk of breast cancer when given as HRT and endogenous oestrogen accounts for the increased risk associated with adiposity after the menopause, as androstenedione, which continues to be produced by the ad- renals, is converted to oestrogen in adipose tissue. It is presumably oestrogens, too, that cause a small increase in risk of breast cancer during and immediately after pregnancy and the oestrogen com- ponent of the steroid contraceptives that causes a similar small in- crease in risk during their use and for a few years after their use is stopped. It is, however, unclear which hormone-​related processes are involved in reducing the long-​term risk for the rest of a woman’s life that occurs some years after the occurrence of each pregnancy, and it is equally unclear why the use of oral contraception and the consequent suppression of ovulation reduces the long-​term risk of ovarian cancer. Sex hormones, it is thought, may also be involved in produ- cing cancers of the testis and prostate in men. For testis cancer, the strongest evidence has been for an effect of maternal oestrogen levels on the developing testis in utero, whereas for prostate cancer the evidence relates to adult androgen levels, but for neither has caus- ation been established. Randomized trials of the effects of physical or medical castration in men who already have prostate cancer have shown that progression of the disease can be slowed substantially, presumably by the reduction of androgenic stimulation. In recent years it has been found that prior raised endogenous levels of insulin-​like growth factor-​1 (IGF-​1) are associated with raised risk of cancers of the breast, colon, and prostate, and there has also been evidence that endogenous and exogenous growth hor- mone may affect the risk of colorectal cancer. Physical inactivity Physical inactivity contributes to the risk of cancer indirectly by increasing the risk of obesity but it may also contribute directly. Associations with colon, breast, and endometrial cancers have fairly consistently been reported, and an association with prostate cancer has been found in most studies. The mechanism of effect is uncertain, but possibilities include the effects of exercise on hormone levels, on immune function, and on intestinal transit time and hence the dur- ation of exposure of the colonic mucosa to faecal carcinogens. Interaction of agents Attribution of the risk of cancer to different causes is complicated by the fact that some agents interact with others to produce effects that are much greater than the sum of the separate effects of each on its own. An example is provided by smoking and asbestos, which multiply each other’s effects so that, compared with non​smokers in general, the incidence of cancer of the lung was increased six- fold among a group of asbestos insulation workers in the United States of America who did not smoke, but were heavily exposed to asbestos dust in the 1940s, 10-​ to 20-​fold among cigarette smokers in general who did not work with asbestos, and nearly 90-​fold among the asbestos workers who also smoked cigarettes regularly. Other examples are provided by smoking and radon (which interact simi- larly, though somewhat less than multiplicatively, to produce cancer of the lung), by smoking and alcohol (which interact to produce cancers of the mouth, pharynx, larynx, and oesophagus), and by in- fection with the hepatitis B virus and aflatoxin (which interact to produce cancer of the liver). Such interactions complicate the attribution of risk, as we may find ourselves appearing to claim that more cancer can be prevented than actually occurs by attributing, say, 80% of lung cancers in men heavily exposed to asbestos to their occupational exposure and 90% of the same cancers to cigarette smoking. Each, separately, is correct, but they cannot be added to show the combined effect. Conclusion Estimates of the proportions of fatal cancers that can be attributed to environmental and behavioural factors, grouped into 11 main categories, are given in Table 5.1.7. The evidence on which these es- timates are based is summarized in this chapter and in greater detail by Doll and Peto (1981), Stewart and Kleihues (2003), IARC (2012) and Thun et al. (2018).

5.1  Epidemiology of cancer 425 The sum of the best estimates in Table 5.1.7 amounts to less than 100%, despite the fact that some of the listed agents interact with one another to augment each other’s effect and that some fatal can- cers are consequently counted twice. The total would be somewhat more than 100%, however, if the true proportions attributable to some of the categories turn out to be nearer the upper end of the acceptable estimates. The estimates in the second and third columns of Table 5.1.7 do not distinguish between factors (such as tobacco) that are suffi- ciently understood to enable specific action to be taken with a guar- antee of success and those (such as diet) that are not. They should not, therefore, be taken as guides to the proportion of cancer deaths that can now be prevented by practicable means. This is illustrated by the fourth column in Table 5.1.7, which shows the proportions of United Kingdom cancer deaths that are reliably known to be avoid- able by practicable means. The percentage attributed to tobacco is more than the sum of the percentages reliably attributable to other specific factors for which practicable preventive measures are avail- able; and tobacco causes about twice as many deaths from other dis- eases as it does from cancer. The position is different in countries such as China, where hepatitis B virus causes are approaching as many cancer deaths as tobacco and the hazard for future generations can be avoided in a cost-​effective way by infant vaccination. Epidemiology of cancer by site of origin In the following account of the epidemiology of cancers arising in specific organs, the description of each type is preceded by notes showing its importance in England. One figure gives the propor- tion of all cancers that arise at the site, from national cancer registra- tions for England in 2014 (Office for National Statistics, 2016) while another gives the proportion of all cancer deaths allocated to the site in the national mortality statistics for England and Wales for 2014 (Office for National Statistics, 2015). A third gives the ratio of the age-​standardized incidence rates in England for each sex in 2014. The way in which the incidence of the disease varies with age is shown for males and females in a series of graphs, using data for England over a five-​year period (2008–​12). Trends in incidence and mortality for each type, along with the trends in possible causative factors, are given by Swerdlow et al. (2001). Trends internationally are commented on in the text, and described more fully in Doll et al. (1994). Comments on the total worldwide frequency of different cancers are based on data from Ferlay et al., (2014); they disregard incidence of non​melanoma skin cancers, for which reliable inter- national statistics are not available. Lip • 0.1% of all cancers and 0.02% of cancer deaths • Sex ratio of rates 1.7:1; age distribution like oesophageal cancer Carcinoma of the lip was one of the first types of cancer to be re- lated to an extrinsic cause when, more than 200 years ago, it was noted to occur characteristically in pipe smokers. Many years later it was realized that the disease could also be produced by smoking cigarettes, although much less readily, so that it must be produced by the chemicals in smoke rather than by the non​specific effect of local heat. It is also much more common in outdoor than in indoor Table 5.1.7  Estimated proportion of United Kingdom cancer deaths in 2005 attributed to previous exposure to different environmental and behavioural factors and proportion of future United Kingdom cancer deaths avoidable by known effects of practicable changes in current exposure levels Factor or class of factors Percentage of UK cancer deaths in 2005 attributed to
previous exposure levels Estimated percentage of future UK cancer deaths avoidable by known effects of practicable changes in current exposure levels Best estimate Range of acceptable estimates Tobacco 28 25–​30 <28a Alcohol 6 4–​8 <6b Ionizing radiation 3c 2–​4 <1 Ultraviolet radiation 1 1 <1 Infection (virus 3%, bacteria 2%) 5 4–​15 <1d Medical drugs <1 0–​1 <1 Occupation 4 3–​5 <1e Pollution 2 1–​5 <1 Diet 25 15–​35 <8f Reproductive and hormonalg 15 10–​20 <1 Other and unknown ? ? ? a The proportion of UK cancer deaths attributed to smoking fell from 34% in 1985 to 28% in 2005 and is still decreasing (as current levels of smoking would cause less than 28% of future UK cancer deaths). b Mostly cancer of the upper aerodigestive tract that could have been avoided by not smoking. c 0.8% diagnostic X-​rays, less than 0.1% all other manmade, 0.7% natural radon in houses, 1.8% other cosmic or terrestrial natural sources. d Cervical cancer currently causes 1.4% of UK female cancer deaths. Without screening it would cause several per cent; with even better screening it would cause a fraction of 1%. e Although asbestos exposure is now strictly controlled, the delayed effects of past exposure probably account for about 3% of current cancer mortality (including more than 1% from mesothelioma), and diesel, coal, and other smoke must also have appreciable effects, especially if they potentiate tobacco smoke. f For those of later middle age living in the United Kingdom, almost half are overweight (BMI 25–​30 kg/​mb) and a quarter are obese (>30 kg/​m2); minimal cancer mortality is at c.25 kg/​m2, and about 8% of current cancer mortality would be avoided if the overweight and obese had a BMI of 25 kg/​m2. (Instead, BMI is increasing by about 1 kg/​m2 per decade.) g Includes other factors affecting the secretion of reproductive hormones.

