The immunology of infection

DEFENCE AGAINST INFECTION

The immunology of infection

Key points C

Pathogen recognition mediated by the innate immune system utilizes germline-encoded pathogen receptors, facilitating rapid immune responses during primary infectious exposure

C

Pathogen recognition mediated by the adaptive immune system utilizes receptors generated by random somatic gene rearrangement and mutation. Recognition of a pathogen by adaptive receptors during a primary infection allows the selection and retention of those receptors for use during a secondary infection, i.e. immunological memory

C

Immune effector responses are highly specific to a given pathogen class. The linkage of an appropriate effector response to a pathogen receptor is a key feature of both the innate immune system and immunological memory in the adaptive immune system

C

The innate and adaptive immune systems operate in a highly cooperative manner. Effective immune responses to infection or vaccination engage both the innate and adaptive immune systems

James J Gilchrist Calman A MacLennan

Abstract The human immune system is composed of a collection of specialized cells and secreted proteins that allows the identification and removal of an invading pathogen, and in doing so limits host injury or death. This system is composed of innate and adaptive branches. It is important to recognize that although the innate and adaptive branches of the immune system differ fundamentally in their mechanisms of pathogen recognition, neither branch functions in isolation. In this article, we address how the innate and adaptive immune systems sense the presence of a pathogen, how the immune system then coordinates anti-pathogen effector functions to remove the pathogen, and finally how immunological memory functions to better protect its host against subsequent exposure to the same pathogen.

Keywords Adaptive immunity; B cells; dendritic cells; immunological memory; innate immunity; macrophages; MRCP; neutrophils; NK cells; T cells

broad mechanisms: through the identification of evolutionarily conserved molecular structures displayed by microbes, or by surveillance for altered distributions of self-antigens, acting as alarm signals indicating infection and tissue damage. The first mechanism employs pattern-recognition receptors (PRRs), which bind microbial structures collectively termed pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs) are examples of these. The structural diversity of PRRs allows detection of a broad range of bacteria, viruses and fungi (Table 2). In addition to PRRs detailed in Table 2, a network of plasma proteins (complement) act as innate pathogen sensors (Figure 1). Natural killer (NK) cells are innate lymphoid cells expressing germline-encoded receptors facilitating the identification of infected, especially virus-infected, cells. The best-characterized family of NK cell-expressed innate receptors are killer cell immunoglobulin-like receptors (KIRs). Depending on the associated intracellular signalling domain, KIRs can act as inhibitory or activating receptors. Inhibitory KIRs bind surface-expressed major histocompatibility complex (MHC) class I molecules monitoring total MHC expression. Viral infection of a cell results in down-regulation of MHC class I expression, which results in loss of NK cell KIR inhibitory signals. By contrast, activating KIRs bind surface-expressed ligands on infected cells, which are expressed as markers of cellular stress that increase in concentration in infected cells. Thus, there is an equilibrium between activating and inhibitory KIR signals when an NK cell interacts with a cell. This determines whether the NK cell identifies that cell as being infected and kills it (see below).

Introduction Infection-associated morbidity and mortality, in particular mortality before reproductive maturity, have made infectious agents among the strongest selective forces driving human evolution.1 The co-evolution of vertebrates alongside their pathogens has directed the emergence and development of the vertebrate immune system. In vertebrates, two complementary branches of the immune system emerged, first an evolutionarily ancient system of innate immunity, followed by more recent emergence of adaptive immunity. Our understanding of immunity to infection in humans has been particularly informed by genetic studies of rare individuals with primary immunodeficiencies2 and population-based studies of infection susceptibility,3 for example genome-wide association studies (Table 1).

Pathogen recognition Innate pathogen recognition Innate pathogen sensors are germline-encoded, and as such their specificities are invariant throughout a person’s lifespan. Innate immune recognition of an invading pathogen proceeds by two

James J Gilchrist DPhil MRCPCH is an Academic Clinical Fellow training in clinical immunology at the Oxford University Hospitals, UK. Competing interests: none declared.

Adaptive pathogen recognition In contrast to innate immune receptors, adaptive immune receptors are generated by random somatic gene rearrangements and mutations. This allows the generation of a highly diverse

Calman A MacLennan DPhil FRCP FRCPath is a MRC Senior Clinical Fellow, Professor of Vaccine Immunology and Consultant Immunologist at the University of Oxford, UK. Competing interests: none declared.

