Cell-Mediated Defense against Infection - Elseviersecure-ecsd.elsevier.com › uk › files ›...

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50 6 Cell-Mediated Defense against Infection Tobias M. Hohl e principal function of the mammalian immune system is to combat infectious diseases. Immune responses to antigens and microbial pathogens are divided into those mediated by cells (i.e., cell-mediated immunity) and those mediated by antibodies (i.e., humoral immunity). e T lymphocyte, the “central player” in cell-mediated immunity, provides antigen specificity and orchestrates the antimicrobial activi- ties of cells that generally lack antigen specificity. “Supporting players” in cell-mediated immune responses include dendritic cells (DCs), which present antigens to T cells and provide contextual information, and monocytes and macrophages, which carry out instructions pro- vided by activated T cells. Antigen specificity is conferred by T cells, which bear T-cell receptors (TCRs) responsible for the detection of pathogen-derived peptides presented on major histocompatibility complex (MHC) molecules. Primary encounter of naïve T cells with a pathogen results in their activation, proliferation, and differentiation into effector T cells. Aſter pathogen clearance, effector T cells contract and give rise to long-term memory T-cell populations. Our under- standing of T-cell responses to microbial pathogens and of the com- plexity of cell-mediated immune responses have increased dramatically. e discussion in this chapter is focused on our current knowledge regarding T-cell responses to microbial pathogens. T-CELL SUBSETS AND PHENOTYPIC DIVERSITY Mammalian T lymphocytes are generally categorized on the basis of TCR and co-receptor expression. Most T lymphocytes that have been implicated in the defense against infectious diseases express a heterodi- meric TCR composed of an α-chain and a β-chain. 1,2 A minor fraction of circulating human T lymphocytes express a TCR composed of a γ- chain and a δ-chain. γδ T cells reside primarily in mucosal tissues and appear poised to amplify inflammatory responses and exert cytolytic activity at portals of microbial invasion, although their role in defense against infection is less well defined. 3 T lymphocytes are also divided on the basis of CD4 or CD8 co-receptor expression. CD4 + and CD8 + T cells differ both in terms of function and specificity, and their roles in defense against infectious diseases are distinct. As a rule, T lymphocytes orchestrate antimicro- bial defense but do not participate directly in microbial killing. e major strategy used by CD4 + T cells to disable microbes is to secrete cytokines and chemokines that activate other cells (e.g., macrophages and B cells) or recruit other cells (e.g., monocytes and neutrophils) to sites of infection. e major strategy used by CD8 + T lymphocytes is to secrete lytic proteins that destroy infected cells, depriving the patho- gen of an environment that is suitable for replication and long-term survival. ese functions, collectively called T-cell effector functions, are expressed selectively by different T-cell subsets, providing a diversity of antimicrobial mechanisms. 1,2,4,5 In the following sections, differences and similarities of T-cell subsets are discussed. CD4 + T Cells CD4 + T cells play a central role in the response to a range of microbial infections. Aſter infection, naïve CD4 + T cells can differentiate into phenotypically distinct effector T cells, a process that is determined by the inflammatory context induced by the microbe. 6 Classic studies of CD4 T-cell priming suggested that differentiation can give rise to 1 cells that produce interferon-γ (IFN-γ) or to the cells that produce interleukin (IL)-4 (IL-4). 7 In the past 20 years, additional helper T-cell () populations have been identified and functionally characterized, resulting in a model of naïve CD4 T-cell differentiation that is both more diverse and flexible than initially anticipated. 6 In general, cytokines produced during infection promote the expression of distinct transcriptional regulators that determine which effector molecules will be expressed by responding T cells. e expression of transcriptional regulators and the secretion of specific cytokines forms the basis for classifying distinct CD4 + T-cell populations that respond to infection (Fig. 6-1). Th1 Cells 1 cells secrete IFN-γ, a signature cytokine that activates macro- phages and DCs and thereby enhances their ability to kill intracellular microbes and to present antigens to T lymphocytes. 1 cells can also secrete tumor necrosis factor (TNF), lymphotoxin, and IL-2, which contribute to antimicrobial defense as well. 6 Naïve T cells are driven to differentiate into 1 cells by early exposure to IFN-γ and IL-12 at the time of T-cell priming. e earliest source of IFN-γ may be natural killer (NK) cells or nonspecific memory T cells, and the sources of IL-12 during priming are DCs at the site of infection. IFN-γ induces signaling via the signal transducer and activator of transcription (STAT) 1 (STAT1) pathway and induces expression of T-bet, 8-10 a tran- scriptional regulator that promotes IFN-γ expression and downregu- lates the expression of IL-4, a cytokine that promotes 2 differentiation. IL-12 binds the IL-12 receptor heterodimer and signals via the STAT4 pathway, amplifying 1 responses and enhancing IFN-γ production by responding 1 cells. 11,12 1 cells play an important role in defense against infections by intracellular bacteria, such as Salmonella typhimurium or Mycobacte- rium tuberculosis. 13 Supportive evidence comes from extensive series of experiments with mice deficient in IFN-γ, T-bet, or IL-12. 14 In humans with genetic defects in receptors for IFN-γ, IL-12, or STAT1, increased susceptibility to infection with strains of Salmonella and Mycobacterium provides support for the essential role of 1 cells in defense against intracellular bacterial infections. 15-18 CD4 + 1 cells also enhance defense against the intracellular protozoal pathogen Leishmania major. 19 In this setting, 1 cell production of IFN-γ acti- vates macrophages to kill intracellular parasites. Th2 Cells 2 cells express a range of cytokines that influence B-cell differentia- tion and antibody production, eosinophil recruitment, and mucus production. e signature cytokines produced by 2 cells are IL-4, IL-5, and IL-13, but 2 cells can also produce IL-9, IL-10, IL-25, and amphiregulin. 20 2 responses are generated when naïve T cells are exposed to IL-4 at the time of T-cell priming. In the setting of low antigen concentrations, IL-4 can be produced by responding T cells. 21 Aſter antigenic challenge, IL-4 can also be produced by mast cells and basophils in the vicinity of T-cell priming. 22,23 IL-4 signals naïve T cells via the STAT6 pathway to express GATA3, the master regulator of 2 differentiation, 24 a process that can be enhanced by IL-4– and STAT6- independent GATA3 activation, 25 all of which drives the expression of additional downstream activators. Although 2 cells are best known for causing or contributing to allergic diseases such as atopic dermatitis, allergic rhinitis, and asthma, 2 cells also contribute to defense against infections, particularly helminth infections of the gastrointestinal tract. 26 In this setting, eosin- ophil recruitment, IgE production, and mucus hypersecretion can enhance parasite expulsion in an IL-4 and IL-13 signaling–dependent manner, a notion that is supported by murine studies of Nippostrongy- lus brasiliensis infection. 27,28 e secretion of amphiregulin by 2 cells can stimulate intestinal epithelial cell proliferation and expulsion of Trichuris muris, a nematode that infects mice. 29 Besides 2 cells,

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6 Cell-Mediated Defense against InfectionTobias M. Hohl

�e principal function of the mammalian immune system is to combat infectious diseases. Immune responses to antigens and microbial pathogens are divided into those mediated by cells (i.e., cell-mediated immunity) and those mediated by antibodies (i.e., humoral immunity). �e T lymphocyte, the “central player” in cell-mediated immunity, provides antigen speci�city and orchestrates the antimicrobial activi-ties of cells that generally lack antigen speci�city. “Supporting players” in cell-mediated immune responses include dendritic cells (DCs), which present antigens to T cells and provide contextual information, and monocytes and macrophages, which carry out instructions pro-vided by activated T cells. Antigen speci�city is conferred by T cells, which bear T-cell receptors (TCRs) responsible for the detection of pathogen-derived peptides presented on major histocompatibility complex (MHC) molecules. Primary encounter of naïve T cells with a pathogen results in their activation, proliferation, and di�erentiation into e�ector T cells. A�er pathogen clearance, e�ector T cells contract and give rise to long-term memory T-cell populations. Our under-standing of T-cell responses to microbial pathogens and of the com-plexity of cell-mediated immune responses have increased dramatically. �e discussion in this chapter is focused on our current knowledge regarding T-cell responses to microbial pathogens.

T-CELL SUBSETS AND PHENOTYPIC DIVERSITYMammalian T lymphocytes are generally categorized on the basis of TCR and co-receptor expression. Most T lymphocytes that have been implicated in the defense against infectious diseases express a heterodi-meric TCR composed of an α-chain and a β-chain.1,2 A minor fraction of circulating human T lymphocytes express a TCR composed of a γ-chain and a δ-chain. γδ T cells reside primarily in mucosal tissues and appear poised to amplify in!ammatory responses and exert cytolytic activity at portals of microbial invasion, although their role in defense against infection is less well de�ned.3

T lymphocytes are also divided on the basis of CD4 or CD8 co-receptor expression. CD4+ and CD8+ T cells di�er both in terms of function and speci�city, and their roles in defense against infectious diseases are distinct. As a rule, T lymphocytes orchestrate antimicro-bial defense but do not participate directly in microbial killing. �e major strategy used by CD4+ T cells to disable microbes is to secrete cytokines and chemokines that activate other cells (e.g., macrophages and B cells) or recruit other cells (e.g., monocytes and neutrophils) to sites of infection. �e major strategy used by CD8+ T lymphocytes is to secrete lytic proteins that destroy infected cells, depriving the patho-gen of an environment that is suitable for replication and long-term survival. �ese functions, collectively called T-cell e�ector functions, are expressed selectively by di�erent T-cell subsets, providing a diversity of antimicrobial mechanisms.1,2,4,5 In the following sections, di�erences and similarities of T-cell subsets are discussed.

CD4+ T CellsCD4+ T cells play a central role in the response to a range of microbial infections. A�er infection, naïve CD4+ T cells can di�erentiate into phenotypically distinct e�ector T cells, a process that is determined by the in!ammatory context induced by the microbe.6 Classic studies of CD4 T-cell priming suggested that di�erentiation can give rise to �1 cells that produce interferon-γ (IFN-γ) or to the cells that produce interleukin (IL)-4 (IL-4).7 In the past 20 years, additional helper T-cell (�) populations have been identi�ed and functionally characterized, resulting in a model of naïve CD4 T-cell di�erentiation that is both more diverse and !exible than initially anticipated.6 In

general, cytokines produced during infection promote the expression of distinct transcriptional regulators that determine which e�ector molecules will be expressed by responding T cells. �e expression of transcriptional regulators and the secretion of speci�c cytokines forms the basis for classifying distinct CD4+ T-cell populations that respond to infection (Fig. 6-1).