426 SECTION 5  Principles of clinical oncology workers and is induced by ultraviolet radiation in the same way as other cancers of the exposed skin. Solar ultraviolet radiation and to- bacco account, between them, for the great majority of all cases in the United Kingdom, probably multiplying each other’s effects. The disease is much less common than it used to be, because of the de- crease in both pipe smoking and outdoor work. Oral cavity and pharynx (excluding salivary glands and nasopharynx) • 2.4% of all cancers and 1.4% of cancer deaths • Sex ratio of rates 2.2:1; age distribution like oesophageal cancer Cancers of the tongue, mouth, and pharynx (other than naso- pharynx) are all related to smoking (of pipes, cigars, and cigarettes) and to the consumption of alcohol. The two factors act synergistic- ally and cancers at these sites are extremely rare in non​smokers who do not drink alcohol. There is also fairly consistent evidence of an association of risk with low intake of fruit and vegetables. Cancer of the tongue is much less common in Britain than it was 100 years ago, but the reason for this sharp decline is unknown. One explanation could be the decrease in syphilis, which was commonly believed to be a predisposing factor because of the clinical associ- ation with syphilitic leucoplakia. Recent increases in oral and pha- ryngeal cancer in the United Kingdom but not the United States are partly due to increased consumption of alcohol and possibly, in the case of pharyngeal cancer, due to human papillomavirus infection. Cancers that occur low in the hypopharynx are distinguished by a tendency to affect women who have suffered from iron-​deficiency anaemia and dysphagia. Cancers of the mouth and pharynx (excluding nasopharynx) are particularly common in Southeast and Central Asia where tobacco smoking was largely replaced by chewing tobacco, betel nut or leaf, and lime (calcium hydroxide). A close association with such chewing habits has been established by studies that have shown that the can- cers tend to originate in the part of the mouth in which the quid is usually held—​a characteristic that varies both between individuals and between areas. The materials chewed differ in different places and, al- though the disease is commonly described as ‘betel chewer’s cancer’, betel is not invariably a component of the quid and the most character- istic constituent seems to be a small amount of lime and, in most cases, some form of tobacco. In parts of Asia, the disease is so common that it accounts for 20% of all cancers and in those populations the abandon- ment of chewing would be the single most effective means of reducing the total incidence of cancer—​so long as the habit was not replaced by an increase in tobacco smoking. Among habitual quid chewers, the risks are particularly elevated in those who both chew and smoke—​ indeed, in parts of India most deaths from betel chewer’s cancer could have been avoided if those affected had not also smoked. The incidence might also be reduced by improved nutrition, as the disease in southern Asia tends to be associated with vitamin A deficiency. In parts of India where women tend to smoke local cigars and cig- arettes with the burning end inside the mouth to prevent them going out, the habit is associated with cancer of the palate. Salivary glands • 0.2% of all cancers and 0.1% of cancer deaths • Sex ratio of rates 1.4:1; age distribution, see Fig. 5.1.3 The salivary glands are not common sites for cancer anywhere. They are, however, relatively more common in circumpolar Inuits than others. No causative factors are known other than ionizing radiation exposure, and no notable changes in incidence over time have been reported. Nasopharynx • 0.1% of all cancers and of cancer deaths • Sex ratio of rates 3.0:1; age distribution, see Fig. 5.1.4 Cancers of the nasopharynx, unlike those in other parts of the pharynx, are not related to alcohol and are only weakly related to tobacco. They are rare in most populations but are common in Southern China, especially so in Cantonese originating from parts of Guangdong, where the disease is among the most common types 0 1 2 3 4 5 6 7 8 9 0– 5– 10– 15– 20– 25– 30– 35– 40– Age group Rate (per 100000) 45– 50– 55– 60– 65– 70– 75– 80– 85+ 10 11 Female Male Fig. 5.1.3  Annual incidence of cancers of the salivary glands, by age and sex. 0– 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Rate (per 100 000) 1.0 1.1 1.2 1.3 1.4 5– 10– 15– 20– 25– 30– 35– 40– Age group 45– 50– 55– 60– 65– 70– 75– 80– 85+ Female Male Fig. 5.1.4  Annual incidence of cancer of the nasopharynx, by age and sex.

5.1  Epidemiology of cancer 427 of cancer. A weak relationship with human leukocyte antigen type has been reported. Moderately high rates have been observed in Eskimos, American Indians, Malays, Filipinos, Thais, and in parts of North Africa. Rates decrease over succeeding generations in Chinese migrants to (low-​risk) Western countries. DNA characteristic of the Epstein–​Barr virus (EBV) has been detected in the nuclei of nasopharyngeal cancer cells and patients with the disease tend to have unusually high antibodies against EBV-​ related antigens. Among adults, sudden increases in certain EBV antigens in the blood often precede the appearance of a cancer by a few years. Infection with the EBV is, however, almost universal and can be only one of several agents that act in combination to produce the disease. One such agent in Southern China occurs in the ‘salted fish’ on which children are commonly weaned. This strongly fla- voured delicacy bears little relation to the salted fish eaten elsewhere, and might better be described as decomposing fish:  it contains various mutagens, and exposure to it in childhood when infection with EBV first occurs may alter the usual lifelong balance between host and virus in some hazardous way. Oesophagus • 2.5% of all cancers and 4.7% of cancer deaths • Sex ratio of rates 2.5:1; age distribution, see Fig. 5.1.5 Cancer of the oesophagus is the eighth most common cancer in the world. It is exceptional among malignancies in the extent of geographical variation in incidence, both internationally and often also over relatively small distances. Like other cancers of the upper respiratory and digestive tracts, cancer of the oesophagus is closely related to prolonged smoking and the consumption of alcohol. All types of smoking have comparable effects as, so it appears, do all alcoholic drinks, although spirits may be slightly more effective per gram of ethyl alcohol than other alcoholic drinks. Alcohol and tobacco act synergistically and, in the absence of either, the incidence of the disease in Western countries would be greatly reduced. Smoking raises the risk of both squamous cell and adeno- carcinoma of the oesophagus, whereas alcohol affects largely or solely the former. A few cases originate from the scars produced by poisoning with corrosive substances and a very few in conjunction with a particular hereditary form of tylosis (presenting with kera- toses of the palms and soles). The relatively small excess in men probably reflects the existence of other unknown causes in women, possibly nutritional in origin and similar to those responsible for cancers of the hypopharynx. Mortality (which, because of the high fatality rate, approaches incidence) fell progressively in men in Britain from the 1920s to the 1960s, in line with the fall in the con- sumption of alcohol, and rose again after 1960 when the trend in the consumption of alcohol reversed, but has stabilized since 2000. Since pipe smoking affects oesophageal cancer risks at least as strongly as cigarette smoking, no large effects on male oesophageal cancer trends could be predicted from the male switch from pipes to cigarettes, although the switch by females from non​smoking to cigarettes should, other things being equal, have produced a large upward trend. It appears, however, that other things were not equal and some other, possibly nutritional, cause of oesophageal cancer seems to have decreased, for the upward trend in oesopha- geal cancer in women has been moderate. Oesophageal cancer is associated in several studies with low fruit and vegetable consump- tion. In men, by contrast, the rates have increased since 1960 when based on smoking they might have been expected to decrease. To some extent this can be accounted for by the increased consump- tion of alcohol and possibly by an increase in the nitrosamine con- tent of tobacco smoke, which has resulted from changes in the method of curing tobacco and which could have a specific effect on the oesophagus. A part of the increase is due to an increased risk of adenocarcinoma at the lower end of the oesophagus, which may be associated with a decreased prevalence of Helicobacter pylori and gastritis, and an increase in gastro-​oesophageal reflux and Barrett’s oesophagus, which is a common precursor of the tumour. Obesity is associated with risk of adenocarcinoma of the oesophagus, per- haps via an effect on reflux. The balance of adenocarcinoma and squamous cell cancer of the oesophagus has altered greatly over time in Western countries, such that the former, which has been increasing, now generally predominates in men in the United Kingdom and United States, although not in much of Continental Europe. (In China, Japan, and Korea, however, the tumours are overwhelmingly squamous cell.) In Africa and Asia, the epidemiological features are quite dif- ferent and present some of the most striking, unsolved problems in the field of cancer epidemiology. In parts of China (particularly in north Henan but also elsewhere), and on the east coast of the Caspian Sea in Turkmenistan and Iran, oesophageal cancer is the most common type of cancer, with incidence rates in both sexes that are equal to the highest rates observed for lung cancer in men in European cities. Within China, the disease varies more than 10-​fold from one county to another. In parts of Africa, particu- larly in the Transkei region of South Africa and on the east coast of Lake Victoria in Kenya, extremely high rates are also observed, sometimes equally in both sexes and sometimes only in men. In these and several other areas, the high-​incidence zones are strictly localized and the incidence tails off rapidly over distances of 200 or 300 miles (c.300–​500 km). 15– 0 10 20 30 40 50 60 70 80 90 100 110 120 Rate (per 100 000) 20– 25– 30– 35– 40– 45– Age group 50– 55– 60– 65– 70– 75– 80– 85+ Female Male Fig. 5.1.5  Annual incidence of cancer of the oesophagus, by age and sex.

428 SECTION 5  Principles of clinical oncology When tobacco and alcohol are used they increase the hazard, but they are not the principal agents in these high-​incidence areas. Many dietary causes have been proposed, including micronutrient deficiencies, contamination of food and pickled vegetables by fungi (particularly by species of fusaria) with the production of carcino- genic metabolites, an agent associated with the production of beer from maize, drinking very hot beverages, and the residues left be- hind in pipes from smoking opium (which are commonly swal- lowed). None, however, is supported by any impressive, consistent epidemiological data. The high-​incidence area in Iran, which has been intensively investigated, is characterized by extreme poverty and a restricted diet consisting chiefly of home-​made bread and tea, with some sheep’s milk and milk products, and very little meat, vegetables, or fruit. In this area the disease has been common for centuries. In southern Africa, however, it seems to have become common only since the First World War. In China, where cancer of the oesophagus was the second most important neoplastic cause of death in the 1970s, incidence is still high, and it currently accounts for 13% of all cancer deaths. Stomach • 1.8% of all cancers and 2.7% of cancer deaths • Sex ratio of rates 2.4:1; age distribution like oesophageal cancer Until about 1980, gastric cancer was responsible for more deaths from malignant disease worldwide than any other; it is now third to lung and liver cancers with over 700 000 deaths per year, mainly in developing countries. Over the last 50 years, the incidence has declined in Western countries (see Fig. 5.1.6), and recently it has begun to do so in South America and Japan. The highest rates now are in parts of Japan, China, and Korea, with moderately high rates in countries in the ex-​Soviet Union and Eastern Europe and in parts of South America, while low rates are found both in North America and Australasia, and in some of the least developed parts of Africa. This contrasts with the strong socioeconomic gradient in incidence of the tumour seen within Western countries. Irrespective of whether the incidence in a country is high or low, the sex ratio is generally between 1.5 and 3 to 1. In migrants from high-​risk to low-​risk countries, for instance, from Japan to the United States of America, risk decreases with longer time since migration, but can take two or more generations to reach local levels. Risk of gastric cancer is raised in relation to gastritis associated with chronic infection by H. pylori (sometimes leading to atrophic gastritis), a diet deficient in fruit and green and yellow vegetables, and a poor diet with large amounts of salt and salt-​preserved food. Chronic infection with H. pylori, which is very common, is a major cause of peptic ulcer, a finding that is of consid- erable practical value in patients with ulcers, because the infection can generally be eliminated from the stomach by a short course of appropriate antibiotic therapy and this provides long-​term protec- tion against recurrence. Whether such treatments will have any ma- terial effect on the incidence of stomach cancer remains, however, to be shown. How these various factors influence the production of the disease is unclear. One possibility is that they encourage or dis- courage the formation of carcinogens in vivo, particularly perhaps the production of nitrosamines; but if they do, the intake of nitrates (which can be converted into nitrites by bacterial enzymes) is not a rate-​limiting factor. Changes in the prevalence of the aforemen- tioned three factors could have contributed to the decline in the in- cidence of the disease, but they could not have brought about such a large and widespread reduction in risk, and it seems probable that the better preservation of food, resulting from the extensive use of refrigeration, has played the major part. No risk has been detected from the consumption of mutagens produced by the different methods of cooking meat and fish, nor from food additives or pesticide residues. Some food additives may, on the contrary, have served to reduce risk (by avoiding food spoilage and hence improving nutrition, by avoiding contamination by carcinogen-​producing microorganisms, or by some antioxidant or other protective effect on the gastric epithelium). Risk of gastric cancer is also raised, moderately, by smoking, and raised by exposure to ionizing radiation. Large bowel • 11.5% of all cancers and 9.9% of cancer deaths • Sex ratio of rates 1.5:1; age distribution like oesophageal cancer Cancers of the colon and rectum ought to be considered separately, as their causes are not identical. Cancer of the colon, for example, tends to occur more often in women than in men, particularly when it occurs on the right side, while cancer of the rectum is nearly twice as common in men. The geographical distribution also differs slightly, colonic cancer varying in incidence more than rectal cancer. Separate consideration may, however, sometimes be misleading as cancers commonly occur at the rectosigmoid junction and the site of origin of these cases is not recorded consistently. Moreover, there is a growing tendency to describe both diseases merely as ‘cancers of the large bowel’, which, according to the internationally agreed coding rules, are classed as cancers of the colon. The two diseases will, there- fore, be considered together. Cancers of the colon and rectum are the third most commonly in- cident cancer, and the fourth most common cause of cancer death, in the world. Over 1.3 million cases occur per year worldwide, mainly in developed countries. The disease is most common in Western countries, but incidence has generally stabilized or decreased in these countries in recent years, especially at younger ages. In Japan, where incidence used to be very low, rates have risen to be similar to those in the United States of America and Western Europe, and indeed in parts of Japan are now almost the highest in the world. Rates in migrants from low-​risk to high-​risk countries, for instance, in previous times from Japan to the United States, tend to gain much or all of the host population risks within the first generation. 1860 0 10 20 30 Age-standardized rate per 100 000 population 40 50 60 70 1880 1900 1920 1940 Year of death 1960 1980 2000 Males Females 80 Fig. 5.1.6  Mortality from cancer of the stomach, England and Wales, 1868–​1997, ages 35 and older, by sex.