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Genetic susceptibility to infectious diseases in individuals and populations Primary immunodeficiencies Immune pathway

Gene(s)

Innate cytosolic pathogen sensing Complement C1QA, C1QB, C1QC, C1QR, C1QS, C2, C4A, C5, C6, C7, C8A, C9, CFD, CFP TLR-mediated IRAK4, MYD88 NF-kB activation TLR-mediated UNC93B1, TLR3, IRF activation TRAF3 Oxidative burst CYBA, CYBB, NCF1, NCF2, NCF4

Th1 responses

Disease

Genetic susceptibility in populations

Infection susceptibility Gene(s)

NOD2

Infection susceptibility Autoimmune disease susceptibility Leprosy

Complement deficiencies

Encapsulated CFH, CFHR3, Meningococcal bacteria, especially C4A disease pathogenic Neisseria

IRAK4/MYD88 deficiency UNC93B1/TLR3/TRAF3 deficiency Chronic granulomatous disease

Pneumococcal disease TLR1

SLE

Leprosy

HSV encephalitis

Catalase-positive microbes, e.g. Staphylococcus aureus, Aspergillus Mendelian Non-tuberculous susceptibility to mycobacteria, mycobacterial disease non-typhoidal Salmonella

IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, NEMO, CYBB, IRF8, TYK2, ISG15 Th17 responses CARD9, IL17RC, IL17F, Familial chronic STAT1 mucocutaneous candidiasis BTK X-linked agammaAntibody globulinaemia production/ function MHC class I TAP1, TAP2, TAPBP Bare lymphocyte antigen syndrome type I presentation MHC class II RFX5, RFXAP, RFXANK, Bare lymphocyte antigen CIITA syndrome type II presentation

Crohn’s disease

Candida albicans

NCF2

SLE

IL12B, STAT1, IFNGR2, TK2

IBD, SLE, MS

IL23R

IGH Sinopulmonary infection and encapsulated bacteria Respiratory tract HLA-A/B/C infections

Leprosy

IBD, psoriasis, MS

Rheumatic heart disease

HIV

Psoriasis, ankylosing spondylitis

Disseminated viral HLA-DP/DQ/ HBV, HCV, typhoid, Rheumatoid arthritis, and fungal infections, DR leprosy, leishmaniasis, IBD, type 1 diabetes Pneumocystis tuberculosis mellitus, MS, SLE

Examples of human primary immunodeficiencies and genetic susceptibility to infectious and autoimmune diseases in populations (as identified by genome-wide association studies). These studies highlight the overlapping genetic factors underlying infectious disease susceptibility and autoimmune disease susceptibility, implicating selection pressure imposed by infectious agents in the evolution of autoimmune diseases. The studies also highlight the pathogen-specificity of genetic risk factors for infection susceptibility, an observation that has greatly facilitated our understanding of the roles of distinct responses of the human immune system in anti-pathogen defence. HBV, hepatitis B virus; HCV, hepatitis C virus, IBD, inflammatory bowel disease; MS, multiple sclerosis; SLE, systemic lupus erythematosus, For other abbreviations, see text.

Table 1

receptor repertoire, further shaped by pathogens encountered during an individual’s lifespan. T and B lymphocytes of the adaptive immune system express two classes of pathogen recognition molecule. B cells express immunoglobulin molecules, as membrane-bound B cell receptors (BCRs) on naive B cells, and secreted immunoglobulin/antibody molecules from effector B cells (Figure 2). T cells express the membrane-bound T cell receptor (TCR), which allows the detection of pathogen-derived peptide presented on cell surfaces associated with MHC molecules (Figure 3).

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Each naive B cell and T cell has a unique antigenic specificity defined by the sequence of its receptor (BCR, TCR). The human antibody repertoire (i.e. the number of potential antigenic specificities) is estimated to be of the order of at least 1011. To generate that degree of receptor diversity, both B and T cells employ random somatic rearrangement of immunoglobulin and TCR-encoding gene segments; this is accompanied by somatic mutation and coupled with clonal expansion of antigen-specific cells on encountering a receptor’s cognate antigen.

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Innate pathogen receptors Class

Molecule

Location

Cognate ligand

Pathogens recognized

Toll-like receptors

TLR1 TLR2 TLR3 TLR4 TLR5 TLR6

Cell surface

Lipoprotein, peptidoglycan

Endosomes Cell surface

dsRNA Lipopolysaccharide Flagellin Lipoprotein, peptidoglycan

TLR7 TLR8 TLR9 NOD1 NOD2 Dectin-1 RIG-I Mannose-binding lectin C-reactive protein

Endosomes

Single-stranded RNA

Gram-positive and Gram-negative bacteria, mycobacteria, fungi Viruses Gram-negative bacteria Flagellated bacteria Gram-positive and Gram-negative bacteria, mycoplasma, fungi RNA viruses