Th1 Cells�1 cells secrete IFN-γ, a signature cytokine that activates macro-phages and DCs and thereby enhances their ability to kill intracellular microbes and to present antigens to T lymphocytes. �1 cells can also secrete tumor necrosis factor (TNF), lymphotoxin, and IL-2, which contribute to antimicrobial defense as well.6 Naïve T cells are driven to di�erentiate into �1 cells by early exposure to IFN-γ and IL-12 at the time of T-cell priming. �e earliest source of IFN-γ may be natural killer (NK) cells or nonspeci�c memory T cells, and the sources of IL-12 during priming are DCs at the site of infection. IFN-γ induces signaling via the signal transducer and activator of transcription (STAT) 1 (STAT1) pathway and induces expression of T-bet,8-10 a tran-scriptional regulator that promotes IFN-γ expression and downregu-lates the expression of IL-4, a cytokine that promotes �2 di�erentiation. IL-12 binds the IL-12 receptor heterodimer and signals via the STAT4 pathway, amplifying �1 responses and enhancing IFN-γ production by responding �1 cells.11,12

�1 cells play an important role in defense against infections by intracellular bacteria, such as Salmonella typhimurium or Mycobacte-rium tuberculosis.13 Supportive evidence comes from extensive series of experiments with mice de�cient in IFN-γ, T-bet, or IL-12.14 In humans with genetic defects in receptors for IFN-γ, IL-12, or STAT1, increased susceptibility to infection with strains of Salmonella and Mycobacterium provides support for the essential role of �1 cells in defense against intracellular bacterial infections.15-18 CD4+ �1 cells also enhance defense against the intracellular protozoal pathogen Leishmania major.19 In this setting, �1 cell production of IFN-γ acti-vates macrophages to kill intracellular parasites.

Th2 Cells�2 cells express a range of cytokines that in!uence B-cell di�erentia-tion and antibody production, eosinophil recruitment, and mucus production. �e signature cytokines produced by �2 cells are IL-4, IL-5, and IL-13, but �2 cells can also produce IL-9, IL-10, IL-25, and amphiregulin.20 �2 responses are generated when naïve T cells are exposed to IL-4 at the time of T-cell priming. In the setting of low antigen concentrations, IL-4 can be produced by responding T cells.21 A�er antigenic challenge, IL-4 can also be produced by mast cells and basophils in the vicinity of T-cell priming.22,23 IL-4 signals naïve T cells via the STAT6 pathway to express GATA3, the master regulator of �2 di�erentiation,24 a process that can be enhanced by IL-4– and STAT6-independent GATA3 activation,25 all of which drives the expression of additional downstream activators.

Although �2 cells are best known for causing or contributing to allergic diseases such as atopic dermatitis, allergic rhinitis, and asthma, �2 cells also contribute to defense against infections, particularly helminth infections of the gastrointestinal tract.26 In this setting, eosin-ophil recruitment, IgE production, and mucus hypersecretion can enhance parasite expulsion in an IL-4 and IL-13 signaling–dependent manner, a notion that is supported by murine studies of Nippostrongy-lus brasiliensis infection.27,28 �e secretion of amphiregulin by �2 cells can stimulate intestinal epithelial cell proliferation and expulsion of Trichuris muris, a nematode that infects mice.29 Besides �2 cells,

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KEYWORDSantigen-presenting cells; antigen processing and presentation; assays of immunologic function; cellular immunity; dendritic cells; !ow cytometry; lymph node architecture; lymph nodes; lymphocyte tra"cking; macrophage; major histocompatibility complex; monocyte; neutrophil; pathogen recognition receptors; spleen; T-cell activation; T-cell di�erentiation; T-cell e�ector functions; T-cell memory; T-cell receptor; thymus

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Di�erentiation of murine and human naïve CD4+ T cells into �17 cells requires early exposure to transforming growth factor-β (TGF-β) and IL-6 at the time of priming, IL-21–driven ampli�cation, and IL-23–driven stabilization.6,44-47,48 �e combination of robust TCR stimulation and IL-6/TGF-β exposure induces IL-23 receptor expres-sion and STAT3-dependent expression of the transcription factor RORγt by naïve CD4+ T cells.49 RORγt, together with RORα,50 pro-motes IL-17A, IL-17F, IL-21, and IL-22 release. Activation of human �17 cells leads to the expression of chemokine receptors (i.e., CCR4, CCR6) that facilitate migration to in!ammatory lesions at mucosal sites, speci�cally the intestines, lung, and skin.51 �us, mucosal and epithelial tissues are enriched in �17 cells.

�17 T cells and IL-17A and IL-17F signaling pathways have important roles in defense against microbial infection, particularly against extracellular bacteria and pathogenic fungi. Clearance of murine pulmonary infection with Klebsiella pneumoniae is dependent on cytokines secreted by �17 cells, including IL-17A and IL-22.52 Both cytokines enhance lung neutrophil recruitment by stimulating che-mokine production, and IL-22 enhances pulmonary epithelial cell regeneration during infection. Patients with hyperimmunoglobulin E–recurrent infection syndrome (hyper-IgE syndrome, HIES, Job’s syndrome) have impaired di�erentiation of �17 cells.53 �is defect results from mutations in STAT3, a signaling molecule that is essential for �17 cell development, and accounts for markedly increased risk for pulmonary infections, mucocutaneous candidiasis, and staphylo-coccal infections. Mendelian susceptibility to chronic mucocutaneous candidiasis and dermatophytosis maps to components of the IL-17A and IL-17F signaling pathway and underscores the importance of these �17-derived cytokines in defense against mucosal fungal infections.54-58

Microbial factors that promote the di�erentiation of �17 T cells include M. tuberculosis–derived complete Freud’s adjuvant, speci�-cally the components trehalose dimycolate (i.e., cord factor) and pep-tidoglycan that synergize to recapitulate �17-promoting adjuvant activity of mycobacteria.59,60 �e fungal cell wall components β-glucan and α-mannan also trigger C-type lectin receptor signaling via Dectin-161 and Dectin-2/Dectin-3 heterodimers,62,63 respectively, and stimulate CARD9-dependent signals in DCs that enhance �17-cell

tissue-resident and �2 cytokine-secreting innate lymphoid cells rep-resent a signi�cant source of IL-13 during the early stages of parasitic infection and promote expulsion.30-32

Aberrant �2 responses to pathogens that require IFN-γ and �1 responses for control can result in progressive infections and lethality. For example, Leishmania major infection of certain mouse strains induces �2 responses that result in progressive in vivo replication and host death.33,34 In contrast, mouse strains that respond to L. major with �1 responses clear and survive experimental infections. �e mecha-nisms that determine whether an L. major–speci�c T-cell response will be predominately �1 or �2 are complex.35 In some mouse strains, �2 responses occur because of T-cell responses to one dominant antigen called LACK (Leishmania analogue of the receptors of acti-vated C kinase).36 In the absence of a T-cell response to this speci�c antigen, the responding CD4+ T cells di�erentiate into �1 cells.

In humans, the type of disease associated with Mycobacterium leprae infection is also tied to CD4+ T-cell di�erentiation. �1 di�er-entiation is associated with tuberculoid leprosy, a paucibacillary infec-tion in which IFN-γ–producing T cells enhance microbial killing. �e induction of type I interferon and IL-10 signaling in innate immune cells during leprosy can antagonize IFN-γ–dependent protection.37 �2 di�erentiation is associated with high tissue densities of M. leprae and more robust, but ine�ective, antibody responses.38,39

Th17 Cells�e discovery of CD4+ �17 cells resulted from a search, in mouse models, for CD4+ T-cell populations that cause in!ammatory diseases, such as experimental autoimmune encephalitis.40,41 Although many autoimmune in!ammatory diseases had been attributed to �1 cells, mounting evidence demonstrated that mice de�cient in IL-12 and IFN-γ, the two cytokines essential for �1 di�erentiation, still devel-oped antigen-driven in!ammatory diseases. �17 cells produce IL-17, a cytokine that promotes recruitment of proin!ammatory cells, includ-ing neutrophils.42 �17 cells are implicated in murine in!ammatory diseases such as experimental autoimmune encephalitis and in!amma-tory bowel disease and in human in!ammatory diseases such as rheu-matoid arthritis and multiple sclerosis.43

FIGURE 6-1 Helper T-cell subsets. Naïve CD4+ T cells proliferate and differentiate into distinct helper T-cell (Th) subsets after T-cell receptor activation by cognate major histocompatibility complex (MHC) class II/peptide antigen (Ag) complexes during infectious and inflammatory states. Cytokine- and STAT-mediated signals drive Th differentiation into distinct Th effector subsets that are maintained by the expression of transcriptional activators (T-bet, GATA3, RORγt, FOXP3) or repressors (BCL6) and secrete distinct effector cytokines. The origin of follicular helper T cells (Tfh) remains controversial; these cells may arise from naïve CD4+ T cells or from Th cells that have previously assumed characteristics of Th1, Th2, or Th17 cells (dashed arrows). There is emerging experimental evidence that individual Th-cell subsets demonstrate plasticity and may interconvert during infectious challenges to facilitate microbial eradication and reduce immune-mediated tissue injury.

MHC II-Ag ondendritic cell

NaïveCD4+ T cell

Th1 Th2 Th17 iTreg Tfh

IFN-γEffectorcytokines

Il-4, IL-5,IL-13

IL-17A, IL-17F,IL-22

IL-10, IL-35,TGF-β

Il-21

STAT1/4 STAT6 STAT3 STAT5 STAT3IFN-γ,IL-12

IL-4IL-6,IL-23

TGF-β

T-bet GATA3 RORγt FOXP3 BCL6

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� subsets. Although not exclusive to T# cells, genetic defects in ICOS-1 lead to a marked reduction in circulating and germinal center T# cells and represent a rare monogenetic cause of common variable immunode�ciency.91

Plasticity of Helper T-Cell Subsets�e paradigm of distinct CD4 �-cell subsets has been critical in understanding CD4 T-cell–dependent immunity to a wide range of microbial pathogens. A limitation of this model is the recognition that the expression of master regulator genes and cytokine expression pat-terns is not as exclusive or enduring as initially proposed.92 For example, IL-4–producing �2 cells speci�c for murine lymphocytic choriomen-ingitis virus switch to IFN-γ production upon adoptive transfer and ensuing infection of mice.93 During helminth infection, in vivo conver-sion of e�ector �1 into �2 cells can occur.94 Similarly, �17 cells can be reprogrammed to secrete �1 cytokines through exposure to IL-12.95 �ese data are consistent with the notion that e�ector T cells remain responsive to external cues during the progression of microbial infections to optimize microbial eradication and to limit immunopa-thology. However, it is less clear whether shi�s in cytokine production and plasticity in e�ector � cell phenotypes represent an important mechanism to control microbial infections in vivo.