5.1  Epidemiology of cancer 429 In most parts of Asia, and in Africa and Eastern Europe, large bowel cancer has been relatively uncommon (except in areas where chronic schistosomal infestation of the large intestine is prevalent; for example, high rectal cancer rates are found in Chinese counties in which Schistosoma japonicum was, until recently, a major cause of death). Rates tend to rise markedly, however, with the introduction of a Western lifestyle. Incidence rates in different countries correlate closely with the per caput consumption of fat and meat and crudely with the con- sumption of processed foods from which the natural fibre has been removed. Ways in which these and other dietary constituents might influence the development of the disease have been discussed under diet. Other factors associated with increased risk are obesity and physical inactivity. A  weak but reasonably consistent association with smoking has been observed. There has been an association with consumption of alcohol in most studies and with a high-​fat diet. Cases in childhood or early adult life occur as a complication of familial adenomatous polyposis and of hereditary non​polyposis colorectal cancer (HNPCC) syndrome. These conditions are de- termined by dominant genes, which increase the susceptibility to the disease so much that, unless prophylactic measures such as colec- tomy are undertaken, cancer is highly likely to develop at or before middle age. Many other cases develop from adenomatous polyps and a few occur as a complication of long-​standing ulcerative col- itis and Crohn’s disease. There is substantial evidence of reduced colorectal cancer risk among long-​term users of non​steroidal anti-​inflammatory drugs. There is also increasing evidence for an association of colorectal cancer risk with hormones of the growth hormone/​IGF-​1 axis:  colorectal cancer risks are increased in pa- tients with acromegaly; raised risks have been found in the general population in relation to prior greater levels of IGF-​1; and raised risks have been found in patients treated with growth hormone. Anal intercourse causing infection with types 16, 18, or some other specific types of the human papillomavirus is a probable cause of some anal carcinomas in both sexes, but patients who have sexu- ally transmitted anal warts that are due to other types of human papillomavirus are not for this reason at special risk of anal cancer. Liver • 1.5% of all cancers and 3.1% of cancer deaths • Sex ratio of rates 2.3:1; age distribution, see Fig. 5.1.7 Liver cancer is the second most common cause of cancer death in the world, with four-​fifths of cases occurring in developing coun- tries, and one-​half occurring in China alone. Incidence is consid- erably greater in men than women almost everywhere. Incidence rates have tended to be overestimated in developed countries be- cause the primary condition is often confused with metastases to the liver from cancer in various other organs, particularly at older ages. In recent decades, however, there has been a large increase in the United Kingdom and the United States from the very low level that existed previously, perhaps due in part to an increased prevalence of infection with hepatitis C. The disease is common in Southeast Asia (the highest recorded rates are in parts of China and Thailand), Korea, and Japan, and rates are also high in tropical Africa. In China it accounts for 15% of all cancer deaths and in sub-​Saharan Africa it is the second most common cancer in men. Most cases derive from the main cells of the organ (hepatocellular carcinomas) and are attributable pri- marily to chronic active infection, established early in life, with the hepatitis B virus, exacerbated by consumption of some spe- cific metabolite (e.g. aflatoxins) of particular types of fungi that contaminate stored foods. Neonatal vaccination against the virus produces a marked decrease in the proportion of children who be- come chronically infected. This has begun in many countries, now including the whole of China and parts of tropical Africa, and has already produced a decreased risk of hepatocarcinoma at young ages. Some cases, however, are caused by chronic infection with hepatitis C (a blood-​borne RNA virus that cannot be avoided by immunization; see Chapter 8.5.22). In developed countries, although some cases are also due to in- fection with hepatitis B and C viruses, more arise as complications of cirrhosis of the liver attributable to heavy and prolonged con- sumption of alcohol or, rarely, to haemochromatosis, certain types of porphyria, α1-​antitrypsin deficiency, and hereditary tyrosinaemia type 1. Occasionally, liver cancer is produced by drugs. A few cases have occurred in young men who have taken androgenic anabolic steroids to increase their muscular strength and a few from the use of steroid contraceptives, either arising de novo or from benign adenomas, which are themselves rare complications of the use of steroid contraceptives. Some can be attributed to smoking, for an association has been observed in parts of China where little alcohol is drunk and case–​control studies in Europe have shown an associ- ation after alcohol consumption has been taken into account. A second histological type (cholangiosarcoma) arises from the intrahepatic bile ducts, tends to occur at a somewhat later age than hepatocellular carcinoma, and, although generally less common than hepatocellular carcinoma, nevertheless accounts for an ap- preciable proportion of cases. In parts of China, Thailand, and elsewhere in south or east Asia it can be produced by chronic in- fection with liver flukes (Clonorchis sinensis or Opisthorchis viver- rini). In northeastern Thailand the latter fluke causes one of the highest rates of liver cancer in the world. In developed countries, primary sclerosing cholangitis is the main known risk factor for cholangiosarcoma. 0– 0 10 20 30 40 50 60 Rate (per 100000) 5– 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.7  Annual incidence of cancer of the liver, by age and sex.

430 SECTION 5  Principles of clinical oncology A third histological type that is extremely uncommon every- where has been variously described as reticuloendothelioma or angiosarcoma. It was first recognized as a complication of the use of Thorotrast as a contrast agent in neuroradiology, a long-​abandoned practice that led to chronic retention of insoluble thorium radio- nuclides in the marrow, spleen, and liver. In 1973, the disease was found to be an occupational hazard for men exposed to vinyl chloride monomer. A few hundred cases have occurred throughout the world in men who were heavily exposed in the manufacture of vinyl chloride polymer, and linear extrapolation suggests that the minute amounts that have leached out of plastic consumer products might have caused only a dozen or so cases altogether in the general public, if indeed they have produced any. A third, and even rarer, cause is prolonged exposure to inorganic arsenic, such as used to result from the medical prescription of Fowler’s solution. Despite these multiple causes only one case of hepatic angiosarcoma nor- mally occurs annually per 10 million people, which is why the rec- ognition of new causes has been easy. The relative rarity of cancer of the liver in most developed coun- tries is intriguing, since most of the carcinogens thus far discovered in experimental animals induce, perhaps with other cancers, tu- mours of the liver. Gallbladder and extrahepatic bile ducts • 0.6% of all cancers and of cancer deaths • Sex ratio of rates 0.9:1; age distribution like oesophageal cancer Cancers of the gallbladder and extrahepatic bile ducts are nearly always classed together, which is unfortunate as the causes differ. The former is more than twice as common in women as in men, is probably associated with obesity, and is usually preceded by (and probably caused by) cholelithiasis. The latter is slightly more common in men and is increased in incidence by liver fluke infection, primary sclerosing cholangitis, and long-​standing ul- cerative colitis. Both types are uncommon, and their aggregate varies only moderately from one population to another. The highest rates are recorded in Korea and Chile, and in women also New Delhi, India. The incidence of cancer of the gallbladder has fallen sharply in women in the United States in recent decades, but levelled off after falling in men. The decreases may be partly due to an increase in the rate of cholecystectomy in people who, having gallstones, are at greatest risk of cancer of the gallbladder. Pancreas • 2.7% of all cancers and 5.5% of cancer deaths • Sex ratio of rates 1.2:1; age distribution like oesophageal cancer Cancer of the pancreas is two to three times more common in regular cigarette smokers than in lifelong non​smokers. The chem- icals in cigarette smoke that specifically cause pancreatic cancer have not been identified, but the volatile nitrosamines in smoke that are absorbed from the alveoli and carried to the pancreas in the blood- stream are likely candidates. The disease is twice as common in dia- betics as in the population as a whole and risk is raised in patients with chronic pancreatitis. Cancer of the pancreas is generally regarded as a disease of the developed world, but the diagnosis is difficult in the absence of a well-​developed medical service and some of the relatively small geographical and temporal variations may be due to variation in diagnostic standards. The greatest reported rates are in US blacks. Mortality rates in the United States have been stable for the past 40 years, and in Britain stable apart from a decrease in men in the 1980s and early 1990s. Nose and nasal sinuses • 0.9% of all cancers and 0.02% of cancer deaths • Sex ratio of rates 1.7:1; age distribution like oesophageal cancer Surprisingly, in view of the widespread exposure of the human nose to tobacco smoke and other airborne toxins, cancers of the nasal cavity itself are extremely rare. Most arise from the paranasal sinuses. Several occupational hazards have been recognized, including the refining of nickel, processes giving rise to exposure to strong sulphuric acid mists, and the manufacture of hardwood fur- niture and leather goods. It would be wrong, however, to conclude that all contact with nickel, hardwood dust, and leather creates a hazard. The hazards have been observed in special occupational situations in which exposure has been intensive and prolonged. The nickel-​refining hazard was first observed in South Wales where the nickel carbonyl process was used, but similar hazards were subse- quently observed with other refining processes in Canada, Norway, and the Soviet Union. In the Welsh refinery the workplace expos- ures were much heavier before the Second World War, and (des- pite the continued use of the nickel carbonyl process in Wales) no hazard of nasal sinus cancer has been observed among men first employed there since 1950. The hazard in furniture workers was first observed in High Wycombe (southern England) and appears to have followed the introduction of high-​speed woodworking ma- chinery early in the 20th century. A hazard certainly affects some other groups of woodworkers, but should not be assumed to affect furniture workers in general. Most nasal and nasal sinus cancers are squamous carcinomas, but the hazard from hardwood dust characteristically produced adenocarcinomas. In some of the groups exposed to this hazard, as many as 5% of the men developed the disease. This meant that the risk of adenocarcinoma was increased 1500 times (as this histo- logical type of the disease is normally very rare) and the hazard was, in consequence, easy to confirm once suspicion had been aroused. Chromate workers are sometimes said to experience a hazard of nasal cancer, but this may be an error due to confusion with the char- acteristic ‘chrome ulcer’ of the nasal septum. Such ulcers have not generally been found to become malignant. A causal excess of nasal sinus cancer has been seen, however, in women employed in the United States in the early 20th century to apply radium-​containing luminescent paint to dials and clocks, who ingested the radium when they licked the brushes to shape their tips. Risk of nasal cancer is also modestly related to smoking. Larynx • 0.6% of all cancers and 0.5% of cancer deaths • Sex ratio of rates 5.6:1; age distribution, see Fig. 5.1.8 Cancers of the larynx, like cancers of the oesophagus and buccal cavity, are closely associated with tobacco smoking and with the