Cytosol Cytosol Cell surface Cytosol Secreted protein Secreted protein

CpG DNA Peptidoglycan Muramyl dipeptide b-Glucan Double-stranded RNA Mannose and fucose Phosphocholine

Bacteria, viruses, protozoa Gram-negative bacteria Gram-positive and Gram-negative bacteria Fungi, mycobacteria RNA viruses Bacteria, viruses, fungi Gram-positive and Gram-negative bacteria

NOD-like receptors C-type lectin receptors RIG-I-like receptors Lectins Pentraxins

Pattern-recognition receptor (PRR) molecules have distinct patterns of cellular expression and subcellular locations, which determine their function in the innate immune response. Membrane-bound PRRs are expressed by immune cells, for example dendritic cells and macrophages, and allow extracellular pathogen surveillance. Cytosolic PRRs detect intracellular pathogens and are expressed more broadly, in both immune and non-immune cells.

Table 2

Both BCR (and antibodies) and TCR chains are germlineencoded by gene segments that require rearrangement to produce a gene that can be transcribed in a lymphocyte. The variable regions in TCR and immunoglobulin chains are encoded by V and J segments, with an additional D segment in TCR b chains and immunoglobulin heavy chains. One copy of each segment is spliced together, along with a gene segment encoding the constant region of the receptor, to form a functional gene. Receptor diversity is generated by virtue of each gene segment having multiple copies, and these are spliced together in a random manner. Through this process, each naive B and T lymphocyte expresses multiple copies of a single receptor of unique antigen specificity. On binding its cognate antigen, a lymphocyte bearing that receptor is then triggered to undergo clonal expansion, allowing highly specific pathogen recognition, but also the capacity for immunological memory. MHC class I presentation of peptide derived from cytosolic, pathogen proteins identifies an infected cell, marking it out for destruction by a CD8þ T cell (see below). Antigen presentation by MHC class I molecules thus acts as surveillance for intracellular infection. MHC class I molecules are expressed by all nucleated cells except neurones. By contrast, expression of MHC class II molecules is restricted to immune cells specializing in immunological surveillance for pathogens, notably dendritic cells, macrophages and B cells. With dendritic cells, sites of likely pathogen entry (e.g. skin, lung, gut subepithelial spaces) are actively monitored for the presence of pathogens by constant sampling of the extracellular milieu by endocytosis. Engagement of innate receptors on the dendritic cell leads to migration of the dendritic cell to secondary lymphoid organs (e.g. lymph nodes), where pathogen-derived

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peptide can be presented to CD4þ T cells. This results in clonal expansion of a naive antigen-specific T cell. Dendritic cell presentation of MHC-associated peptide thus serves to activate naive antigen-specific T cells. B cells and macrophages also interact with CD4þ T cells via MHC class IIassociated peptide presentation, but do so with activated (not naive) T cells. B cells engage activated CD4þ T cells via MHC class II presentation of peptide processed following BCRmediated endocytosis of pathogen proteins. This allows T cell help in directing B cell differentiation following antigen recognition (see below). In common with dendritic cells, macrophages act as scavenger cells, sampling the extracellular environment. The presentation of MHC class II-associated peptide by macrophages to activated CD4þ T cells has two functions. First, it provides re-stimulation for activated CD4þ T cells as they migrate from lymphoid organs to the site of infection. Second, macrophages act as a site of infection for several intracellular bacteria, for example mycobacteria and Salmonella. By engaging activated CD4þ T cells, they facilitate upregulation of macrophage antibacterial effector mechanisms (see below).

Effector functions Innate effector functions Complement and secreted antimicrobial molecules: effector mechanisms of the complement cascade are outlined in Figure 1. In addition to complement, many secreted antimicrobial molecules have direct antimicrobial activity. Lysozyme and phospholipase A2 are secreted at mucosal surfaces (including oral mucosa, conjunctiva and gut) and by phagocytes; they result in disruption of peptidoglycan-containing cell walls (i.e. bacterial

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The complement cascade Lectin pathway C4 C4b

Classical pathway

C4a

C4 C4b

+

Alternative pathway B

C4a

Bb

+

C2 C2a + C2b

Ba +

C2 C2a + C2b

C3 C3b + C3a

C1

MBL

Pathogen

IgG

Complement proteins identify extracellular pathogens via one of three pathways, the lectin, classical and alternative pathways. • The lectin pathway used secreted pattern-recognition receptors (PRRs), e.g. mannose-binding lectin (MBL), to directly ligate carbohydrate pathogen-associated molecular patterns (PAMPs) on pathogen surfaces. • The classical pathway used the C1 protein complex to bind pathogen surfaces opsonized by other serum proteins, e.g. antibody or C-reactive protein (CRP – binds phosphocholine of lipopolysaccharide). • Alternative complement pathway components are constantly deposited on cell surfaces. On host cell surfaces, these activated alternative pathway complement components are rapidly removed by surface-expressed host cell enzymes (e.g. decay-accelerating factors), but microbial surfaces, lacking immune effectors.