In Vivo CD4+ T-Cell Responses to Microbial Infection: Lessons from Mouse ModelsExtensive studies, primarily using mouse models, have characterized in vivo CD4+ T-cell responses to microbial infection. Priming of naïve T cells generally occurs within lymph nodes that are draining sites of infection and likely occurs in a stepwise fashion, with sequential inter-actions with distinct antigen-presenting cells that each contribute to the phenotype of the e�ector CD4+ T-cell response.96 Studies of in vivo CD4+ T-cell priming and tra"cking have been facilitated by the devel-opment of mice in which T-cell speci�city is engineered by transgenic expression of de�ned TCRs. Adoptive transfer of T cells of de�ned speci�city (e.g., to S. typhimurium, M. tuberculosis, Aspergillus fumiga-tus, or in!uenza virus) into normal recipient mice, followed by infec-tion, has allowed investigators to determine the kinetics of CD4+ T-cell activation and to measure tra"cking from sites of T-cell priming to sites of infection. CD4+ T-cell priming a�er infection with in!uenza virus, for example, is very rapid and is accompanied by di�erentiation into �1 cells.97 Along similar lines, pulmonary infection with A. fumigatus or Blastomyces dermatitidis results in rapid activation of naïve fungus-speci�c CD4+ T cells in draining pulmonary lymph nodes, followed by tra"cking to the lung parenchyma.98,99 Interest-ingly, complete di�erentiation of fungus-speci�c CD4+ T cells does not occur until CD4+ T cells arrive in the lung. In contrast to these infec-tions, CD4+ T cells responding to inhaled M. tuberculosis infection are not activated until at least 1 week a�er infection100-102 and do not reach the lung until 8 to 12 days a�er infection. Di�erences in the kinetics of antigen transport to draining lymph nodes and the action of regula-tory CD4+ T-cell populations may account for these �ndings. Adoptive transfer of M. tuberculosis–speci�c �1 CD4+ T cells before infection can provide substantial immunity, but, remarkably, not until the infec-tion has progressed for roughly 1 week.102,103

CD8+ T CellsIn contrast to CD4+ T cells, which can di�erentiate along di�erent lines, naïve CD8+ T cells typically di�erentiate into cytolytic T lympho-cytes (CTLs).5 �e principal e�ector function of CD8+ T cells is lysis of pathogen-infected cells, a process triggered by MHC class I–dependent presentation of microbial antigens. �is function is particu-larly e�ective in defense against viral infections, in which case lysis of infected host cells prevents further viral replication.104 �e most rapid cytolytic mechanism involves release of perforin granules by antigen-activated CD8+ T lymphocytes. Perforin lyses the target cell membrane and enables granzymes, which also are released by cytolytic T cells, to enter the target cell and initiate the apoptotic pathway.105 In addition to perforin/granzyme–mediated lysis, CD8+ T cells can also mediate target cell death by expressing Fas ligand, which engages Fas on the target cell surface, resulting in Fas-mediated death. Cytolysis mediated by CD8+ T cells generally targets the host cell and not the microbe.

di�erentiation in mice and humans.64,65 Humans with autosomal recessive mutations that impair Dectin-1 and CARD9 function are susceptible to mucosal candidal infections.61,65 In the intestine, coloni-zation of germ-free mice with commensal segmented �lamentous bac-teria is su"cient to induce the appearance of �17 cells in the lamina propria,66 suggesting that intestinal commensal microbes can stimu-late �17-mediated mucosal protection against pathogenic microbes.

T-Regulatory CellsIn contrast to �1, �2, and �17 cells, which derive from naïve T cells during priming, T-regulatory cells (Tregs) can either emerge from the thymus as nTregs or they can be induced during T-cell priming of naïve T cells, in which case they are referred to as iTregs.67 Tregs were �rst identi�ed as a CD4+CD25+ T-cell subset in normal mice and consti-tuted roughly 10% of the CD4+ T-cell population.68 �is T-cell popula-tion had the remarkable ability to prevent autoimmune in!ammatory bowel disease when transferred into susceptible mice. Both nTregs and iTregs collaborate to suppress the activation of autoimmune T lympho-cytes and mediate tolerance. A recent study demonstrated that tolerance-inducing nTregs and iTregs each contribute to and expand the TCR repertoire implicated in regulatory T-cell function.69 Both TGF-β and IL-10 have been implicated in this process, and, depending on the organ site, both can limit the development of in!ammatory disease. Similar to conventional CD4+ and CD8+ T lymphocytes, nTregs are selected in the thymus on epithelial cells.70 iTregs, on the other hand, are induced in the periphery when naïve T cells are primed in the presence of TGF-β, which induces expression of FOXP3, a tran-scriptional regulator that induces iTreg di�erentiation.71 nTregs emerg-ing from the thymus also express FOXP3.70 �e essential role of FOXP3 in the development of Tregs is revealed by the human disease consist-ing of immune dysregulation, polyendocrinopathy, and enteropathy that is X-linked (IPEX), a profoundly in!ammatory disease that results from a mutation in the gene encoding FOXP3.72-74

�e role of Tregs in defense against microbial infection is signi�-cant. Depending on the microbial pathogen and the type of the elicited in!ammatory response, Tregs can play a positive or a negative role. In murine infection with L. major, for example, Tregs at the site of parasite inoculation prevent complete, �1-mediated microbial clearance, which enables long-term CD4+ T-cell memory to be maintained.75 Along similar lines, depletion of Tregs during murine herpes simplex virus and respiratory syncytial virus infection resulted in adverse outcomes as a result of disorganized early innate immune cell recruit-ment76 or immunopathology linked to respiratory syncytial virus–speci�c CD8+ T-cell responses.77

Conversely, during murine malaria infection, Tregs can su"ciently restrict immune responses to create a lethal outcome and depletion of Tregs enhances survival.78 Similarly, depletion of Tregs in murine �lar-ial infection models markedly enhanced in vivo pathogen clearance.79 In murine tuberculosis, pathogen-speci�c Tregs expand early during pulmonary infection and delay the arrival of e�ector CD4+ and CD8+ T cells in the lung80 yet undergo eventual elimination in response to IL-12–mediated signals.81 In humans, Tregs have been identi�ed during human immunode�ciency virus (HIV), hepatitis B, and hepa-titis C infection.82-84 Treg frequencies in duodenal and hepatic biopsies, but not peripheral blood, are increased in patients with untreated HIV infection or chronic hepatitis C,82,85 suggesting that functional studies of human Tregs will likely require analysis of infected tissue samples.

Helper T-Follicular CellsHuman and murine helper T-follicular (T#) cells represent a distinct migratory CD4+ T-cell population that regulates B-cell antibody responses to T-cell–dependent antigens in germinal centers, a special-ized B-cell zone within secondary lymphoid organs.86,87 T# cells produce IL-21 and promote immunoglobulin class switching and a"n-ity maturation. T# cells are characterized by high expression of the surface markers programmed cell death protein 1 (PD-1), inducible co-stimulator (ICOS), and the chemokine receptor CXCR5 for target-ing to B-cell zones. ICOS-dependent signals regulate the induction of a transcriptional repressor and T# master regulator, BCL6.88-90 It remains unclear whether T# cells arise directly from naïve CD4+ T cells or represent a distinct state of one or more of the de�ned

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priming antigen, antigen-presenting cell, and time of CD8+ T-cell priming. Global transcriptional responses that govern CD8+ T-cell memory formation appear conserved in the context of systemic infec-tion with lymphocytic choriomeningitis virus and Listeria monocyto-genes, despite di�erences in in!ammatory responses elicited by viral and bacterial pathogens.126 In the case of CD8+ T cells, the size of the memory compartment appears to expand with immunologic experience.127

During early stages of the immune response against microbial pathogens, a subset of T cells maintains the capacity to undergo more extended proliferation.128 IL-7 receptor expression marks e�ector CD8+ T cells that give rise to long-term memory T cells,129 although forced expression of this receptor does not promote memory formation.130,131 Memory CD8+ T cells speci�c for a viral pathogen diminish in fre-quency in the absence of IL-15 or if they do not express the IL-15 receptor.132-134 Memory CD4+ T-cell populations show a similar depen-dency on IL-7 and IL-15 signals.135 Despite these similarities, antiviral CD8+ memory T-cell populations typically persist at relatively constant levels over time, whereas virus-speci�c CD4+ memory T cells decrease in frequency.136 Antigen persistence is required for long-term mainte-nance of L. major–speci�c CD4+ memory T cells.75

Several models of memory T-cell generation have been proposed, including the idea that memory T cells rapidly distinguish themselves from conventional e�ector T cells during the primary immune response.137 Most data support an alternate model, however, of naïve T cells di�erentiating into e�ector T cells, then further di�erentiating into memory T cells.118 In support of this notion, loss of cytokine signals via IL-21, IL-10, and STAT3 signaling disrupted the formation of virus-speci�c CD8+ memory T cells while retaining CD8+ terminal e�ector di�erentiation states.138 Comparison of the TCR repertoire of pathogen-speci�c e�ector and memory T cells suggested that these two populations shared a common ancestry.139 Memory T cells appear to have expressed genes encoding e�ector proteins, further supporting the notion that memory T cells derive from e�ector T cells.140

�e interaction between CD4+ and CD8+ T cells during immune responses to infection has also been investigated. CD8+ T-cell priming depends on the presence of CD4+ T-cell help a�er immunization in the absence of in!ammation. CD4+ T cells can provide help through two mechanisms. First, they can activate antigen-presenting DCs through CD40/CD40L interactions, upregulating the expression of co-stimulatory molecules on antigen-presenting cells.141-143 Under priming conditions associated with greater innate in!ammation, CD8+ T-cell priming and expansion can occur without CD4+ T-cell help. However, long-term memory responses of CD8+ T cells primed in the absence of CD4+ T cells are compromised.144 Several studies indicate that CD8+ T-cell memory is programmed during the priming process through CD4+ T-cell–mediated stimuli that are distinct from the stimuli required for proliferation and e�ector di�erentiation.144-146 A recent study demonstrated that IL-2 stimulation of CD8+ T cells during priming has long-term e�ects on their survival,147 re�ning the old notion that CD4+ T cells produce IL-2 to enhance CD8+ T-cell prolif-eration and di�erentiation.

Generation and maintenance of CD8+ T-cell memory a�er micro-bial infection is in!uenced by the chronicity of infection. �us, infec-tions that persist eventually result in CD8+ T-cell populations that lose e�ector functions in a process referred to as exhaustion, a process that may be driven by sustained antigen presentation.148-150 Studies in mice infected with a strain of lymphocytic choriomeningitis virus that causes chronic infection demonstrated that exhausted CD8+ T cells expressed PD-1, a surface protein that transmits an inhibitory signal to proliferat-ing cells. Blocking PD-1 signaling with a speci�c antibody rescued exhausted T cells, enabling them to reexpress e�ector functions.151 Studies of patients with persistent HIV infection also demonstrated PD-1 expression on exhausted CD8+ T cells, which was associated with disease progression.152 Whether blockade of PD-1 in HIV-infected patients can reverse CD8+ T-cell exhaustion is not known.