5.1  Epidemiology of cancer 431 consumption of alcohol. The two agents act synergistically and in the absence of either, the disease is rare. The different parts of this small organ are, however, related to the two agents differently. Cancers of the glottis are strongly related to smoking, particularly to cigarette smoking, and only weakly to alcohol, while cancers of the epilarynx resemble cancers of the neighbouring hypopharynx and are strongly related to both agents and to pipe and cigar smoking equally with cigarette smoking. The highest reported incidence of laryngeal cancer in men is in the Azores and in parts of Spain and Cuba. Rates in women are relatively low everywhere and generally a small proportion of those are in men in the same place. Trends with time vary consid- erably between countries and between the sexes, reflecting trends in smoking and alcohol consumption, and probably some other aetiological factor, perhaps nutritional in character. There has been fairly consistent evidence for an inverse association of risk with fruit and vegetable consumption. That there are other causal factors is evident from the relatively high incidence rates in parts of India, Turkey, North Africa, and Brazil, which cannot be ac- counted for by tobacco and alcohol. The disease has also occurred as an occupational risk in the manu- facture of mustard gas and in processes that cause exposure to strong sulphuric acid mists. Lung • 12.6% of all cancers and 21.5% of cancer deaths • Sex ratio of rates 1.4:1; age distribution, see Fig. 5.1.9 Nearly all lung cancers are bronchial carcinomas and should prop- erly be so described. The term ‘lung cancer’ is, however, in such common use that it is used here as synonymous with bronchial carcinoma, although it actually includes a very small proportion of alveolar cell carcinomas and other rare types of cancer with dif- ferent characteristics. Lung cancer is the most common cancer in the world, with over 1.8 million new cases per year, and the most common cause of cancer death. Until the 1920s, lung cancer was uniformly rare (except in the Hartz mountains, see ‘Occupation’, next). In the next two dec- ades, German and then British pathologists began to comment on an apparent increase, but this tended to be dismissed as an artefact of the greatly improving methods of diagnosis and the establishment of special centres for thoracic disease. Gradually, however, the increase became so pronounced and the change in the sex ratio so marked that the increase could no longer be dismissed as wholly artefactual and by the late 1940s, when the age-​standardized mortality rate in men in the United Kingdom had increased 20 times, it was clear that the developed world had begun to see an epidemic of lung cancer comparable in severity to the epidemics of infectious disease of the past, though with a longer time scale. Until the 1940s, the increase among British women was largely a diagnostic artefact. Since 1950, however, diagnostic standards in middle age have changed very little, the increase in British men has been replaced by a decrease, while the increase among middle-​aged women has continued for longer, be- fore flattening (Fig. 5.1.10). As a result, the sex ratio (male rate divided by female rate), at for example 50–​54 years of age, which 0 10 20 30 Rate (per 100000) 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.8  Annual incidence of cancer of the larynx, by age and sex. 0– 0 100 200 300 400 500 600 Rate (per 100000) 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.9  Annual incidence of cancer of the lung, by age and sex. 6000 5000 4000 3000 2000 1000 0 1910 1920 1930 1940 1950 1960 1970 1980 1990 2000 2010 2020 Percentage of 1911–15 rate Males Females Fig. 5.1.10  Mortality from lung and pleural cancers, England and Wales, 1911–​2014, ages 0–​84, by sex. (Lung and pleural cancers aggregated because the data do not allow their separation before 1960. The trends are virtually for lung cancer, however, as pleural cancer has been so much less common.)

432 SECTION 5  Principles of clinical oncology rose from 1.8 after the First World War to 8.9 after the Second World War, was reduced to 1.1 in 2014. Changes in treatment have had little effect on the fatality rate, which remains extremely high, and real changes in mortality closely reflect real changes in incidence. Smoking These time trends can be explained almost entirely by the effect of smoking tobacco, particularly in the form of cigarettes, which caused more than 90% of all lung cancers in the United Kingdom in the early 1990s. Evidence of this effect was first obtained in the middle of the last century by comparing the smoking histories of patients with different diseases (case–​control studies). It was found that the proportion of patients who had never smoked was much smaller if they had lung cancer (the ‘cases’) than if they had some other disease (the ‘controls’), and the proportion who had smoked heavily was correspondingly greater. Further evidence was obtained by asking large numbers of ap- parently healthy men and women what they smoked and then fol- lowing them up to determine the causes of death of those who had died. Cohort studies of this type, in the United States, in doc- tors in the United Kingdom, and in other groups, have all shown similar results, the risk increasing with the amount smoked, and varying with the length of time cigarettes had been smoked. If attention is restricted to populations in which most cigarette smokers had been smoking cigarettes regularly since early adult life, lung cancer is about 20 times more common in regular cig- arette smokers than in lifelong non​smokers and up to 40 times more common in very heavy smokers. At first the relationship was less marked in women than in men, but this was because female smokers who were old enough to have a high risk of cancer ei- ther had not begun smoking cigarettes so early in adult life or had smoked them less intensively when they began, and the sex differ- ences in behaviour and risk have both been progressively elimin- ated with the passage of time. Further studies have found that the relative risk of lung cancer has increased with decreasing age of starting to smoke and de- creased with the number of years that smoking has been stopped (detectable at 5 years after stopping, but never reaching the risk of a lifelong non​smoker); that the national increases in incidence have appeared at appropriate times after the increase in cigarette sales (after due allowance is made for a spurious increase due to improved diagnosis and appropriate differences in consumption by men and women); and that there is a general parallelism between the incidence of the disease in different countries and social and religious groups and their prolonged consumption of cigarettes. Finally, and most encouragingly, the trend in mortality has reversed following reduction in smoking. By 2014, the mortality from lung cancer among men in their thirties in Britain was only one-​eighth of that of men of the same ages 60 years earlier, corresponding to the earlier changes in the prevalence of smoking. The reduction in tar delivery between 1939 and 1965 contributed to the reduction in lung cancer in young men after the war, but the later reduction had little effect because of changes in the way cigarettes were manufac- tured and in the way they were smoked to ensure an adequate intake of nicotine. At older ages the decreases are less striking, but they are now seen at all ages in British men. In British women, however, although peaks were followed by decreases at ages under 75 some years ago, there has been a stabilization or increase in rates in the last 10–​15 years. In recent years, it has been shown that indoor air pollution with tobacco smoke—​‘passive smoking’—​increases lung cancer risk, by about 15–​30% from long-​term adult exposure as a never-​smoking spouse of a smoker. Occupation Several other causes of lung cancer have been discovered as a result of observations in industry. Many thousands of men and women have experienced significant hazards from exposure to asbestos or to polycyclic hydrocarbons (from the combustion of fossil fuel). The former has given rise to hazards in asbestos mines, asbestos textile works, and insulation work in the shipbuilding and con- struction industries and the latter to specific hazards in the manu- facture of coal gas in coking ovens, in steel works, in aluminium foundries, and wherever substantial amounts of incompletely com- busted fumes were released into the working environment. Much smaller numbers of men have experienced substantial hazards from radon in the air of mines (not only when mining radioactive materials, but also when mining haematite and fluorspar under conditions in which radon seeped into the mine air from streams and the surrounding rock); from the manufacture of chromates and chrome pigments; from the refining of nickel, beryllium, and cadmium; from arsenic (in the manufacture of arsenical pesticides and in the refining of copper, which is always contaminated with arsenic); from exposure to bischloromethyl ether in the chemical industry; from exposure to vinyl chloride; from the manufacture of mustard gas, to a small extent from exposure to silica if sufficient to cause silicosis; and from painting work and in rubber manufac- ture. In one extreme situation (in the cobalt mines of the Hartz mountains in central Europe, which were subsequently mined for radium and uranium), the absolute risk of contracting lung cancer due to the occupational hazard of radon was so large that more than half the workers contracted the disease. In several other situ- ations with heavy exposure to asbestos or the early stages of nickel refining, the occupational hazard has affected as many as 20–​30% of the exposed men. Atmospheric pollution Some of the materials responsible for these occupational hazards—​ particularly the combustion products of fossil fuels—​are or have been widely distributed in the air of towns and it is still uncertain how far they have, in this way, contributed to the production of the disease in the general population. That lung cancer was more common in big towns than in small towns and rural areas is cer- tain, but this held as strongly for Oslo and Helsinki, two relatively unpolluted cities, as for more polluted ones. Differences between the largest towns and the least populated areas have seldom been more than threefold and much of the difference can be accounted for by past differences in cigarette smoking. Attempts to ‘allow for’ cigarette smoking have usually been inadequate, as it is impos- sible to take full account of such factors as the age of starting to smoke cigarettes, the amount smoked daily at different periods, and the method of smoking (number of puffs, depth of inhaling, and so on).