C3 convertase C3 C3b

C3a

C3 C3b

+

C4b

C3a +

Bb C3b

C2a

Pathogen surface

facilitating immune cell recruitment to the site of infection.

C5 convertase C5 C5b

C5a

C5 C5b

+

C5a +

C3b

C3b C4b

Complement activation results in formation of a proteolytic enzyme complex – the C3 convertase, which cleaves C3 into C3a and C3b. • The lectin and classical pathway C3 convertases are composed of C4b and C2a. • The alternative pathway C3 convertase is composed of C3b and Bb. • C3a is not retained on the pathogen surface, and functions as a signalling molecule via endothelial and mast cells, supporting

Bb C3b

C2a

Addition of the C3b to the C3 convertase results in formation of a second proteolytic enzyme complex – the C5 convertase, which cleaves C5 into C5a and C5b. • C3b on pathogen surfaces acts as an opsonin for pathogen uptake by phagocytes, e.g. macrophages. • In common with C3a, C5a is released from the pathogen surface, resulting in immune cell recruitment.

Pathogen surface

Membrane attack complex C9

C6 C5b

C7 C8

Pathogen surface

C5b deposition on a pathogen surface triggers recruitment of the terminal complement components, C6, C7, C8 and C9. • Polymerization of C9 on the pathogen surface results in the formation of membrane-spanning pore structures: the membrane attack complex. • Membrane attack complex disrupts the integrity of the pathogen outer membrane, resulting in direct pathogen destruction.

Figure 1 The complement cascade functions to identify the surfaces of extracellular pathogens. On identification of a pathogen surface, the sequential formation of proteolytic multi-protein complexes direct the production of activated complement components. Activated complement components function to recruit immune cells to the site of infection, opsonise pathogens for phagocytosis, and form multi-protein membranespanning pores to directly damage pathogens.

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The structure and function of antibody Class Structure

Constant region

n ai ch in vy ha ea t c gh Li

H

Variable region

Antigen-binding surface

Subclass Major functions

IgA

Dimeric

Neutralizing antibody at epithelial surfaces

IgD

Monomeric

Unclear

IgE

Monomeric

Mast cell and basophil sensitization

IgG

Monomeric

IgG1

IgG2 IgG3

IgG4 IgM

Pentameric

Neutralization Opsonization NK cell sensitization Complement activation Placental transfer Neutralization Neutralization Opsonization NK cell sensitization Complement activation Placental transfer Neutralization Complement activation

Antibodies (in their monomeric form) are composed of two heavy and two light chains, each comprising immunoglobulin protein domains (two in the case of light chains, four for heavy chains). Each chain’s amino terminus contributes to two variable regions of the antibody molecule, which forms the antigen-binding surface. The carboxy terminus of each chain contributes to the constant (non-variable ) regions of the antibody molecule, which determines interaction of the pathogen-bound antibody with cells and molecules of the immune ding on the invading pathogen and the appropriate immune response, this can be subsequently changed to another antibody class in a process termed class-switching. The structure and function of the major antibody classes are depicted in the table.

Figure 2

cell walls) and cell membranes. Similarly, antimicrobial peptides, such as defensins, are secreted by phagocytes and epithelial cells at mucosal surfaces, and form membrane-disrupting pores in microbes.

facilitates microbial killing by acidifying the phagolysosome, recruiting NADPH oxidase, resulting in the delivery of reactive oxygen species with direct microbial toxicity, delivery of antimicrobial molecules (e.g. antimicrobial peptides, lysozyme) and restriction of key molecules required for microbial survival and growth (e.g. iron). In addition to phagocytosis, neutrophils also control invasive, extracellular pathogens by undergoing a form of programmed cell death, resulting in release of chromatin and DNA, which form extracellular webs (neutrophil extracellular traps). These act to trap extracellular microbes, limiting their dissemination and facilitating clearance.