Detailed characterization of memory T lymphocytes revealed that they can di�er with respect to surface expression of activation markers. �is discovery has led to the division of memory T lymphocytes into two populations: e�ector memory T cells and central memory T cells.122,153 �e former population expresses low levels of CD62L and

Studies have shown, however, that human T cells produce a cytolytic protein called granulysin that can kill bacteria directly, including M. tuberculosis.106 Granulysin is produced by CD8+ and CD4+ T cells and has been implicated in defense against M. leprae.107

In most infections, pathogen-speci�c CD8+ T cells secrete IFN-γ and TNF, which enhances macrophage- and neutrophil-mediated microbial killing. In addition to cytokines, CD8+ T cells produce che-mokines, such as RANTES (regulated on activation, normal T-cell expressed and secreted) and macrophage in!ammatory proteins 1α and 1β. Chemokine secretion by T cells contributes to the recruitment of in!ammatory cells to sites of infection. Expression of these chemo-kines by CD8+ T cells is suggested to play a role in reducing HIV infectivity.108

Studies in animal models have provided the clearest picture of pathogen-speci�c T-cell responses. CD8+ T-cell responses are induced rapidly a�er infection with viruses, such as in!uenza and lymphocytic choriomeningitis virus, and bacteria, such as L. monocytogenes.109,110 Pathogen-speci�c T cells are detectable 5 days a�er infection and expand rapidly to peak frequencies approximately 8 days a�er infec-tion. Studies using adoptively transferred T cells of de�ned speci�city, labeled with a !uorescent dye, have shown that antigen-speci�c T cells undergo rapid proliferation during this phase of the immune response, dividing at a rate of approximately once every 6 hours.111,112 In a matter of a few days, the frequency of antigen-speci�c T cells can increase from 1 in 100,000 CD8+ T cells to 1 in 2 CD8+ T cells in some viral infections.

During the early immune response, a rapid expansion phase of activated, antigen-speci�c CD8+ T cells occurs and this process involves a metabolic switch from oxidative phosphorylation to anaerobic gly-colysis to meet the demand for nucleic acid, protein, and lipid synthe-sis.113 In addition, some cytokine receptors, such as the IL-7 receptor, which transmits homeostatic signals to naïve T cells, are downregu-lated whereas other receptors, such as the high-a"nity IL-2 receptor, are upregulated.114 On initial stimulation, responding T cells are pro-grammed and undergo proliferation and di�erentiation independent of further encounters with antigens or with the in!ammation associ-ated with infection.115,116 Although innate in!ammation and T-cell priming are interwoven processes, when primed, T cells follow a pathway that is remarkably independent of in!ammation. When CD8+ T cells complete the expansion phase, the frequency of pathogen-speci�c CD8+ T cells decreases, ultimately giving rise to a residual memory T-cell population. Similar to the expansion phase, the contrac-tion phase of CD8+ T-cell responses is antigen and in!ammation inde-pendent.117 Characterization of gene expression by CD8+ T cells during an immune response to infection indicates that molecules associated with cell survival, prominently BCL2, are regulated in a fashion that leaves e�ector T cells vulnerable to apoptosis.118 Downregulation of BCL2 and upregulation of Fas on the cell surface enhances CD8+ T-cell death and contributes to contraction of antigen-speci�c T-cell popula-tions. More recent studies demonstrate that T-cell survival re!ects the balance of Bim, a proapoptotic molecule, and antiapoptotic BCL2 pro-teins.119 For example, glucocorticoid-induced TNF receptor–related protein (GITR), a TNF receptor family expressed on CD8+ T cells, provides an antiapoptotic signal that promotes CD8+ T cell expansion during acute in!uenza.120

T-Cell MemoryA hallmark of the adaptive T-lymphocyte response is its capacity for antigen-speci!c memory.121,122 T lymphocytes with speci�city for a par-ticular pathogen can persist in the host for many years a�er the patho-gen has been eliminated. CD8+ T lymphocytes can persist without reexposure to antigen, either from exogenous sources or from antigen depots that might be maintained a�er the resolution of primary infec-tion. Whereas naïve T cells depend on the presence of MHC molecules for long-term survival, memory T cells survive and undergo homeo-static proliferation in the absence of any MHC molecules.123 �us, in vivo maintenance of naïve and memory T cells is fundamentally di�erent.

A single naïve CD8+ T cell can give rise to both e�ector and memory T-cell populations in a broad range of infection models,124,125 indicating that memory fate decisions and formation occur independently of the

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high circulating IgM levels, and are susceptible to infections with Pneu-mocystis jirovecii, indicating defective T-cell–mediated immunity. In mice de�cient for CD40 or CD40L, T-cell activation and maintenance of memory T-cell populations are defective, accounting for increased susceptibility to some infections.141,142 Other receptors, such as 4-1BB and OX40, also have been implicated as playing a positive role in the generation of pathogen-speci�c T-cell responses.176

�e interface between T lymphocytes and antigen-presenting cells is a highly organized structure termed the immunologic synapse.177 �is structure, also referred to as the supramolecular activation complex (SMAC), contains a central region (i.e., cSMAC) that contains TCR and MHC contacts and a peripheral region (i.e., pSMAC) that contains lymphocyte function–associated antigen 1 (LFA-1) and intracellular adhesion molecule (ICAM) contacts.177 Co-stimulatory molecules, such as CD28, and essential signaling molecules, such as PKC-θ, are present in the SMAC as well.178 Synapse formation occurs when an antigen-speci�c T cell encounters an antigen-presenting cell, enabling these cells to engage each other for several hours. �e role of the SMAC in T-cell activation remains somewhat controversial, however, because T-cell signaling can precede synapse development.179 A currently favored hypothesis is that SMAC formation may play a more signi�-cant role in the response to infrequent peptide/MHC complexes or perhaps to lower a"nity ligands for the TCR.180

Antigen Presentation to T LymphocytesSeveral aspects of T lymphocyte recognition of antigens distinguish this process from antibody-mediated antigen recognition. First, T lym-phocytes recognize antigens only in the context of MHC haplotype-matched, antigen-presenting cells.181 Early studies indicated that antigen-presenting cells degrade pathogen-derived proteins in a time-dependent process that requires antigen internalization followed by transport to the cell surface.182 Further characterization of this process revealed that T cells detect peptide fragments of pathogen-derived proteins and that short synthetic peptides corresponding to fragments of pathogen-derived proteins can stimulate T lymphocytes.183,184

�e precise nature of MHC involvement in antigen presentation became clear with the crystallization of human leukocyte antigen (HLA)-A2, which showed a globular protein with a central groove that precisely accommodates a solitary peptide.185,186 �e other important development that allowed a complete picture of the T-cell recognition process was the identi�cation of the TCR.187 �is heterodimeric protein, consisting of two transmembrane chains, provides diversity and speci�city by a gene recombination process that is mechanistically similar to the generation of antibody diversity in B cells.188 �e follow-ing sections describe the structure of MHC molecules and the process by which pathogen-derived peptides are presented to TCRs of CD4+ and CD8+ T cells.189

Major Histocompatibility Complex Structure and Peptide BindingMHC Class I StructureMHC class I molecules present peptide antigens to CD8+ T lympho-cytes. Most nucleated cells express MHC class I molecules, although the amount on the cell surface varies dramatically among cell types and under di�erent in!ammatory conditions.190 MHC class I mole-cules are transmembrane proteins that consist of a single α-chain and associate with β2-microglobulin for proper folding and tra"cking to the cell surface. �e structure of an MHC class I molecule binding a virally derived peptide is shown in Figure 6-2.

�e characteristic structural features of MHC class I molecules are α1, α2, and α3 domains; they create a globular protein in which a β-pleated sheet forms the !oor of the peptide-binding groove and is bounded by two helical regions that form the sides of the groove.185,186,191 An important feature of the MHC class I groove is the restricted size of the peptide, typically 9 amino acids in length, that can be accom-modated, since the groove is closed at both ends.192

MHC class I molecules are highly polymorphic. Although di�erent protein products (allomorphs) encoded by MHC class I genes are structurally similar and bind many di�erent peptides, the peptides that bind to di�erent allelic forms of MHC class I are distinct.192 Each allomorph of MHC class I binds a distinct subset of peptides because

CCR7, actively expresses e�ector functions (e.g., cytokine synthesis and cytolytic activity), and tra"cs to peripheral rather than secondary lymphoid tissues. Recent studies suggest that e�ector memory cell populations are heterogeneous and include tissue-resident memory T cells that reside within the gut, skin, and other mucosal tissues with minimal turnover.154,155 For example, noncirculating, skin-resident CD8αα+ T cells persist in human genital mucosa at the dermoepider-mal junction, the site of herpes simplex virus type 2 neuronal release, and are critical to contain episodes of viral reactivation.156 Central memory T cells express high levels of CD62L and CCR7, do not actively express e�ector functions, and tra"c preferentially to second-ary lymphoid tissues. �ese two populations of memory T cells are postulated to play di�erent roles in providing protective immunity. E�ector memory T cells are present in tissues and are ready to engage the pathogen, should invasion occur. Central memory T cells are believed to provide a source of e�ector memory cells and, although they would be anticipated to require more time for activation, can play a role in protective immunity.

NKT Cells and T Cells with a Restricted TCR ResponseAs discussed earlier, T-lymphocyte populations bear diverse TCRs that determine antigen-binding speci�city. A small subset of T lympho-cytes, generally termed natural killer T cells (NKT cells), expresses a limited repertoire of TCRs that includes a single TCR α-chain (Vα14) and a limited number of Vβ chains. In addition to the TCR, these cells also express NK-cell markers and are either CD4+ or double nega-tive (CD4−/CD8−). NKT cells are of particular interest because they respond to de�ned synthetic (α-galactosyl ceramide), bacterial,157,158 and fungal159 glycolipid antigens presented on CD1d. Alternatively, NKT-cell activation can occur in a cytokine-driven fashion, as has been observed in the context of viral infections.160 NKT cells may play a role in immune defense against Borrelia burgdorferi,161 Streptococcus pneu-moniae,162 in!uenza, and viral hepatitis.163

Two additional populations of human T lymphocytes with a restricted TCR α-chain repertoire have been identi�ed. Mucosal asso-ciated invariant T (MAIT) cells predominately reside in the gut, express an invariant TCR α-chain (Vα7.2Jα33 in humans) coupled to a limited repertoire of TCR β-chains, and are activated by microbial ribo!avin metabolites presented on the MHC class I–related molecule MR1.164,165 Human CD4 germline-encoded mycolyl lipid–reactive (GEM) T cells express a limited TCR α-chain diversity and respond to M. tuberculosis–derived mycolic acids restricted by CD1b (see later section “CD1”).166 �ese populations of TCR α-chain–restricted lymphocytes, γδ T cells, and the growing subsets of innate lymphoid cells, including NK cells and lymphoid tissue inducer cells,167 straddle the traditional boundar-ies between innate and adaptive immune cells.