5.1  Epidemiology of cancer 433 It is clear, however, that in the absence of cigarette smoking any effect of urban pollution in developed countries is relatively small. Estimates, based on extrapolation from the heavy pollution with coal smoke that used to occur in large towns, suggest that in such towns it may have contributed, in synergism with smoking, to as much as 10% of the risk of lung cancer, but would have caused very little risk in non​smokers. On this basis, the present levels of pollution with benzo[a]pyrene and the other known lung carcino- gens in town air can be only very small. Modern pollution with ultrafine particles (<10 µm diameter) may, however, be more haz- ardous. Study of residents in six contrasting cities in the United States in which information about personal smoking habits had been obtained suggests that the risk in the most polluted city com- pared with that in the least polluted could be increased by about one-​quarter in both smokers and non​smokers. The position in some developing countries is different: notably in parts of China, where intense indoor pollution with smoke and fumes from heating and cooking more than doubles the risk of lung cancer in non​smokers. Radon The effect of another form of pollution—​that of indoor air with radon arising from naturally occurring radium in rock and soil—​ has been estimated by extrapolation from the effects of the much larger doses to which some groups of underground miners have been exposed, and by direct observation in studies of people with and without lung cancer. These studies suggest that indoor radon may contribute to about 3% of lung cancers in the United Kingdom and about twice as much in the United States. The absolute effects are far greater in smokers than in lifelong non​smokers, so that in the absence of smoking few cases would be produced. Geographical differences The development of the male lung cancer epidemic and the early signs of its departure have been most prominent in the United Kingdom and Finland, since the switch of young men to cigar- ettes was largely complete in these countries by the 1920s. In the United States, where cigarette consumption doubled during the Second World War, the peak of mortality occurred a little later. In some other developed countries, the development of the epi- demic is still further behind and it is only just beginning to ap- pear in many developing countries. For example, Chinese males, who now consume about a third of the world’s cigarettes, experi- enced a 10-​fold increase in cigarette consumption per head be- tween the 1950s and 1990s that may well eventually cause almost a million cancer deaths a year when the young men of today reach middle age. In women, development of the epidemic has generally been later than in men. (Only in the Maori population of New Zealand did it occur at the same time.) In the United Kingdom, the United States, and a few other developed countries, the female lung cancer rates from smoking are already substantial, but in others, such as Spain and France, the epidemic in women has scarcely begun. The greatest recorded incidence rates in men world- wide are in parts of Turkey, and in blacks in parts of the United States, and the greatest in women in American Indians in parts of the United States. A relatively high risk has long been noted in Chinese women who are non​smokers, irrespective of their country of residence, which is probably due to their exposure to mutagens in the fumes from oils used in cooking with a wok and from the coal smoke with which many Chinese homes have been heavily polluted. Mesothelioma (of the pleura and peritoneum) • 0.8% of all cancers and 1.6% of cancer deaths • Sex ratio of rates 6.0. Age distribution like laryngeal cancer The existence of a specific type of tumour arising from the pleura, or less commonly the peritoneum, was debated by pathologists until 1960 when Wagner and his colleagues reported that six African patients with a similar type of ‘peripheral lung cancer’ had all lived in villages that were heavily polluted with dust produced by the mining of blue asbestos (i.e. crocidolite). Since then, occu- pational asbestos exposure (in asbestos mines, shipyards, building construction, asbestos product manufacture, and other work) has been shown to be responsible for the great majority of mesotheli- omas, which are the predominant cancers of the pleura and peri- toneum. They are much less likely to be produced by white asbestos (chrysotile) than by brown asbestos (amosite) or blue, as the two latter persist for longer in the lungs. A few cases arise from neigh- bourhood pollution with asbestos or secondary contamination (e.g. from household contact with asbestos workers) and some in Turkish villages are due to the weathering into the general at- mosphere of erionite fibres in local rock and houses; these fibres are physically similar to asbestos although chemically different. A few cases have been caused by radiotherapy, and natural ion- izing radiations may be responsible for most of those that are not associated with asbestos. An SV40-​like virus has been found in some tumours, but it is uncertain whether it plays a part in causing the disease. Mesotheliomas seldom occur less than 15  years after first ex- posure to asbestos, commonly occur 25–​30 years afterwards, and may be delayed for 50 years or more. Hence cessation of use of as- bestos (peak imports to the United Kingdom, and peak production worldwide, were in the 1970s) will only lead to decreasing meso- thelioma rates several decades later. In the last two decades, the recorded mortality in men under age 65 in Britain has begun to decrease. Almost all mesotheliomas are fatal. Due to confusion with lung or other types of cancer, it is still uncertain how many cases have occurred each year and some of the large increase in Western coun- tries since 1960 may be artefactual. The highest recorded rates of mesothelioma incidence now are in men in Bremen, Germany and Genoa, Italy and rates are unusually high in the United Kingdom compared with other countries. Rates in women in Western coun- tries tend to be much lower than in men. Pleural mesothelioma is not related to cigarette smoking and the occupational hazard affects smokers and non​smokers alike. Bone • 0.2% of all cancers and of cancer deaths • Sex ratio of rates 1.2:1; age distribution, see Fig. 5.1.11 Sarcomas can affect any bone, but characteristically affect the long bones in adolescence. After 45 years of age they occur

434 SECTION 5  Principles of clinical oncology most commonly in bones affected by Paget’s disease (osteitis deformans), which predisposes to sarcoma so strongly that as many as 1% of all people affected by the disease eventually develop a bone tumour. Many different histological varieties occur, some of which appear to have different causes. Osteogenic sarcomas and chondrosarcomas are the most common, the former account­ ing for nearly all the adolescent peak. One rare type (Ewing’s tu- mour) occurs only in children and young adults and is almost unknown in black people, irrespective of the society in which they live. Ionizing radiation is the main known extrinsic cause. Cases have been produced after high-​dose radiotherapy, especially such radio- therapy to childhood cancer patients, and after internal radiation from radionuclides including thorium in Thorotrast, an erstwhile contrast medium, radium in ‘luminizers’, once used to paint clocks and dials, and radium-​224 therapy. Bone cancer risk has also been found raised after alkylating agent treatment of childhood cancers, and osteosarcoma risk is raised in several rare cancer syndromes, for example, Li–​Fraumeni syndrome (OMIM 151623), and in retino- blastoma patients. National statistics in Britain record no substantial change in inci- dence over the last 30 years. Internationally, bone cancer is relatively rare everywhere, with less geographical variation than for most cancers. Connective tissues • 0.6% of all cancers and of cancer deaths • Sex ratio of rates 1.5:1; age distribution, see Fig. 5.1.12 Sarcomas of the soft tissues include a variety of different diseases, all of which are rare everywhere. Some occur in genetic syndromes, for example, Li–​Fraumeni syndrome and neurofibromatosis type 1 (OMIM 162200), and others are caused by ionizing radiation. A  few might be caused by intensive immunosuppression or ex- posure to chlorophenols and related compounds, but the evidence is inconclusive. Melanoma of the skin • 4.4% of all cancers and 1.6% of cancer deaths • Sex ratio of rates 1.1:1; age distribution, see Fig. 5.1.13 Melanoma accounts for a small proportion of incident skin cancers but for most skin cancer deaths. Incidence, and to a lesser extent mortality, rates have been increasing in white populations as far back as data are available. In recently born generations in several popula- tions, however, this trend has stabilized or reversed. The incidence of the disease varies inversely with the amount of skin pigmentation, both comparing whites with non​whites, and when comparing within whites, in whom skin sensitivity to sun- shine (ease of burning and tanning) and fair or redhead complexion 0– 0 1 2 3 Rate (per 100000) 5– 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.11  Annual incidence of cancer of bone, by age and sex. 0– 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Rate (per 100000) 5– 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.12  Annual incidence of cancer of connective and other soft tissue, by age and sex. 0– 0 10 20 30 40 50 60 70 80 90 100 Rate (per 100000) 5– 10– 15– 20– 25– 30– Age group 35– 40– 45– 50– 55– 60– 65– 75– 80– 70– 85+ Female Male Fig. 5.1.13  Annual incidence of melanoma of the skin, by age and sex.