Clearance of extracellular bacteria by phagocytes: on invasion of a tissue, a pathogen is likely to initially encounter tissueresident macrophages. Engagement of PAMPs by membranebound PRRs on macrophages induces cytoskeletal changes, which result in the engulfment of the pathogen into a membranebound intracellular vesicle e a phagosome. Phagocytes, activated by detecting PAMPs, also release soluble chemoattractants, cytokines and chemokines, which act to recruit immune cells to the infection site. Immune cells recruited include the other principal phagocytes in this context, neutrophils, followed by inflammatory monocytes and cells of the adaptive immune system. Once phagocytosed by a macrophage or neutrophil, pathogens within phagosomes are targeted for killing. The phagosome fuses with pre-formed vesicles (e.g. lysosomes) and recruits antimicrobial effectors, to form a mature phagolysosome. This

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Inflammatory and antiviral responses: Toll-like receptor (TLR) signalling in innate immune cells results in activation of nuclear factor-kB (NF-kB) or interferon regulator factor transcription factors. NF-kB activation results in the production and secretion of a range of proinflammatory cytokines, including interleukin (IL)-1b, tumour necrosis factor (TNF), IL-6, IL-8 and IL-12. These cytokines direct immune cell recruitment and activation at the infection site. IL-1b, TNF and IL-6 also have systemic effects, supporting the

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TCR antigen recognition CD8+ T cell

CD4+ T cell

TCR CD8

TCR CD4

Peptide

Peptide Extracellular pathogen/antigen

MHC class I

Intracellular pathogen/ antigen

MHC class II

Antigen-presenting cell

Infected cell

T cell receptors (TCRs) ligate pathogen-derived peptide presented by major histocompatibility complex (MHC) molecules expressed on host cell surfaces. • TCRs are composed of two protein chains (most T cells expressing TCRs composed of α and β chains). In common with antibody molecules, each chain contributes to an amino terminus variable region, which forms the antigen-binding surface, and to a carboxy terminus, which is constant and anchors the TCR to the T cell plasma membrane. • Two classes of MHC present pathogen-derived peptides: MHC classes I and II. Both are composed of two polypeptide chains, are membrane-bound and present peptide at the cell surface in a highly polymorphic binding groove. MHC class I molecules are composed of a polymorphic chain, which makes up the peptide-binding groove, and an invariant β2-microglobulin chain. MHC class II molecules, by contrast, are composed of two polymorphic polypeptide chains (α and β), both of which contribute to the peptide-binding groove. • Peptide presented by MHC class I molecules (left panel) is derived from cytosolic pathogen proteins by the cytosolic proteasome. Pathogen-derived peptides can then be transported to the endoplasmic reticulum, where they are loaded onto MHC class I molecules. MHC class I-associated peptide is recognized by TCRs on CD8+ T cells. • MHC class II molecules present peptides derived from pathogen antigen in endosomes of antigen-presenting cells (right panel). Extracellular antigen is taken up by antigen-presenting cells either via receptor-mediated endocytosis of opsonized pathogens (e.g. via complement or Fc receptors), or by random sampling of the environment (termed macropinocytosis), before loading onto MHC class II molecules and presentation at the cell surface. MHC class II-associated peptide is recognized by TCRs on CD4+ T cells.

Figure 3

acute-phase response, triggering fever, neutrophil egress from the bone marrow and hepatic production of acute-phase proteins. A second, independent mechanism of inflammatory cytokine induction by innate immune cells is pyroptosis. Nucleotidebinding oligomerization domain (NOD)-like receptors, on sensing cytosolic PAMPs, activate the formation of multiprotein scaffolds, inflammasomes, which activate proinflammatory caspases. This triggers pyroptosis (proinflammatory programmed cell death), which includes the production and release of IL-1b and IL-18.

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Interferon regulator factor activation is induced by innate sensors of viral PAMPs (e.g. TLRs 3, 7, 8 and 9, and RIG-I). This results in type 1 IFN production (IFN-a, IFN-b), of which plasmacytoid dendritic cells are particularly potent producers. Type 1 IFN induces viral resistance in host cells, activating enzymes that degrade viral RNA and suppressing protein translation (and thus viral replication). Type 1 IFN also upregulates IFN-induced transmembrane (IFITM) proteins that suppress viral membrane fusion with endosomal membranes (and thus inhibit viral invasion), and MHC class I expression.

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markers of cytosolic infection, which are surface-expressed MHC class I molecules bearing pathogen-derived peptide. CTLs induce apoptosis via release of pre-formed granules onto the surface of infected host cells. These granules contain perforin and granzymes. Perforin forms pores in the host cell membrane, allowing granzymes to be delivered into the infected cell cytosol. Granzymes induce apoptosis by directly damaging mitochondria and initiating caspase activation. A key feature of CTL-induced cell death is that it is immunologically silent: it does not induce inflammation, minimizing neighbouring tissue damage.