T-CELL ACTIVATIONT-cell activation is a remarkably complex process and begins when the TCR binds to cognate MHC peptide complexes,168 triggering a highly complex signaling cascade. �e most important signal a�er engage-ment of the TCR with the MHC/peptide complex is mediated by the co-receptor CD28 on the T cell and B7.1 and B7.2 on the antigen-presenting cell.169 �is interaction induces IL-2 production by T lym-phocytes, which promotes their proliferation. On activation, T lymphocytes express CTLA-4, which also binds B7.1 and B7.2, but instead of stimulating proliferation, this signaling molecule inhibits proliferation and acts as a brake on expansion. Studies have identi�ed many other related B7 molecules that seem to play a role in the activa-tion of T lymphocytes in peripheral tissues.170 One of these molecules, B7h, interacts with the inducible molecule ICOS on the surface of activated T cells and promotes their di�erentiation toward the �2 phenotype.171-173

Many receptors that belong in the TNF receptor superfamily also play a role in the activation and di�erentiation of T lymphocytes.174 Among these, the role of CD40 and its agonist CD40L are perhaps the most completely investigated. �ese genes play a critical role in T-cell and B-cell immunity, since genetic defects in CD40L and CD40 under-lie the hyperimmunoglobulin M (hyper-IgM) syndrome.175 A�ected individuals have defects in antibody isotype switching, accounting for

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�e MHC class I antigen-processing pathway begins in the cytosol with the degradation of a protein that usually represents an endogenous self-protein. Intracellular microbial-derived proteins enter the MHC class I processing pathway a�er microbial infection.189 Extracellular host or microbial proteins that are internalized into membrane-bound compartments by the process of endocytosis or phagocytosis can enter the MHC class I processing pathway by a mechanism referred to as cross-presentation.

Proteasomes constitutively degrade misfolded or unneeded pro-teins by proteolysis in the cytosol and thereby regulate cellular protein composition. �ese barrel-shaped multicomponent proteases consist of four stacked rings that each contain seven subunits.200 Proteasomes target proteins for degradation by a variety of mechanisms. Misfolded proteins and defective ribosomal products, both of which fail to assume native conformation states, may reveal peptide sequences that are rec-ognized by proteasomes, leading to their rapid degradation.201 Alter-natively, many proteins targeted for rapid proteasomal degradation undergo conjugation to ubiquitin by enzymes that recognize the phos-phorylation of speci�c amino acids.201

During microbial infection, proteasomal degradation of microbial antigens is critical for the display of microbial peptides by MHC class I proteins. �e proteasomal composition changes in activated cells or a�er exposure to IFN-γ, with some proteasomal subunits replaced by others that enhance the generation of antigenic peptides and with additional components added to the ends of the barrel to alter the e"ciency and speci�city of protein degradation.189

In the setting of infection, it is unclear whether pathogen-derived polypeptides and proteins are degraded selectively and targeted for presentation by MHC class I molecules. Bacterial proteins that �nd their way into the cytosol are degraded rapidly because they express unique amino-terminal amino acids202 or contain internal amino-acid sequences that promote rapid degradation.203 In most circumstances, pathogen-derived antigens are probably degraded nonselectively along with endogenous proteins, and pathogen-derived peptides compete with more prevalent endogenous peptides for a place in an MHC class I peptide–binding groove.

Proteasomes generate peptides that, by virtue of the length of the proteasome channel, are 9 to 12 amino acids in length.204 Proteasome-generated peptides are bound to the peptide transporter named trans-porter associated with antigen processing (TAP). �is heterodimeric, adenosine triphosphate (ATP)-dependent transporter e"ciently moves peptides from the cytosol into the lumen of the endoplasmic reticulum. �e transport e"ciency of TAP as a transporter is not mediated by primary sequence (i.e., most peptide sequences are well transported by TAP) but rather by peptide length, with peptides shorter than 6 amino acids or longer than 14 amino acids being poorly transported. TAP is the major peptide transporter involved in the generation of peptide/

of structural di�erences between the peptide-binding grooves. Most MHC class I molecules have a groove with two or three deeper pockets that provide space for bulky amino-acid side chains. �e peptide sequences that a particular MHC class I molecule can bind are deter-mined by the shape, depth, and charge of the groove and the binding pockets. Because most individuals express six di�erent MHC class I alleles (two each of HLA-A, HLA-B, and HLA-C), a diversity of pathogen-derived peptide sequences can be presented to T lympho-cytes. Elution of peptides from the HLA-A2 MHC class I molecule and analysis by mass spectrometry revealed that HLA-A2 can bind thou-sands of distinct peptides that share a common motif but derive from a plethora of cellular proteins.193

Although binding of peptides and interaction with TCRs is medi-ated entirely by the MHC class I protein, stable surface expression of peptide/MHC class I complexes requires the association with β2-microglobulin. �is 12-kDa protein binds to membrane-bound MHC class I molecules, providing structural stability. In the absence of β2-microglobulin, most MHC class I molecules fold improperly and are destroyed before leaving the endoplasmic reticulum.194

MHC Class II StructureMHC class II molecules present peptides to CD4+ T cells. In contrast to MHC class I molecules, MHC class II expression is restricted to only a few cell types: macrophages, DCs, and B lymphocytes. On activation, endothelial cells can also express MHC class II molecules.195 Regula-tion of MHC class II expression is mediated in part by the class II transactivator protein (CIITA), a transcription factor that enhances expression of MHC class II molecules, the associated invariant chain, and other molecules associated with MHC class II antigen process-ing.196 Mutations in the CIITA gene give rise to bare lymphocyte syn-drome, an important human primary immunode�ciency syndrome associated with profound immunosuppression, because circulating peripheral blood mononuclear cells do not express surface MHC class II molecules.197

�e folded MHC class II molecule consists of two transmembrane proteins, an α-chain and a β-chain, which together form a protein with an open-ended peptide-binding groove.198 �e open-endedness of the MHC class II groove accounts for the binding of substantially longer peptides than seen with MHC class I molecules.199 Peptides bound by MHC class II molecules typically are longer than 10 amino acids and occasionally more than 20 amino acids. �e structure of an MHC class II molecule binding an antigenic peptide is shown in Figure 6-3.

Mechanisms of Antigen ProcessingMHC Class I Antigen-Processing PathwayAntigens presented by MHC class I and class II molecules generally derive from di�erent cellular compartments (see Fig. 6-4 for details).

FIGURE 6-2 Class I major histocompatibility complex. MHC class I molecules bind short antigenic peptides in a central groove formed by two α-helices of the heavy chain. The T-cell receptor binds the peptide but also associates with the MHC class I molecule, in order to transmit an activation signal to the T lymphocyte. The structures that are depicted represent the murine H2-Kb MHC class I molecule (purple) binding a peptide derived from vesicular stomatitis virus (green). β2-microglobulin is shown in pink. The left panel shows a side view of the structure, and the right view of the complex is from the top, as it would be detected by the T-cell receptor on a CD8+ T cell. (Courtesy Dr. Chris Garcia, Stanford University, Palo Alto, CA.)

FIGURE 6-3 Class II major histocompatibility complex. MHC class II molecules also bind antigenic peptides in a groove formed by the α- and β-chains. In contrast to peptides bound by MHC class I molecules, which are of defined length, the MHC class II structure accommodates peptides of various lengths. The MHC class II α- and β-chains are shown in pink and purple, whereas the antigenic peptide is shown in green. The left panel shows a view of the complex from the side, and the right panel shows a view from the top, as it would be detected by a T-cell receptor on a CD4+ T cell. (Courtesy Dr. Chris Garcia, Stanford University, Palo Alto, CA.)

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that modify proteasome e"ciency and speci�city, and prominent among these is PA28, an activator that consists of six subunits that form rings that can cap the ends of the proteasome.219 PA28 can increase the e"ciency with which virus-derived, MHC class I–restricted epitopes are presented to CD8+ T cells.220 In the setting of infection, the MHC class I antigen-processing pathway is enhanced, allowing more e"cient presentation of pathogen-derived peptides to CD8+ T lymphocytes. �e MHC class I antigen-processing pathway is outlined in Figure 6-4.

Viral Intervention with the MHC Class I Antigen-Processing PathwayCD8+ T lymphocytes and the MHC class I antigen-processing pathway are involved principally in defense against viral infection. �e length to which viral pathogens have gone to subvert the MHC class I antigen-processing pathway is fascinating and indicates the importance of this process in antiviral defense. It was an early observation that cells infected with herpesviruses downregulate MHC class I expression. Exploration of the mechanism behind this observation uncovered many viral proteins that block distinct steps along the MHC class I antigen-processing pathway.221 ICP47 is encoded by herpes simplex virus and blocks human TAP by plugging the peptide transport channel from the cytosolic side.222,223 Using a similar strategy, but with a com-pletely distinct protein, the human cytomegalovirus-encoded protein US6 blocks TAP transport by obstructing the peptide transporter from the endoplasmic reticulum luminal side.224,225 Human cytomegalovirus also uses several other mechanisms to prevent MHC class I molecules from reaching the cell surface. �e US3 protein binds MHC class I molecules in the endoplasmic reticulum and prevents their tra"cking to the cell surface.226,227 A similar strategy also is used by adenoviruses, which encode the type I membrane protein E3-19K.228,229 �is protein binds MHC class I molecules in the endoplasmic reticulum lumen and prevents their egress from the endoplasmic reticulum by expressing an endoplasmic reticulum retention motif on its cytoplasmic tail. Another strategy for downregulating surface MHC class I retention is to dis-place endoplasmic reticulum–resident MHC class I molecules into the cytoplasm, where they are ubiquitinated rapidly and degraded by

MHC class I complexes; mice with genetic deletions of TAP have mark-edly decreased levels of surface MHC class I and markedly diminished numbers of CD8+ T cells.205 TAP de�ciency has been identi�ed rarely in humans and is associated with markedly decreased numbers of circulating CD8+ T cells and modest immunode�ciency.206,207

�e TAP1 and TAP2 molecules each contain seven transmembrane regions and an ATP binding site and together transport peptides from the cytosol into the endoplasmic reticulum lumen.208 Newly synthe-sized MHC class I molecules associate with TAP in the endoplasmic reticulum, and the recruitment of several other endoplasmic reticulum resident proteins and chaperones leads to the formation of the peptide loading complex (PLC). �e PLC includes the MHC class I/β2-microglobulin complexes bound to tapasin, which serves as a molecu-lar adaptor for TAP as well as the chaperones calreticulin and the thiol reductase ERp57.189,209-211 �e key role of β2-microglobulin and of the PLC is to maintain the MHC class I peptide–binding groove in a con-formation that favors the binding of high-a"nity peptides. Because TAP delivers many peptides into the endoplasmic reticulum lumen that are too long to �t into the MHC class I peptide–binding groove, the endoplasmic reticulum resident proteases ERAP1 and ERAP2 trim peptides before their �nal integration into the MHC class I groove.212,213 If the a"nity of the peptide-MHC class I interaction is su"ciently high, the PLC releases the MHC class I/β2-microglobulin/peptide complex, enabling it to tra"c via the Golgi complex to the cell surface. If the a"nity of the peptide/MHC class I interaction is low, the MHC class I heavy chain undergoes re-glycosylation of an N-linked glycan,214 a reaction that redirects MHC class I molecules into the PLC for peptide exchange and prevents release into the secretory pathway.