5.1  Epidemiology of cancer 435 predict risk. Risk is also related strongly to numbers of benign moles and atypical moles on the skin, and less strongly to markers of cu- taneous ultraviolet damage such as solar keratoses. There is a par- ticularly great risk for patients with giant congenital naevi and those with xeroderma pigmentosum. In white people the tumour occurs most commonly on the legs in women and the trunk in men, and is least common on the buttocks and soles of the feet (areas not ex- posed to the sun). In blacks, in whom melanoma is rare, a high pro- portion occur on the soles of the feet. Incidence rates in white people vary roughly in proportion to the flux of sunshine (ultraviolet radiation) in the countries in which they live, although the reverse is true across Europe, probably reflecting darker complexions as one goes south. Risks rise in white migrants from countries with low insolation to those with higher insolation, especially if migration is at a young age. The greatest recorded inci- dence is in Queensland, Australia, and in whites in Hawaii, where melanoma is the second (Queensland) or third (Hawaii) most common cancer (disregarding non​melanoma skin cancer). For all skin sites combined, the incidence is not, however, greater in out- door than indoor workers (rather the reverse, in fact, perhaps due to the protective effects of a semi-​permanent suntan). The totality of the evidence suggests that recreational intermittent exposure of un- tanned skin to solar ultraviolet radiation, such as when sunbathing, is the principal cause of melanoma and the reason for the rising rates. The relationship is not simple, however, and indeed melanomas of the head and neck occur typically in elderly outdoor workers and appear to relate to chronic ultraviolet exposure. There is suggestive, but not decisive, evidence that use of sunbeds increases melanoma risk, and inconsistent evidence that PUVA (methoxypsoralen UVA) treatment can do so. Skin (non​melanoma) • 38.0% of all cancers and 0.5% of cancer deaths • Sex ratio of rates 1.6:1; age distribution like oesophageal cancer Non​melanoma skin cancers are the most common cancers in fair-​skinned populations, although rarely fatal. The predominant cause is sunshine (ultraviolet) exposure, and correspondingly the highest reported rates of incidence are in Australia. Rates have been rising in white populations across the world for many years, and tend to be greater in men than in women. The tumours are of two main types: basal cell and squamous cell carcinomas. The former, also known as rodent ulcers, have a causation that appears to relate to both cumulative ultraviolet exposure and intermittent intense exposures such as sunbathing. They occur mainly on parts of the body that are regularly exposed to the sun and, in particular, on the face, head, and neck. They are more common in outdoor workers, such as seamen and farmers, than in indoor workers; more common in fair-​skinned (and blond and red-​haired) than in dark-​skinned (and dark-​haired) people; and are almost un- known in blacks (except those who suffer from albinism). Some few cases have been produced by exposure to X-​rays, but the risk is very small unless the dose is very large and they seldom occur after normal courses of radiotherapy. People who suffer from xeroderma pigmentosum, a hereditary condition in which there is a defect in the enzyme responsible for the repair of the damage done to DNA by ultraviolet radiation, develop large numbers of skin tumours at an early age in response to even quite mild sun exposure (see Chapter 23.9). Squamous cell carcinoma is also produced by ultraviolet radi- ation, risk being proportional to cumulative sun exposure, and in PUVA-​treated patients proportional to cumulative PUVA dose. It accounts for about 20% of cancers on ultraviolet-​exposed skin. It is, however, the principal type of skin cancer produced by various carcinogenic chemicals, and particularly by polycyclic hydrocar- bons in the combustion products of coal. These chemicals have been responsible for the scrotal cancers of chimney sweeps, who accu- mulated soot in the folds of the scrotum; of mule spinners, whose clothes were saturated with carcinogenic oils; and of various other groups of workers whose clothes were contaminated with tar. They have caused (and still do cause) cancers of the forearm in industrial workers whose arms are regularly splashed with tar or carcinogenic oils, cancers of the groin in India, localized by the continued friction of the dhoti cloth, and cancers of the abdomen in Kashmir associated with the habit of carrying a kangri, or small stove, inside the clothes in winter to keep warm. Squamous cell carcinoma has also been due to prolonged ex- posure to arsenic, which is excreted by the skin and in the hair, when it may be accompanied by arsenical pigmentation and keratoses. All these conditions have been produced by prolonged medical treat- ment with inorganic arsenic, which used to be prescribed for a var- iety of chronic conditions, by the consumption of well water from arsenic-​rich soils, and by occupational exposure in the smelting of copper and cobalt (the ores of which often contain arsenic) and in the manufacture of arsenical pesticides. How large a part human papillomaviruses play in the devel- opment of squamous carcinoma of the skin is unclear. The type 5 virus is responsible for the warty lesions of epidermodysplasia verruciformis, some of which progress to cancer, and other types of the virus may contribute to the greatly increased risk that follows the intensive immunosuppression given to enable the survival of organ transplants. A third type is Kaposi’s sarcoma. It is associated with AIDS when AIDS results from homosexual intercourse. Frequent at first, par- ticularly in the United States, the association has become progres- sively less common. Before the advent of AIDS, Kaposi’s sarcoma was common in some parts of Central Africa, where it occasionally affected children, progressed rapidly, and could account for as many as 10% of all hospital patients with cancer. Elsewhere it was rare, but indolent cases occurred occasionally in developed countries, princi- pally on the legs of middle-​aged and elderly men. The disease is ini- tiated by infection with the human herpesvirus type 8, but cofactors are required for tumour development. Breast • 15.6% of all cancers and 7.1% of cancer deaths • Sex ratio of rates 0.01:1; age distribution, see Fig. 5.1.14 Cancer of the breast is the second most common cancer in the world and the most common in women, with over 1.6 million cases occurring per year. Incidence rates in women are greatest in Western countries (greatest in Belgium), somewhat lower in Eastern Europe, and lower again in Asia, Africa, and parts of South America. The geographical differences are unlikely to be chiefly due to genetic

436 SECTION 5  Principles of clinical oncology factors, as rates in migrants from low-​ to high-​incidence countries rise considerably, to levels intermediate between the two, and there is a further rise in succeeding generations. In many countries in- cidence rates have tended to rise slowly over several decades, but mortality rates have started to decrease in recent years in Western countries because of more effective treatments and probably the ef- fect of screening (Fig. 5.1.15). Hormonal factors, particularly oestrogens, are important in the production of the disease. The duration of ovarian activity is rele- vant, as the disease is particularly common in women who have an early menarche and a late menopause (the former being more important than the latter). Pregnancy produces a short-​term in- crease in risk, followed after several years by a lifelong decrease, particularly after teenage or early adult pregnancies. The incidence in later life increases progressively with a woman’s age at the time of her first full-​term pregnancy, being about three times greater when the first birth occurs after 35 years of age than when it occurs before 18 years. Full-​term pregnancies after the first have an add- itional protective effect. Pregnancies that end in abortion have little or no effect, however. The duration of lactation has an additional protective effect but is not marked unless it continues for a year or more. Risk of breast cancer is raised in women with benign breast dis- ease, the degree of risk varying according to the type of disease. Risk is also raised by alcohol consumption, by lack of physical exercise, and by ionizing radiation exposure at young ages, with particularly high risks in women given high-​dose mantle radiation for Hodgkin’s disease. There is not, however, good evidence for causation by any form of environmental pollution. Parity and menstrual differences are insufficient to account for the large variations in the incidence of the disease between different countries, which seem to be correlated with a high standard of living (i.e. with life in a developed country). Diet might play a part, but the evidence is complex and inconclusive. Obesity is associated with a reduced risk before the menopause, for uncertain reasons, and with a raised risk after the menopause. Height is associated with increased incidence both for pre-​ and postmenopausal women. Oestrogens prescribed medically, as HRT after the menopause, increase the risk by about 2% for each year of use; combined with progestogens in the contraceptive pill they increase it by about 25% during use, but the increased risk gradually disappears over 10 years, when use is stopped, as it does after HRT is stopped. Diethylstilboestrol, pre- scribed to pregnant women from the 1940s to the 1970s, increased breast cancer risk in these women as well as causing clear cell adeno- carcinoma of the vagina and cervix in daughters exposed in utero. Tamoxifen, an antioestrogen, reduces the incidence of the disease in the unaffected breast when prescribed for the treatment of breast cancer, and reduces incidence in high-​risk women. As well as the re- lation to sex hormone levels, there is evidence that risk of premeno- pausal breast cancer relates to prior endogenous levels of insulin-​like growth factor-​1 (IGF-​1). Breast cancer has been a particularly fertile area of genetic epi- demiology in recent years, with the identification of several high-​ risk genes, some related to clinical syndromes (e.g. Cowden’s disease, and carriage of an ataxia telangiectasia mutation) but most related only to cancer risk, and with the discovery of more than 150 single nucleotide polymorphisms associated with susceptibility. Uterine cervix • 0.9% of all cancers and 0.5% of cancer deaths • Confined to women; age distribution, see Fig. 5.1.16 Carcinoma of the cervix is the fourth most common cancer in women worldwide, and the most common in parts of Africa; it used also to be common in Europe and North America. It has always been rare in Jewish women and has tended to be less common in Muslim women than in women of other faiths living in the same country (e.g. Hindus in India). Changes in incidence over time have been difficult to assess, partly because mortality data have not always distinguished be- tween deaths due to cancer of the cervix and those due to cancer of the corpus (or endometrium), partly because the introduction of screening programmes has made it possible to diagnose and treat premalignant lesions (see next), and partly because hysterectomy for benign conditions has become progressively more common, with a corresponding reduction in the number of uteri in which the disease could occur. Despite these complications there can be no doubt that the disease has become substantially less common in Europe and North America than it was before the Second World War. 5– 0 100 200 300 400 500 10– 15– 20– 25– 30– 35– 40– Age group Rate (per 100000) 45– 50– 55– 60– 65– 70– 75– 80– 85+ Female Male Fig. 5.1.14  Annual incidence of breast cancer, by age and sex. 80 60 40 20 0 1860 1880 1900 1920 1940 Year of death Age-standardized rate per 100000 population 1960 1980 2000 Fig. 5.1.15  Mortality from breast cancer in women, England and Wales, 1868–​1997, ages 35 and older.