Natural killer cells: NK cells perform two distinct effector functions in the innate immune response to infection. First, infected cells are triggered to undergo apoptosis by NK cell release of perforin and granzymes onto the surface of infected cells (see CD8þ T cell effector functions, below). Second, activated NK cells act as an innate source of IFN-g. Following uptake into macrophages by phagocytosis, some bacteria (e.g. Salmonella, mycobacteria) are adapted to perturb maturation of the phagosome and establish intracellular infection. To clear these intracellular bacteria, macrophages require additional activation to upregulate their antibacterial effector mechanisms. This is driven by NK cell-derived IFN-g during the initial innate phase of an infection.

Effector functions of CD4þ T cells: naive CD4þ T cells, on binding to their cognate MHCepeptide pair, proliferate and then differentiate into one of five effector subsets. Effector CD4þ T cells are defined by the profile of cytokines they produce, which determines the immune cells they recruit and activate, and thus the type of infection they are specialized to clear. These five main effector subsets are designated T helper 1 (Th1), Th2 and Th17 (Figure 4), follicular helper T (Tfh) and regulatory T (Treg) cells.

Adaptive effector functions Effector functions of CD8þ T cells: on binding to its cognate MHCepeptide pair, a naive CD8þ T cell proliferates and differentiates into a cytotoxic CD8þ T cell. Cytotoxic CD8þ T lymphocytes (CTLs) function to induce apoptosis in host cells with

Effector functions of CD4+ Th cells

IFNγ

Macrophage activation intracellular bacteria clearance

Th1 CD4+ T cell Dendritic cell

IFNγ IL-12 Th2 CD4+ T cell IL-4 Naïve CD4+ T cell IL-6 TGFβ IL-23

IL-17 IL-22

IL-4 IL-5 IL-13

Mast cell, eosinophil, basophil recruitment. Mucus production and smooth muscle contraction

Helminth clearance

Th17 CD4+ T cell

Epithelial cell: IL-8 and AMP production

Neutrophil recruitment

Extracellular bacteria and fungi clearance

• Th1 cells produce IFN- γ and support macrophage activation to control intracellular bacteria. Th2 cells produce IL-4, IL-5 and IL-13, recruiting and activating mast cells, eosinophils and basophils, facilitating defence against extracellular parasites. Th17 cells produce IL-17 and IL-22, stimulating epithelial cells to release chemokines, which recruit neutrophils, and antimicrobial peptides facilitating host defence at epithelial surfaces. • The fate of a naive CD4+ T cell during priming is determined by the local cytokine environment, with IL-12 and IFN- γ supporting Th1 development, IL-4 supporting Th2 development, and IL-6, TGF-β and IL-23 supporting Th17 development. These cytokines are initially provided by innate cells (e.g. NK cells providing an initial IFN-γ source), but the overlap between cytokines determining differentiation and effector cytokines (in the case of Th1 and Th2 cells) mean that differentiated CD4+ T cells support their own differentiation by positive feedback. • Effector cytokines from Th1, Th2 and Th17 cells inhibit differentiation of naive CD4+ T cells into effector Th cells other than their own.

Figure 4

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Tfh cells develop in parallel to Th1/Th2/Th17 responses, providing support for maturation of an antibody response (see below). CD4þ Treg cells, in contrast to the other four effector subsets, are inhibitory and act to limit autoreactivity (and thus autoimmune disease).

The generation of long-lived plasma cells and memory B cells from affinity-matured B cells means that antibodies produced by these cells have had their antigen affinity optimized during the first infectious exposure, which is then retained for subsequent exposures. In addition, during a secondary infectious challenge, memory B cells can re-enter germinal centres, allowing further rounds of somatic hypermutation and affinity maturation, resulting in the iterative improvement of antibody affinity for a given pathogen after each exposure. Similarly, as long-lived plasma cells and memory B cells develop from class-switched B cells, this links a given pathogen-specific receptor to a set of effector functions defined by the antibody class, which are appropriate to clear that pathogen.