�e MHC class I antigen-processing pathway is regulated by in!ammation. IFN-γ, in particular, is a cytokine that impacts the MHC class I pathway at multiple levels.215 First, IFN-γ enhances the tran-scription of many components of the MHC class I pathway, including MHC class I molecules, TAP, tapasin, and several components of the proteasome. Speci�cally, three subunits of the proteasome, LMP-2, LMP-7, and MECL, are induced and replace three subunits of the core proteasome complex.216-218 IFN-γ induces additional accessory proteins

FIGURE 6-4 The major histocompatibility complex class I antigen-processing pathway. The MHC class I antigen-processing pathway begins in the cytoplasm with the degradation of proteins by proteasomes. Peptides are transported into the lumen of the endoplasmic reticulum, where they are bound by MHC class I molecules and transported to the cell surface. β2m, β2-macroglobulin; TAP, transporter associated with antigen processing. (Courtesy Anne Ackerman and Peter Cresswell, Yale University, New Haven, CT.)

Calreticulin

ERp57

Tapasin

Class I

Calnexin ProteasomeTAP I TAP II TAP I TAP II

Peptides

Endoplasmic reticulum

Golgi apparatus

Plasma membrane

β2m

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and is not restricted to endogenous cytosolic proteins. Because they o�en are not infected directly, the major route for CD8+ T-cell priming involves uptake of debris from infected cells by DCs and re-presentation of pathogen-derived peptides243 by an antigen-processing pathway that involves endocytosis and TAP-mediated transport of antigen into the endoplasmic reticulum.244 �e CD8+ subset of DCs is particularly e"cient at taking up and delivering exogenous antigens for presenta-tion by the MHC class I antigen-processing pathway.245 Antigen-containing phagosomes in DCs fuse with endoplasmic reticulum membranes,246 resulting in the recruitment of a retrotranslocation machinery that shuttles misfolded proteins or antigens from the phagosome lumen into the cytosol, where proteins are degraded by proteasomes and enter the conventional MHC class I processing pathway.247 �is circuitous route for CD8+ T-cell priming ensures that CD8+ T-cell priming occurs in a regulated fashion and likely represents an essential hurdle to prevent overly robust CD8+ T-cell responses to systemic viral infections.

MHC Class II Antigen-Processing Pathway�e MHC class II antigen-processing pathway presents peptides to CD4+ T lymphocytes (Fig. 6-5). Although similarities to the MHC class I antigen-processing pathway exist, there are some key distinctions. First, most peptides presented by MHC class II molecules derive from extracellular proteins that have been endocytosed by MHC class II–expressing cells.199 MHC class II molecules also present peptides from membrane or secretory proteins that are degraded in endosomal com-partments during transport to the cell surface. With respect to anti-microbial responses, the MHC class II antigen-processing pathway has been implicated principally in the response to extracellular patho-gens and pathogens that reside within vacuolar compartments, such as S. typhimurium and M. tuberculosis.13,248 Because CD4+ T-cell responses also are essential for complete priming, activation, and di�erentiation of CD8+ T-cell responses, however, it is di"cult to attribute immune susceptibility directly to CD4+ T-cell de�ciency.148 �e implications and complexities of CD4+ T-cell de�ciency have been made clear by the HIV epidemic.249,250

�e �rst step in the MHC class II antigen-processing pathway is the translocation and assembly of MHC class II α- and β-chains in the endoplasmic reticulum, a reaction mediated by a dedicated chaperone, the invariant chain. By and large, MHC class II molecules do not bind antigenic peptides in the endoplasmic reticulum. �e α- and β-chains

proteasomes. Human cytomegalovirus encodes two proteins, US2 and US11, which mediate this process.229,230 Remarkably, transport of pro-teins from the endoplasmic reticulum lumen back to the cytosol via the Sec61 translocon is a normal process that usually is restricted to misfolded or otherwise nonfunctional proteins. US2 and US11 seem to accelerate this process selectively for MHC class I molecules. Crys-tallographic studies have characterized the binding of US2 to MHC class I molecules.231

�e Kaposi sarcoma herpesvirus also downregulates surface MHC class I expression, but by another mechanism. Two Kaposi sarcoma herpesvirus–encoded proteins, K3 and K5, function as ubiquitin ligases that selectively conjugate ubiquitin to the cytoplasmic tail of MHC class I molecules and B7.2 co-stimulatory molecules.232,233 On ubiquitination, surface MHC class I molecules are internalized rapidly and targeted for lysosomal degradation. HIV also has evolved mecha-nisms to downregulate surface expression of MHC class I molecules.234-236 In this case, the retrovirally encoded Nef protein selectively downregu-lates the expression of HLA-A and HLA-B molecules by associating with the clathrin adaptor complex.

An important consequence of MHC class I downregulation is that a�ected cells become susceptible to NK cell–mediated lysis. NK cells express receptors that inhibit NK cell activation on contact with MHC class I molecules. To prevent NK cell–mediated lysis of virally infected cells, human CMV encodes an MHC class I–like molecule, UL18,237,238 which acts as a decoy for the NK cell inhibitory receptor LIR-1,239 providing yet another level of camou!age to the viral pathogen.

MHC Class I Cross-Priming�e MHC class I antigen-processing pathway performs two principal functions. First, it presents antigens to naïve CD8+ T cells in a manner that promotes their activation, proliferation, and di�erentiation. Second, it presents antigens to activated CD8+ T cells as a signal of cellular infection. �e �rst function is predominantly, if not exclusively, mediated by DCs.240,241 Any MHC class I–expressing cell that becomes infected can perform the second function. �e rules of antigen pro-cessing di�er in these two circumstances. �e conventional MHC class I antigen-processing pathway, as described in the preceding sec-tions, applies to the second function. When cells become directly infected, pathogen-derived antigens are degraded by the infected cell and presented on the cell surface in the context of MHC class I mol-ecules.242 MHC class I antigen presentation by DCs is more complex

FIGURE 6-5 The major histocompatibility complex (MHC) class II antigen-processing pathway. The MHC class II antigen-processing pathway presents peptides derived from extracellular proteins to CD4+ T lymphocytes. Peptides are generated in an endosomal compartment. The peptide editor HLA-DM promotes dissociation of the place holder CLIP from MHC class II molecules, allowing the exogenous peptide to associate with MHC class II. The MHC class II/peptide complexes are then transported to the cell surface. (Courtesy Anne Ackerman and Peter Cresswell, Yale University, New Haven, CT.)

Endoplasmic reticulum

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DR αβ αβI

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aliphatic chain length (see Fig. 6-6). In contrast to the �ve CD1 iso-forms found in humans, mice lack CD1a, CD1b, and CD1c and have a duplicated CD1d gene. �is important di�erence between mice and human CD1 complicates the experimental analysis of CD1 function because the powerful tools of genetically altered mice are not available to analyze the function of CD1a, CD1b, or CD1c.

Antigens Presented by CD1CD1 presents lipid and glycolipid antigens from a variety of bacteria and fungi to T cells. Beyond the well-described role of CD1 in myco-bacterial antigen presentation discussed in the ensuing paragraph, CD1-presented antigens include diacylglycerols from S. pneumoniae,157 a fungal glycosphingolipid (i.e., asperamide B),159 and a synthetic gly-colipid, α-galactosyl ceramide, which is derived from marine sponges. �ese latter three compounds are presented in a CD1-restricted manner to NKT cells, and α-galactosyl ceramide in particular has been used widely to study the e�ect of CD1d-mediated NKT-cell activation on host defense against infections and tumors.

CD1 plays an important role in presenting mycobacterial lipids to T cells,267,268 speci�cally glycosylated and free mycolic acids and lipo-arabinomannan, two major lipid and glycolipid components of the M. tuberculosis cell envelope. Further analyses of the structure of CD1-presented lipids revealed that T-cell recognition of CD1b-presented glycolipids was exquisitely sensitive to the �ne structure of the carbohydrate head group but relatively insensitive to alterations in the structure of the lipid tail.269 �ese observations, along with later characterization of CD1c-presented mycobacterial isoprenoid glyco-lipids,269 have led to a model of CD1 lipid antigen presentation in which the hydrophilic head group is exposed and available for TCR interac-tions, whereas the hydrophobic lipid tails of the antigen are bound within the hydrophobic pockets of the CD1 protein.

Cell Biology of CD1 Antigen Processing and Loading�e CD1 isoforms are enriched in di�erent intracellular compart-ments, suggesting that each isoform of CD1 has evolved to survey the microbial antigens that appear in distinct parts of the endosomal-lysosomal network.270 Figure 6-7 summarizes CD1 tra"cking path-ways. All CD1 isoforms are found at the cell surface and are internalized during endocytosis. CD1a is found predominantly in early endosomes, CD1c in late endosomes, and CD1b/d in late endosomes and lyso-somes. Speci�c amino-acid residues in their short intracellular tails target CD1 isoforms to these various compartments by binding cyto-solic adaptor molecules that mediate organelle tra"cking.271-273 Although the precise functional implications of this tra"cking pattern for the immune response are still being elucidated, each CD1 isoform may survey a distinct intracellular compartment for distinct lipid structures from distinct pathogens.

fold, using the invariant chain, a membrane-bound protein, as a sub-stitute for peptide.251 A portion of the invariant chain occupies the MHC class II groove as the complex exits the endoplasmic reticulum, traverses the Golgi complex, and tra"cs to the endosomal compart-ments.252,253 On acidi�cation of the endosomal compartment, proteases such as cathepsin D and cathepsin B are activated and degrade all parts of the invariant chain except for the portion protected by the MHC class II groove.254,255 It is in the acidi�ed endosome, in a compartment called MIIC, that MHC class II molecules can intersect with endocy-tosed antigens in the process of degradation.256,257 In a complex, topo-logically challenging series of events, the invariant chain fragment is replaced by a proteolytically generated peptide. Although MHC class II molecules, similar to MHC class I molecules, are selective with respect to peptide binding, because the groove is open and can accom-modate peptides in various registers, the range of peptides that are bound is greater and the binding stringency relaxed. �e mechanics of MHC class II peptide binding are accelerated by the presence of another MHC-encoded, MHC class II–like molecule that resides in MIICs named HLA-DM.258 HLA-DM catalyzes the extraction of the invariant chain peptide fragment from the MHC class II molecule, and recent crystallographic studies reveal that HLA-DM stabilizes empty MHC class II proteins in a conformation that promotes the insertion of a high-a"nity peptide.259,260 In addition to proteases, a thiol reduc-tase called GILT (gamma interferon–inducible lysosomal thioreduc-tase) also is involved in the denaturation of some antigens before their degradation and presentation by MHC class II molecules.261 GILT has also been implicated in the activation of the major secreted virulence factor listeriolysin-O, of the bacterial pathogen L. monocytogenes, pro-viding a remarkable example of microbial exploitation of antigen-processing pathways.262

On peptide binding in the MIIC, MHC class II molecules tra"c to the cell surface, where the MHC class II/peptide complex can be detected by CD4+ T cells. MHC class II molecules are re-internalized, and it is possible that these complexes recycle to the endosomal com-partments and acquire new peptides before returning to the cell surface. �e extent to which this pathway contributes to the MHC class II antigen-processing pathway during immune responses to infection is incompletely de�ned.263

CD1�e CD1 family comprises antigen-presenting molecules that resemble MHC class I molecules in general structure and in their association with β2-microglobulin.264 �e human CD1 locus on chromosome 1 contains �ve distinct genes that encode for proteins named CD1a through CD1e. Consistent with their structural similarity to MHC class I, CD1 molecules generally are highly expressed on antigen-presenting cells, present antigens for recognition by the TCR, and interact with T lymphocytes. CD1 molecules di�er dramatically from the MHC in the class of antigens presented, however. In contrast to the peptide antigens presented by the MHC, CD1 proteins present lipid and glycolipid antigens to T cells, a discovery that radically expanded the universe of antigens recognized by T lymphocytes. Figure 6-6 shows the structure of the human CD1b molecule presenting the gan-glioside GM2.265 �is section reviews the antigens presented by CD1, the lymphocytes that recognize these antigens, and the importance of this antigen-presenting system in host defense against speci�c infec-tious diseases.