5.1  Epidemiology of cancer 437 The rarity of the disease in Jewish women and its relative rarity in Muslim women suggest that male circumcision may reduce the risk of its development, but this is unlikely as the state of circum- cision of her husband has no substantial effect on a woman’s risk of developing the disease in communities in which only some men are circumcised. Cleanliness is likely to be protective, as the disease is relatively uncommon in communities that practise ritual ablution before and after intercourse and, within each community, it becomes less common with rising socioeconomic status. Squamous carcinoma, which constitutes the vast majority of all cases, is intimately connected with sexual activity. It almost never oc- curs in virgins and increases in frequency with the number of sexual partners that a woman or her partner has had and with younger age at first sexual intercourse. Almost all cases are attributable in part to infection with certain types of the human papillomavirus, most notably types 16 and 18. A vaccine effective against these two types has been developed, and vaccination should greatly reduce future incidence. The development of squamous carcinoma is preceded by patho- logical changes limited to the epithelium, known as cervical intraepithelial neoplasia (CIN) types I, II, and III. CIN III is associ- ated with the same types of virus as squamous carcinoma, but CIN I and CIN II generally are not. The changes may progress from one to another, finally leading to carcinoma, but the early lesions (CIN I and II) commonly regress and even CIN III (previously known as carcinoma in situ) may do so occasionally. The lesions can be recog- nized in cervical smears and destroyed by lasers or extensive biopsy and the occurrence of clinical disease can be greatly reduced by the examination of all sexually active women every two or three years and the treatment of advanced CIN lesions. Other factors associated with the production of the disease are high parity, the use of oral contraceptives, and cigarette smoking. Both of the latter tend to be associated with behaviour conducive to venereal infection, but it appears that this tendency cannot wholly account for their association with the disease. That smoking may be responsible for some cases is suggested by the presence of mutagens in the cervical mucus of smokers that are not present in the secre- tions of non​smokers. Adenocarcinoma of the uterine cervix is generally uncommon, but has become somewhat more common recently in several coun- tries. It is related to human papillomavirus infection, but also appears to relate to factors similar to those for endometrial adenocarcinoma. Endometrium (corpus uteri) • 2.6% of all cancers and 1.3% of cancer deaths • Confined to women; age distribution like cancer of ovary The epidemiological features of endometrial cancer are in many re- spects the opposite of those of cervical cancer. Histologically, it is nearly always an adenocarcinoma. It is common in developed coun- tries, especially parts of the United States of America, and rare in poor populations. It is inversely related to parity, but not otherwise related to coitus, and is unaffected by the number of sexual partners. Like cancer of the breast, it is positively associated with late meno- pause, and perhaps with early menarche. Incidence in the United Kingdom and most Western countries has increased markedly in the last 20 years. In the United States there was a peak in the 1970s followed by a decline, as discussed next, but rates have been fairly stable since 1980. The one factor known to produce the disease is regular exposure to oestrogens, unopposed by progestogens. This leads to endometrial hyperplasia and eventually, in some cases, to cancer. Known causes include oestrogen-​secreting tumours of the ovary, the use of oral contraceptives in which oestrogens and progestogens are prescribed sequentially (types that have now been abandoned), the use of un- opposed oestrogens to relieve menopausal and postmenopausal symptoms, and adiposity. The last causes the disease because oes- trogens are produced in the body after the menopause in adipose tissue from the adrenal hormone, androstenedione. Tamoxifen, an analogue of natural oestrogens, which blocks oestrogen receptors in the breast and hence acts as an antioestrogen, can, due to dif- ferences between the hormone receptors in different tissues, have a pro-​oestrogenic effect in some other organs, and increases the in- cidence of endometrial cancer in proportion to the length of treat- ment. Endometrial cancer risks are reduced in users of combined (concurrent oestrogen and progestogen) oral contraceptives. It is improbable that oestrogens are initiating agents. They are not mutagens in vitro and the changes that took place in the incidence of the disease in the United States following the increase and sub- sequent reduction in the use of unopposed Premarin (a conjugated oestrogen) for the treatment of menopausal symptoms occurred so quickly that they make sense only if oestrogens act on some late stage(s) of the carcinogenic process. Endometrial cancer risk is raised in women who are physically inactive and in women with dia- betes, in each instance with evidence that the relation may be more than just a consequence of obesity. Endometrial cancer risk, at least postmenopausally, appears to be reduced among smokers. Ovary • 2.0% of all cancers and 2.5% of cancer deaths • Confined to women; age distribution, see Fig. 5.1.17 About 90% of ovarian cancers are of the surface epithelium, and the causes of the tumour that have been recognized may refer only 15– 20– 25– 30– 35– 40– Age group Rate (per 100000) 45– 50– 55– 60– 65– 75– 70– 85+ 80– 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Female Fig. 5.1.16  Annual incidence of cancer of the uterine cervix, by age.

438 SECTION 5  Principles of clinical oncology to these. International variation in ovarian cancer incidence is less than for most cancers, with high rates generally in Western coun- tries, but also, for instance, in the Philippines. The highest recorded rates are in Latvia and parts of England. Incidence decreases pro- gressively with increasing number of children. There is no strong relation to age at menarche or age at menopause, however. Risk of the disease is reduced by the use of oral contraceptives, more greatly with longer use, and seems to depend on the lifetime number of ovulations. Risk is decreased by tubal ligation and perhaps by hys- terectomy, and increased by mutations in the BRCA1 and BRCA2 genes. There is also an association of mucinous ovarian cancers with smoking. Prostate • 13.4% of all cancers and 7.1% of cancer deaths • Confined to men; age distribution, see Fig. 5.1.18 Cancer of the prostate is the second most common cancer in men worldwide and is found mainly in Western countries. Rates are par- ticularly low in much of Asia and parts of North Africa. It is more characteristically a disease of old age than any other cancer, so that it comes to play a much larger part in clinical experience as the pro- portion of old people in the population increases. It is unusual in that foci of cells resembling cancer can be found in a high propor- tion of clinically normal prostates, so that the recorded incidence is drastically increased by increasing the number of prostatic biopsies. Increases in incidence have been recorded in many Western coun- tries. The introduction of prostate-​specific antigen (PSA) testing has given rise to considerable artefacts in recorded rates in the United States. Some increase in mortality had been recorded in Britain and the United States, up to the early 1990s but in the last 10 years there has been a decrease by a half in the United States. The weight of evi- dence suggests that the disease is principally due to factors that have affected society for many years, but what these factors are remains obscure. Associations have been reported with both increased and decreased sexual activity and there is some evidence, not conclusive, for a reduced risk in men who eat more tomatoes and tomato prod- ucts, the main source of lycopene. There is also some evidence for reduced risk in men who take considerable physical exercise. On general grounds it seems likely that the disease is dependent on sex hormone imbalance (particularly as castration or oestrogen admin- istration slows the progression of clinical disease) but the nature of the imbalance is unknown. Decreased risks of prostate cancer have been found in patients with Klinefelter’s syndrome and those with diabetes; the former, and there is some evidence that also the latter, have reduced androgen levels. An association of prostate cancer risk has been found with prior raised circulating levels of IGF-​1. Vasectomy was thought to increase the incidence of the disease, but probably does not. Two epidemiological observations stand out:  the exceptionally high incidence in black populations in the United States (the highest recorded), and the low (although increasing) incidence in parts of Japan in contrast with other developed countries. Both may be partly due to genetic factors, but they are not wholly so, as Japanese and blacks have much higher rates in the United States than they have in Japan and Africa, respectively. Testis • 0.7% of all cancers and 0.03% of cancer deaths • Confined to men; age distribution, see Fig. 5.1.19 Testicular cancers are of two main types. Seminomas, which are the more common, have a peak incidence at about 35 years of age and teratomas, commonly called embryonal carcinomas in the United States, have a peak incidence about 10 years younger. Testicular cancer is the most common cancer in young white men in many countries, but much less common in non​white groups living in the same areas, except Polynesians, and is comparatively rare in most of Asia. Tumours after 50 years of age are mostly lymphomas and are now classed as such. Both genetic and environmental factors are important. On the one hand, the disease is uniformly rare in black populations, whether in Africa or in the United States. On 0– 5– 10–15–20–25–30–35– Age group Rate (per 100 000) 40–45–50–55–60– 75–80– 65–70– 85+ 0 10 20 30 40 50 60 70 Female Fig. 5.1.17  Annual incidence of cancer of the ovary, by age. 0– 10–15–20–25–30–35–40– Age group Rate (per 100 000) 45–50–55–60–65– 80– 70–75– 85+ 0 100 200 300 400 500 600 700 800 900 Male Fig. 5.1.18  Annual incidence of cancer of the prostate, by age.

5.1  Epidemiology of cancer 439 the other, it has increased in incidence over many decades in white populations around the world. In Britain, the increase began in the 1920s and affected first the higher socioeconomic groups. The in- crease trebled the mortality at 15–​34 years of age and produced a sharp peak in young adult life that had not previously been pre- sent. Mortality has greatly decreased in recent decades, however, as treatment has improved (Fig. 5.1.20), although in the United States the decrease has stalled in the last 15 years. The disease is generally more common in more prosperous populations. Greatest incidence rates are in parts of Chile and of Switzerland, and in New Zealand Maoris. Testicular cancer risk is greatly raised in men with XY go- nadal dysgenesis, and in brothers, and to a lesser extent fathers, of cases. The cancer is much more likely to occur in an undescended than in a normal testis (c.10% of cases in whites are in men who have had maldescent), and in a testis opposite one that has been cancerous, and is also associated with prior inguinal hernia, but otherwise its causes are unknown. The leading hypothesis, in part because of the age distribution and association with cryptorchidism, has been that the aetiology is prenatal, due to exposure in utero to raised maternal oestrogen levels during the first trimester of preg- nancy. Potential prenatal factors have been extensively investigated, with the strongest evidence for reduced risk with late birth order and raised risk for dizygous twins and boys born prematurely, but none are established. Penis • 0.2% of all cancers and 0.1% of cancer deaths • Confined to men; age distribution like cancer of the salivary glands Carcinoma of the penis has not been common in the past few dec- ades except in some parts of tropical Africa and Brazil, where it has accounted for 10% of all cancers in men. Recorded rates now are low everywhere, although greatest still in tropical Africa and Brazil. It is avoided almost entirely by circumcision at birth and is very rare if circumcision is carried out in boyhood. Phimosis is a risk factor. In developed countries penile cancer is rare even in the absence of cir- cumcision if the glans, coronary sulcus, and foreskin are kept clean. The oncogenic types of the human papillomavirus (principally types 16 and 18) can usually be identified in the malignant cells and are important causes of the disease. Kidney • 3.5% of all cancers and 2.6% of cancer deaths • Sex ratio of rates 1.9:1; age distribution like liver cancer Cancers of the kidney are of three main types: nephroblastomas (or Wilms’ tumours), adenocarcinomas (or hypernephromas) of the renal parenchyma, and transitional and squamous cell carcinomas of the renal pelvis. The first are limited to childhood, occur with al- most equal frequency everywhere, and apart from a few of genetic origin, are of unknown aetiology. The second constitute by far the majority of all cases, are more common in Europe (greatest in the Czech Republic) and North America (greatest in some American Indians) than in Africa and Asia, and have been increasing in inci- dence in many Western countries. Cigarette smoking is one cause, but the association is weak and it does not account for more than about one-​quarter of the cases. Obesity is also a risk factor, and phenacetin-​containing analgesics may have been, but the evidence is less clear than it is for renal pelvis cancers. The third type of renal cancer (carcinoma of the pelvis) constitutes some 10% of all cases. Three established causes are occupational exposure to the chemicals that cause cancer of the bladder, cigar- ette smoking, and the consumption of phenacetin in large enough amounts to produce analgesic nephropathy. In all three cases, the hazards are relatively small (two-​ to threefold). Cancer of the renal pelvis is also caused by ingestion of plants containing aristolochic 0– 10– 5– 15–20–25–30–35–40– Age group Rate (per 100 000) 45–50–55–60–65– 80– 70–75– 85+ 0 2 3 5 7 9 11 13 16 15 18 1 4 6 8 10 12 14 17 19 Male Fig. 5.1.19  Annual incidence of cancer of the testis, by age. 300 250 200 150 100 50 0 1910 1920 1930 1940 1950 1960 1970 1980 15–49 50–84 1990 2000 2010 2020 Percentage of 1911–15 rate Year of death Fig. 5.1.20  Mortality from cancer of the testis, England and Wales, 1911–​2014, by age.