Effector functions of B cells: antibody binds to pathogens and pathogen-derived molecules, functioning to neutralize pathogens and toxins (e.g. binding viral particles preventing host cell invasion), and opsonize pathogens for destruction by immune cells and complement. The constant region of the immunoglobulin heavy chain determines the class of that antibody, and thus its function (Figure 2). For a naive B cell to differentiate into a mature antibodysecreting plasma cell or memory B cell, it needs to recognize cognate antigen via its surface-bound BCRs and also receive costimulatory signals. With a protein antigen, this co-stimulation is provided by Tfh cells. Antigen internalized by the B cell is presented on MHC class II molecules and then recognized by Tfh cells. Tfh co-stimulation supports B cell proliferation and survival, somatic hypermutation (a second round of somatic mutation of immunoglobulin variable regions that results in enhanced antigeneantibody affinity) and antibody classswitching. The antibody class to which the B cell switches, by analogy to CD4þ T cell differentiation, is determined by the local cytokine environment, again largely determined by Tfh cells. Tfh secretion of IL-5 leads to production of immunoglobulin IgE, secretion of IFN-g to IgG2a and IgG3 production, and secretion of transforming growth factor-b (TGF-b) to IgA and IgG2b production. The function of these classes mirrors the CD4þ T cell effector functions induced by these cytokines, producing a coordinated T and B cell response to a given pathogen.

T cell memory Following resolution of a primary infection, a proportion of antigen-specific CD4þ and CD8þ T cells persist as memory T cells. Both CD4þ and CD8þ T cells have three classes of effector memory cell, defined according to the tissues to which they home. Central memory T (TCM) cells circulate in the peripheral blood, homing to secondary lymphoid organs. Effector memory T (TEM) cells also circulate in the peripheral blood, but lack lymphoid homing markers and instead home to peripheral tissue. Finally, tissue-resident T (TRM) cells do not circulate in blood, but instead remain in situ in peripheral tissues. This distribution of memory T cells allows three waves of memory T cell effector functions on re-encountering a pathogen. First, TRM cells act to monitor peripheral tissue for invading pathogens, allowing immediate T cell-mediated immunity on invasion of a pathogen. Second, peripheral TEM cells are rapidly recruited to sites of inflammation, supplementing the TRM response. Third, TCM cells encountering cognate antigen in secondary lymphoid tissue rapidly generate secondary effector T cells, further supplementing the TEM and TRM responses.

Immunological memory

Memory and innate immunity Whereas immunological memory is largely the preserve of the adaptive immune system, memory-like characteristics are exhibited by innate immune cells. This has been termed ‘trained immunity’.4 Pathogen recognition by innate cells is restricted to germline-encoded receptors, which cannot be altered by pathogen exposure. Innate cells can, however, alter their effector responses. NK cells and macrophages, following a primary infectious exposure, undergo epigenetic modification that results in enhanced effector function in response to a secondary infectious exposure, months after resolution of the primary infection. These effector functions are enhanced during a secondary challenge with the same infectious agent encountered during the primary infection, but are also enhanced in response to secondary challenge with an unrelated pathogen. That these effects are heterologous, i.e. not specific to the primary pathogen or vaccine, reflects the fact that innate receptors are invariant and not altered by pathogen exposure. Interestingly, however, NK cell memory can also function in an antigen-specific manner. This is best characterized in cytomegalovirus (CMV) infection, in which NK cells bearing innate receptors identifying CMV-infected cells expand in response to a primary infection, and persist following resolution.

B cell memory B cell immunity results in long-lasting, pathogen-specific memory by two principal mechanisms. First, following a primary infection, a proportion of antigen-specific B cells form long-lived plasma cells. After somatic hypermutation and class-switching, a proportion of primed B cells (plasmablasts) migrate from secondary lymphoid tissue to bone marrow, where they form longlived plasma cells. These cells are capable of long-term antibody production in the absence of continuing antigenic stimulation. In doing so, they give rise to serological immunity, that is, steadystate production of antigen-specific antibodies in serum, which are ready to act in a neutralizing capacity at the point of pathogen invasion. The second mechanism by which B cell immunity results in immunological memory is through the formation of memory B cells. These cells, also derived from primed B cells, reside in secondary lymphoid organs in a quiescent state before reencountering their cognate antigen during a second (or subsequent) infectious challenge. Memory B cells express large amounts of cell-surface, class-switched immunoglobulin and MHC class II molecules. This facilitates the rapid production of class-switched plasma cells producing antibody of high affinity, immediately following a secondary infectious challenge.