CD1 Protein StructureCD1 proteins are single-pass transmembrane proteins that have a short intracellular domain. �e extracellular portion of CD1 is composed of three domains that mediate antigen binding, whereas speci�c motifs in the intracellular domain control CD1 tra"cking to intracellular compartments (see later discussion). �e extracellular domains of CD1 molecules form an antigen-binding groove that is structurally analo-gous to the peptide-binding groove of the MHC. Consistent with the lipid presentation function of CD1, the antigen-binding groove con-tains multiple hydrophobic pockets that can accommodate the ali-phatic chains of lipid antigens. �e three-dimensional structure of mouse CD1266 and human CD1b265 reveals a complicated network of hydrophobic channels that can accommodate diverse lipids of varying

FIGURE 6-6 Structure of CD1b in complex with a lipidic antigen. The structure of CD1 molecules differs from the structure of MHC class I molecules but also shares substantial similarities. This figure demonstrates the structure of the human CD1b molecule binding the lipid ganglioside GM2. The CD1 chain is shown in blue and β2-microglobulin is shown in pink. The lipid is shown in green. The left panel is a side view of the complex, and the right panel shows a view from the top, as it would be detected by a T-cell receptor. (Courtesy Dr. Chris Garcia, Stanford Univer-sity, Palo Alto, CA.)

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classes of microbial molecules that serve as a general signal of infec-tion. �is recognition event, rapid but relatively nonspeci�c, plays an essential role in the generation of pathogen-speci�c immunity through the generation of cytokines and chemokines that recruit antigen-speci�c cells to the site of infection and shape the ensuing adaptive immune response. �is section brie!y details the major known recep-tors of the innate immune system and links the function of these receptors to antigen-speci�c cellular immunity.

Molecular Recognition of Microbial ProductsAlthough the concept that microbial products are recognized by speci�c receptors of the innate immune system is not new, recent investigations have dramatically expanded our understanding of the receptors involved in this process. Several outstanding reviews pro-vide a comprehensive overview on the molecular recognition of microbial products and its role on human susceptibility to infectious diseases.274-279

Toll-like and C-Type Lectin ReceptorsToll-like receptors (TLRs) are a family of at least 10 distinct transmem-brane proteins that mediate the recognition of extracellular and endo-somal microbial products. �e TLR ectodomains contain leucine-rich repeats that bind to a broad range of microbial products and an intra-cellular Toll-IL-1 (TIR) domain that activates downstream signal adap-tors and transducers, typically resulting in the activation of nuclear factor kappa β (NF-κB), a transcription factor that promotes the expression of genes associated with immune defense.274 TLR-mediated

INNATE IMMUNE RECOGNITION: SETTING THE STAGE FOR T-CELL RESPONSES�e adaptive immune system consists of B and T lymphocytes that express distinct, somatically recombined receptors with exquisite spec-i�city, at the cost of low frequency. In contrast, the innate system consists of leukocytes (i.e., neutrophils, monocytes, macrophages, and DCs) that typically express families of germline-encoded receptors that bind to molecules of microbial origin. An extensive family of innate immune receptors recognizes microbial molecules that range from bacterial lipopolysaccharide and !agellin, mycobacterial lipoarabino-mannan, malaria parasite–derived hemozoin, and fungal glucans and mannans to viral DNA and RNA motifs. It is increasingly clear from studies on the genetic basis of human susceptibility to infectious dis-eases and from experimental studies in animal models that innate immune responses to microbial molecules, by promoting the expres-sion of co-stimulatory molecules and the secretion of speci�c cyto-kines, form the foundation for the adaptive immune response.

As detailed in this chapter, the molecular basis for antigen-speci�c immune responses derives from combinatorial receptors (αβ TCRs) that have almost in�nitely diverse speci�city. �is system ensures that a great diversity of antigens from pathogenic organisms can be recog-nized, but, because pathogen-speci�c cells are infrequent, it requires time for expansion of these cells to numbers that are su"cient to combat the infection. In the hours a�er a pathogen breach of an ana-tomic barrier, the most rapid pathogen recognition events are medi-ated by cells bearing innate immune receptors. �ese receptors are not combinatorial but recognize conserved structural elements in broad

FIGURE 6-7 Intracellular trafficking of CD1 isoforms. Each panel depicts a distinct pattern of CD1 trafficking. All CD1 isoforms associate with β2-microglobulin (β2m), and all isoforms except CD1e are targeted to the cell surface via the secretory pathway. CD1 is internalized and targeted to distinct locations in the lysosomal-endosomal network based on the interaction of the CD1 cytoplasmic tails with adaptor proteins (AP2, AP3). The varying compartments in the endosomal/lysosomal network are early endosomes/recycling endosomes (EE/RE) associated with CD1a, late endosomes (LE) associ-ated with CD1c, and MHC class II compartment/lysosomes (MIIC/Ly) associated with Cd1b/d. The different subcellular distributions of Cd1 isoforms may reflect surveillance of these compartments for distinct lipid antigens. ER, endoplasmic reticulum; PM, plasma membrane. (Courtesy Steven Porcelli of Albert Einstein Medical College, Bronx, NY.)

PM

Golgi

ER

EE/RE

LE

MIIC/Lysβ2m

Clathrin-coated pit

PM

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AP-2

AP-2

AP-3

AP-3

CD1c CD1e

CD1a CD1b/d

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moieties that originate in a wide range of pathogens, including M. tuberculosis, Mycobacterium kansasii, Candida albicans, P. jirovecii, HIV-1, and Francisella tularensis, and mediates phagocytic responses in vitro. However, the role of the mannose receptor in mediating host defense through innate immune recognition remains unclear because a gene-de�cient mouse strain does not appear susceptible to C. albicans.297

A subset of CLRs signal via tyrosine-based motifs that are embed-ded in their intracellular domain or that are present in adaptor mole-cules that associate with CLRs. Other important components of this signaling pathway are spleen tyrosine kinase and CARD9. As discussed previously, this pathway is central to the recognition of fungal β-glucans (via the CLR Dectin-1), fungal mannans (via the CLR Dectin-2), and Malassezia and M. tuberculosis ligands (via the CLR Mincle). Dectin-1 activation promotes macrophages and DC cytokine and chemokine release and favors the development of �17 responses.298 Humans with autosomal recessive mutations in Dectin-1 and CARD9 have defects in mucosal antifungal immunity.61,65

Links between Innate Immune Recognition and Adaptive Immune ResponsesAntibody and T-cell responses to inoculation of protein antigens are minimal unless an innate in!ammatory response is elicited concur-rently. �us, adjuvants that induce innate in!ammatory responses are co-administered with some vaccines. In early work on the functional link between DC and T-cell activation, immunization with model anti-gens and Freund’s adjuvant led to impaired T-cell responses and abnor-mal isotype-speci�c antibody production in MyD88-de�cient mice,299 suggesting that TLR recognition is essential for proper �1 adjuvant e�ects. DCs that directly engage in TLR signaling a�er stimulation with microbial products can promote the proliferation and di�erentia-tion of naïve T cells. In contrast, DCs that undergo activation by exposure to cytokines elicited by TLR signaling only support T-cell proliferation but not di�erentiation.300,301 Newer studies have expanded this view and demonstrated that T-cell priming and antibody genera-tion can occur in the absence of TLR signaling, provided that non- TLR microbial recognition pathways are activated in DCs. Collectively, these studies argue that other innate immune signaling pathways provide adjuvant e�ects.98,302,303 One commonly used adjuvant for human vaccines, alum, enhances antibody production by stimulating the NLRP3 component of in!ammasomes to produce IL-1 and IL-18.304 �us, NLR and CLR signaling are likely to provide important innate signals that enhance adaptive immune responses.

Antigen-Presenting CellsActivation of naïve T cells requires antigen processing, antigen presen-tation, and co-stimulation. Most mammalian cells are incapable of ful�lling these functions, and thus dendritic cells (DCs) predominately mediate the priming of naïve T lymphocytes.305 In vivo elimination of DCs, as has been performed experimentally in a mouse model, com-pletely abrogates priming of naïve T cells that respond to microbial infections.241 A major function of DCs is to present antigens to T lym-phocytes and promote their proliferation and di�erentiation. High surface levels of MHC class I and class II and the expression of an array of co-stimulatory molecules enable DCs to stimulate naïve T cells e�ectively. DCs are a highly complex and heterogeneous population of cells that reside in both lymphoid and nonlymphoid tissues and share phenotypic and functional characteristics that distinguish these cells from other classes of lymphocytes.