440 SECTION 5  Principles of clinical oncology acid, which are used in Chinese herbal remedies and is responsible for Balkan nephropathy (Chapter  21.9.2), which increases renal pelvis cancer risk several hundredfold. Bladder • 2.9% of all cancers and 3.4% of cancer deaths • Sex ratio of rates 3.6:1; age distribution like oesophageal cancer Cancer of the bladder is almost universally several times more common in men than women. Its greatest recorded incidence in men is in parts of Europe, with low rates in much of Africa, Asia, and South America, and in non​whites compared with whites in the United States of America. The tumour can be produced by cigarette smoking, occupational exposure to aromatic amines, infection of the bladder with Schistosoma haematobium, the use of phenacetin-​ containing analgesics, the medical prescription of chlornaphazine (N,N′-​bis(2-​chloroethyl)-​2-​naphthylamine) and cyclophospha- mide, and ionizing radiation. There has been evidence for a rela- tion to chronic consumption of inorganic arsenic, a contaminant of the water supply in parts of Taiwan and other countries. There has also been some evidence for an association with urinary tract in- fection, supported by raised risks in paraplegics, who tend to have frequent urinary tract infections. Most bladder cancers are transi- tional cell carcinomas, but those associated with schistosomiasis are characteristically squamous carcinomas. It is not surprising that the bladder should be affected by many chemicals, as any noxious small molecules in the blood will tend to be found at greatly increased concentration in the urinary tract. Cigarette smoke contains sev- eral mutagenic chemicals that enter the bloodstream and thence the bladder, so that when tested in vitro on bacterial DNA the urine of cigarette smokers is found to be mutagenic, while that of non-​ smokers is barely active. Occupation An occupational cause was first suspected in 1895 in Germany, when Rehn commented on a cluster of cases in men using aniline for the manufacture of dyes. Aniline, however, is not carcinogenic in experimental animals; more recent studies have failed to in- criminate it epidemiologically, and it seems likely that other car- cinogenic chemicals were present as impurities. Four aromatic amines that are carcinogenic in experimental animals have been shown to cause bladder cancer in humans:  2-​naphthylamine, benzidine, 3,3′-​dichlorobenzidine, and 4-​aminobiphenyl. The first is one of the most powerful human carcinogens yet known and was responsible for the development of bladder cancer in all the 19 men who were employed in distilling it in a British fac- tory. Its manufacture in Britain was stopped in 1949, but small amounts continued to be imported until the 1960s. Other aro- matic amines that may cause bladder cancer include auramine, magenta, and, perhaps, 1-​naphthylamine. The last is dubiously carcinogenic in experimental animals and it seems probable that the cases associated with its use have been due to a small pro- portion of 2-​naphthylamine present as an impurity in the com- mercial material. These chemicals were used in the manufacture of dyes, in the rubber industry as antioxidants (1-​naphthylamine and 4-​aminobiphenyl) and hardeners (benzidine), and in labora- tories as a reagent (benzidine). 2-​Naphthylamine is also found in the combustion products of coal and may have been responsible for the risk of bladder cancer in men who made coal gas. Various other occupational associations, including leatherworking and aluminium work, have been reported, but are less clearly aetio- logical. As many as 10% of cases were, at one time, attributable to occupational causes in Britain and North America, but the pro- portion should now be much less. Smoking The most important cause numerically is cigarette smoking, which probably accounts for about half the total number of cases in Britain and North America. 2-​Naphthylamine and 4-​aminobiphenyl are present in cigarette smoke, but whether the amounts are sufficient to account for the carcinogenic effect is uncertain. Medicines The medicinal causes have, by contrast, been responsible for rela- tively few cases. Chlornaphazine was used briefly for the treat- ment of myelomatosis, until it was found to be metabolized into 2-​naphthylamine. Cyclophosphamide is used primarily for the treatment of malignant disease, but it is also used as an immuno- suppressant. In large doses it may cause sloughing of the bladder mucosa and, occasionally, cancer. High levels of consumption of phenacetin-​containing analgesics led to bladder cancer as well as renal cancer risk, but these drugs have been banned in Western countries for the last 30 years. Parasitic infection Heavy infection of the bladder with Schistosoma haematobium has been found to be a cause of the disease, most notably in Egypt and Tanzania. Artificial sweeteners Artificial sweeteners came under suspicion as potential bladder carcinogens because of the results of animal experiments in which, first, mixtures of cyclamates and saccharin and then saccharin alone were shown to cause bladder cancer in rats. The human use of cyc- lamates was banned before saccharin came under suspicion and it now appears that the ‘positive’ results of animal experiments with cyclamates alone were due to impurities. Saccharin has been shown to cause bladder cancer in rats but the quantities that had to be given were large, constituting a few per cent of the feed. The human evi- dence is extensive and could hardly be more negative, except that it does not cover lifelong use. Brain and other central nervous system • 1.5% of all cancers and 2.6% of cancer deaths • Sex ratio of rates 1.5:1; age distribution, see Fig. 5.1.21 Tumours of the brain and nervous system are of several different histological types, some of which may not be clearly either be- nign or malignant. One type occurs characteristically in child- hood (medulloblastoma), another in adult life (glioblastoma), and a third (astrocytoma) at all ages. Despite the overall male pre- dominance, one type (meningioma) is more common in women. Recorded incidence of brain and other central nervous system tumours tends to be greatest in white populations in Western countries, but geographical variations are less marked than for most tumours.

5.1  Epidemiology of cancer 441 A moderately large secular increase in incidence in old age has been recorded in many countries, which might be attributable to improved diagnosis with CT scans and MRI. Little or no increase in mortality has been reported in or before middle age and the re- corded increases in incidence are certainly largely, and possibly wholly, artefactual. The only established external cause is ionizing radiation. No new environmental cause has been established, but many have been suspected without convincing evidence, including electromagnetic fields associated with the use of electri- city (50–​60 Hz) and mobile telephones (cell phones). A small pro- portion of brain tumours are attributable to high-​risk hereditary syndromes, most commonly neurofibromatosis. The presence of allergy is associated with reduced glioma risk, but the reason is unknown. Thyroid • 1.0% of all cancers and 0.2% of cancer deaths • Sex ratio of rates 0.4:1; age distribution, see Fig. 5.1.22 The thyroid is particularly sensitive to ionizing radiation in child- hood, but risks after adult exposures are relatively small. Substantial numbers of cases have occurred among the survivors of the atomic explosions in Hiroshima and Nagasaki, children who were exposed to large amounts of radioactive iodine following the Chernobyl ac- cident, and young people whose necks were irradiated in infancy for the treatment of an enlarged thymus (a condition now con- sidered to be perfectly normal, but at one time thought to be a cause of sudden death). Fortunately, the thyroid tumours produced by ionizing radiation are nearly all of the papillary and follicular types, which respond well to treatment. No external causes are known of the medullary and anaplastic types, which have a high fatality and occur only in adult life. Many medullary thyroid cancers, however, are inherited as an autosomal dominant, alone or as part of the multiple endocrine neoplasia type 2 (MEN2) syndrome. Papillary and follicular thyroid cancers are less often genetic. Associations of thyroid cancer risk have been found with various benign thyroid conditions, but causality remains unclear. The disease is most common in women in South Korea. Increases in recorded incidence have been seen in recent decades in the United States and several other countries; it is unclear how much of this is due to changes in diagnostic completeness and criteria. Hodgkin’s disease (Hodgkin’s lymphoma) • 0.6% of all cancers and 0.2% of cancer deaths • Sex ratio of rates 1.4:1; age distribution, see Fig. 5.1.23 Hodgkin’s disease is best thought of as at least two diseases, one affecting primarily youths and young adults, the other primarily middle-​aged and older people. This division is suggested partly by the existence of two peaks in the age-​specific incidence rates, partly by the histological appearances (younger patients tending to have the nodular sclerotic form of the disease and older patients the mixed cellular form), and partly by the clinical distinction that young patients show mediastinal involvement in more than 50% of cases and infradiaphragmatic involvement in less than 5%, while the reverse tends to be true in older people. There are several reasons for thinking that the characteristic type in young people is infective in origin. In developing countries, Hodgkin’s disease occurs in childhood, but as the standard of living rises, the childhood cases disappear and are replaced by a larger number, and a peak of incidence, in young adults. This is reminis- cent of what happened to poliomyelitis in the first half of the 20th century and suggests that the disease may be due to a ubiquitous infective agent that tends to be contracted at older ages as hygiene improves, rarely causes Hodgkin’s disease, but is more likely to do so if infection is at an older age. Risk in young adults decreases with factors likely to facilitate early infection (e.g. late birth order). That the infectious agent is likely to be the Epstein–​Barr virus (EBV, human herpesvirus type 4) is suggested by the findings that the incidence is increased 5–​20 years after a clinical attack of in- fectious mononucleosis, that abnormal EBV antibody profiles can 0– 0 10 20 30 40 5– 10–15–20–25–30–35–40–45– Age group Rate (per 100 000) 50–55–60–65–70–75–80–85+ Female Male Fig. 5.1.21  Annual incidence of cancer of the brain and other central nervous system (CNS), by age and sex. 0– 0 1 2 3 4 5 6 7 8 9 10 5– 10–15–20–25–30–35–40–45– Age group Rate (per 100 000) 50–55–60–65–70–75–80–85+ Female Male Fig. 5.1.22  Annual incidence of cancer of the thyroid, by age and sex.