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Hib conjugate vaccines). Delivery of purified pathogen molecule alone does not typically engage innate receptors in sufficient quantity to induce robust, long-lived immunity. For this reason, non-live vaccines often require repeat dosing and administration with adjuvants. Adjuvants are not pathogen-specific, and act partly by engaging innate receptors, inducing more robust immunological memory. A

Induction of memory responses by vaccination By safely inducing immunological memory, vaccination prevents millions of infectious deaths annually.5 Modern vaccines can be grouped into two broad groups. Live vaccines are attenuated, and thus lack the capacity to induce invasive infection, but remain highly related to the disease-causing pathogen. Their attenuation allows live vaccines such as measles, rubella, smallpox and BCG to be safely administered to human populations; in addition, their high degree of relatedness to the wild-type pathogen allows induction of immunological memory that provides effective, specific protection against the target infectious disease. Immunological memory induced by live vaccines has a number of important properties. Vaccination with a whole microbe allows full engagement of innate receptors. This allows live vaccines to be highly immunogenic following a single dose of vaccine, and allows the resultant immunological memory to be linked to an appropriate pathogen-directed effector response. By contrast, non-live vaccines are designed to generate immunological memory either to the whole pathogen (e.g. inactivated polio vaccine, cholera vaccine) or, more commonly, to a specific portion of a pathogen. Examples of such subunit vaccines include recombinant protein vaccines (e.g. hepatitis B), pure carbohydrate vaccines (e.g. typhoid Vi vaccine), toxoid vaccines (e.g. tetanus, diphtheria) and proteinepolysaccharide glycoconjugate vaccines (e.g. pneumococcal, meningococcal and

KEY REFERENCES 1 Karlsson EK, Kwiatkowski DP, Sabeti PC. Natural selection and infectious disease in human populations. Nat Rev Genet 2014; 15: 379e93. 2 Milner JD, Holland SM. The cup runneth over: lessons from the ever-expanding pool of primary immunodeficiency diseases. Nat Rev Immunol 2013; 13: 635e48. 3 Chapman SJ, Hill AVS. Human genetic susceptibility to infectious disease. Nat Rev Genet 2012; 13: 175e88. 4 Netea MG, Quintin J, van der Meer JWM. Trained immunity: a memory for innate host defense. Cell Host Microbe 2011; 9: 355e61. 5 Plotkin SA. Vaccines: the fourth century. Clin Vaccine Immunol 2009; 16: 1709e19.

TEST YOURSELF To test your knowledge based on the article you have just read, please complete the questions below. The answers can be found at the end of the issue or online here.

Question 1

Question 2

A 35-year-old man presented with a 4-month history of fever, night sweats and weight loss, accompanied by a productive, persistent cough. He had a past history of Crohn’s disease treated with an anti-tumour necrosis factor agent (adalimumab) for 1 year. He had been born in Pakistan, before moving to the UK at the age of 15 years. Clinical examination was unremarkable.

A 14-year-old boy presented with a 10-year history of recurrent oral candidiasis despite treatment with appropriate antifungal agents. On examination, he had onychomycosis involving five of his fingernails and all of his toenails. He had a rash consistent with dermatophyte infection on both feet. There was no family history of fungal infection, or other infection susceptibility. Genetic investigations demonstrated a gain-of-function mutation in STAT1.

Investigation  Sputum microscopy showed acidealcohol-fast bacilli

Risk of which infectious agent (at a population level) is increased in individuals with variation in the same immunological pathway as that causing this primary immunodeficiency? A. Salmonella typhi B. Mycobacterium leprae C. Streptococcus pneumoniae D. HIV-1 E. Plasmodium falciparum

What T cell effector function is likely to have facilitated control of his latent infection before his presentation? A. CD4+ Th17 cell secretion of IL-17, leading to neutrophil recruitment and activation B. CD4+ Th1 cell secretion of IFN-g, leading to macrophage activation C. CD8+ T cell cytotoxicity of infected host cells D. CD4+ Tfh cell support of anti-pathogen neutralizing antibody E. CD4+ Th2 secretion of IL-5, leading eosinophil recruitment and activation

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DEFENCE AGAINST INFECTION

C-reactive protein (CRP) is commonly used as a clinical marker of infection and inflammation. In this patient, what is the immunological function of the raised concentration of CRP? A. Complement activation and opsonization of extracellular bacteria B. Recruitment and activation of macrophages at the site of infection C. Recruitment and activation of neutrophils at the site of infection D. Direct killing of extracellular bacteria through the formation of membrane-spanning pores E. Pyrogen activity, increasing body temperature

Question 3 An 18-year-old woman presented with a 2-day history of fever, cough and shortness of breath. On clinical examination, her temperature was 38.5  C, heart rate 122 beats/minute and blood pressure 123/68 mmHg. The chest was clinically clear. Investigations  Haemoglobin 105 g/litre (115e165)  White cell count 18.6  109/litre (4.0e11.0)  Neutrophils 14.4  109/litre (1.5e7.0)  Lymphocytes 4.2  109/litre (1.5e4.0)  Platelets 455  109/litre (150e400)  C-reactive protein 80 mg/litre (<10)  Chest X-ray showed patchy consolidation of the right middle lobe  Blood culture: Gram-positive cocci

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