DCs derive from progressively committed pluripotent precursors in the bone marrow, a pathway that has been the focus of intense study.306 In mice, a common DC precursor gives rise to circulating plasmacytoid DCs and to pre-DCs. Plasmacytoid DCs307,308 express high levels of TLR7 and TLR9 and, on stimulation, secrete very high levels of type I interferons, cytokines that are critical to antiviral defense. Pre-DCs gives rise to CD8α+ and CD11b+ DC populations in lymphoid organs and to CD11b+ and CD103+ DCs in peripheral tissues. A combination of transcriptomic and functional studies revealed that murine lymphoid-tissue resident CD8α+ DCs and non-lymphoid tissue-resident CD103+ DCs are the likely counterparts of

signals induce the secretion of proin!ammatory cytokines, such as TNF and IL-12, and induce the maturation of DCs, enabling them to activate naïve, pathogen-speci�c T lymphocytes. In addition, stimula-tion of TLRs can directly stimulate antimicrobial e�ector mechanisms of the host cell, limiting pathogen replication until adaptive immune cells are recruited to the site of infection.280

Each TLR recognizes speci�c microbial products, and well-de�ned ligands include bacterial lipoproteins (TLR1, TLR2, TLR6), double-stranded RNA (TLR3), lipopolysaccharide (TLR4), the bacterial peptide !agellin (TLR5), single-stranded RNA (TLR7 and TLR8), and CpG motifs within DNA (TLR9). TLR heterodimerization can mediate novel antigen-binding functions and increase the microbial speci�city of individual TLR family members. For example, TLR2 het-erodimerizes with TLR1 or TLR6 to recognize triacylated or diacylated lipoproteins, respectively.281 Structural studies have demonstrated how microbial ligands associate with di�erent TLR proteins, revealing a common pattern of TLR dimerization induced by association with microbial ligands.282,283

Most TLRs, with the exception of TLR3 and TLR4, signal through the adaptor protein MyD88. Children who have autosomal recessive defects in MyD88-mediated signal transduction, a very rare primary immunode�ciency, lack input from most TLRs and from interleukin-1 receptors and develop severe and life-threatening pyogenic bacterial infections, particularly invasive streptococcal, staphylococcal, and pseudomonal infections.284 More commonly, polymorphisms in TLR coding sequences and regulatory elements can confer susceptibility to infectious diseases,285 as illustrated by the association of a common TLR5 stop codon polymorphism with susceptibility to the !agellated bacterium Legionella pneumophila.286 Patients with autosomal recessive defects in TLR3,287 which associates with the adaptor molecule Trif,288 and downstream signaling components289 develop herpes simplex encephalitis, illustrating pathogen speci�city of and host tissue require-ments290 for individual TLRs in host defense.

NOD-like Receptors and the InflammasomeWhereas TLRs predominantly respond to extracellular or endosomal microbial ligands, nucleotide-binding, oligomerization domain (NOD)-like receptor (NLR) proteins detect microbial ligands in the cytosol.275 Among the best characterized of these proteins, NOD1 and NOD2 respond to fragments of bacterial peptidoglycan, whereas NLRC4 responds to bacterial !agellin in the cytosol.291,292 NLRs can activate NF-κB signaling and assemble into multicomponent struc-tures called in!ammasomes that consist of an NLR, an adaptor protein, and caspase subunits.293 In!ammasomes activate host cell apoptotic pathway and the expression of IL-1 and IL-18 by activating caspase-1. Macrophage caspase-1 activity is also important for phagosomal matu-ration.294 �us, detection of microbial molecules in the cytosol induces an in!ammatory response that has consequences for the adaptive �1 and �2 cell responses.295

�e RIG-I-like receptors (RLRs) RIG-I, MDA5, and LGP2 are members of a family of molecules that detect cytosolic nucleic acids from RNA viruses; these include measles virus, in!uenza virus, hepa-titis C virus, and West Nile virus.278 RLRs signal via the adaptor protein IPS-1 and collaborate with other pattern recognition receptors to induce antiviral responses. In!uenza and many other viruses can subvert cytosolic detection systems by directly inhibiting RLR signal transduction and targeting components of this pathway for degrada-tion. Detection of cytosolic DNA, speci�cally to cyclic dinucleotides, occurs via receptors that signal through the adaptor molecule STING.296 RLR- and STING-dependent signaling events turn on NF-κB and type I interferon signaling, although the role of cytosolic sensors of viral nucleic acids in shaping T-cell responses remains less well de�ned.

C-Type Lectins and Other Receptors Implicated in Innate Immune RecognitionC-type lectin receptors (CLRs) bind to carbohydrate moieties of endogenous and exogenous origin, trigger phagocytic responses, and activate innate immune cells, particularly of myeloid origin. �e binding speci�city for some of these receptors has been elucidated and includes fungal, bacterial, mycobacterial, viral, and parasitic ligands. For example, the mannose receptor binds to high mannose and fucose

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stages of disease, these patients progressively accumulate activated NK cells at sites of infection, leading to chronic granulomatous lesions in the skin and respiratory tract.334 �ese �ndings support the notion that NK cells provide functional redundancy in the context of diminished CD8+ T-cell–dependent cytolytic activity, yet cause tissue damage. Although the clinical manifestations of de�cient CD8+ T-cell function and understanding from animal models are not reconciled easily, studies of MHC class I polymorphisms and susceptibility to HIV infection support a role for CD8+ T cells in control of viral infections.335,336

Some viruses, such as human herpesviruses, become transcription-ally inactive a�er host cell infection and represent a particular chal-lenge for the cellular immune system. Although primary active infection with these viruses is controlled, herpesviruses establish clini-cal latency and can cause intermittent disease or disease in the setting of impaired immunity. Transcriptionally inactive proviral DNA is invisible to host T cells because viral proteins are not presented by MHC class I molecules and therefore these viruses cannot be detected by antigen-speci�c T lymphocytes. Although immunologic elimina-tion of the infection is o�en impossible, immunologic control of inter-mittent reactivation is the rule.156

Beyond interfering directly with the MHC class I antigen- processing pathway, many viral pathogens encode proteins that can interfere with cytokine signaling or chemokine-mediated cell recruit-ment. Epstein-Barr virus contains a gene for an IL-10 homologue that can bind the IL-10 receptor and induce immunosuppressive e�ects of this pleiotropic cytokine. Poxviruses express an IL-18–binding protein that can bind IL-18 with high a"nity, interfering with early IFN-γ–mediated in!ammatory responses. Poxviruses also express proteins that bind –CC– chemokines with high a"nity, preventing their asso-ciation with chemokine receptors on the surface of in!ammatory cells and disrupting the recruitment of monocytes, NK cells, and activated T cells to sites of viral infection.

Intracellular BacteriaMany important human pathogens have evolved mechanisms to escape antibody-mediated, complement-mediated, and neutrophil-mediated immune defense. One of the most e�ective escape mechanisms involves entering host cells, o�en phagocytic cells. In this circumstance, the host cell becomes a protective barrier from extracellular microbicidal defenses. Bacterial pathogens have used many clever strategies to exploit the interior of host cells to their bene�t, by manipulating intra-cellular tra"cking pathways or targeting speci�c intracellular niches. �e challenge facing the cell-mediated immune system is to detect and eliminate these pathogens.

Phagosomal PathogensMultiple bacterial pathogens that parasitize phagocytic cells, such as macrophages, reside within the endosomal-phagosomal network. �ese pathogens o�en are accessible to the MHC class II antigen pre-sentation network and can co-localize with the antimicrobial e�ector molecules of macrophages that are delivered to the phagolysosome. Prototypical pathogens of this type include Mycobacterium and Salmo-nella species. Control of these pathogens depends mostly on CD4+ T-cell activation of antimicrobial killing through IFN-γ. IFN-γ secreted by T cells activates macrophage e�ector mechanisms to kill pathogens through nitric oxide, reactive oxygen species, and lysosomal enzymes. Some controversy exists about whether the essential macrophage e�ec-tor molecules that control intracellular bacteria di�er between mice and humans. IFN-γ–induced nitric oxide is essential for control of intracellular pathogens in the mouse, whereas its role in human defense is less clear. Mice rendered de�cient in IFN-γ are highly susceptible to intracellular bacterial pathogens, including M. tuberculosis and Salmo-nella, providing support for the importance of the �1-mediated stim-ulation of antimicrobial killing of phagosomal pathogens.

In many parts of the world, infants are exposed parenterally to live mycobacteria in the form of bacillus Calmette-Guérin (BCG) vaccina-tion. Although highly attenuated compared with its parent M. bovis strain, BCG can replicate within human hosts with impaired host immunity. �is massive cohort of mycobacteria-exposed infants has revealed several inherited defects in antimycobacterial immunity. �e

human CD141+ DCs, all of which are highly e�ective at antigen cross-presentation to T cells a�er viral and bacterial infections.309-313 A recent study suggested that murine CD11b+ DCs in nonlymphoid tissues may be the homologue of human CD1c+ DCs and demonstrated inter-species conservation of mucosal �17 responses mediated by these DC subsets.314

�e role of DCs in antimicrobial defense extends beyond stimula-tion of T cells. In mice and humans, circulating blood monocytes can give rise to in!ammatory DCs at portals of infection or sites of in!am-mation in mice and humans.315-320 Monocyte-derived in!ammatory DCs express a range of TLRs, CLRs, and NLRs that induce their activa-tion and result in their production of cytokines that can orchestrate �-cell di�erentiation.298,316,321,322

DCs are also primarily infected in some settings. For example, CD11b+ DCs and monocyte-derived DCs are major infected cell popu-lations in murine tuberculosis and leishmaniasis.316,323 In pulmonary aspergillosis and tuberculosis and in a vaccine model of blastomycosis, monocyte-derived DCs and CD11b+ DCs play an important role in transporting microbial antigen to draining lymph nodes,100,317,324 in concert with previous studies on the transport of particulate antigens injected into skin, which are taken up by monocytes that are recruited from the bloodstream to the site of injection and migrate to skin-draining lymph nodes via lymphatic channels.325 Tra"cking of acti-vated DCs into lymph nodes involves the upregulation of CCR7 on DCs, enabling them to respond to CCL19 and CCL21, which are expressed in the lymph node paracortex.326 DC tra"cking to draining lymph nodes has also been demonstrated a�er respiratory327 and cuta-neous viral infection,328 and from the marginal zone to the T-cell zone of spleen a�er systemic bacterial infection.329 With respect to systemic bacterial infections, DCs may play an important role in localizing bacteria to the spleen.330,331

Microbial Pathogenesis and the Cellular Immune System�e diverse properties of microbial pathogens provide a challenge to the cellular immune system. Although the details of cellular immune responses to di�erent pathogens vary substantially, some broad gener-alizations regarding cellular immune responses to microbial pathogens can be justi�ed. In most cases, the subcellular anatomic location of a pathogen (extracellular, intracellular/vacuolar, intracellular cytoplas-mic) predicts the arm of the cellular immune response that is necessary to contain infection by that particular pathogen. In many cases, the most de�nitive information about the important arms of antimicrobial defense for a particular pathogen has come from natural or acquired de�ciency states, in particular, defects in immune receptors or signal-ing molecules. In addition, because di�erent classes of pathogens are associated with a particular type of cellular immune response, many pathogens have developed sophisticated molecular countermeasures to avoid elimination.

Viral InfectionsIn general, cellular immune responses against viral infections involve MHC class I presentation of virally derived peptide antigens to CD8+ T cells. Because viral pathogens replicate within their host cell, their elimination by the cellular immune system involves either cytolytic destruction of the infected host cell by virus-speci�c lymphocytes or inhibition of viral replication in host cells.332 Because viruses, by neces-sity, use the host cell for protein synthesis, viral proteins are ready substrates for the MHC class I antigen-processing pathway.

�ere are several reports of congenital de�ciency of CD8+ T cells or MHC class I function. De�ciency in TAP, the antigen transporter necessary for MHC class I peptide loading, was documented in several families.206,207 �ese patients have dramatically reduced levels of MHC class I surface expression and CD8+ T cells. In contrast to the canoni-cal function presented for CD8+ T cells in antiviral defense, TAP1-de�cient patients and a patient de�cient in CD8+ α-chain did not manifest increased susceptibility to viral infection and instead pre-sented with recurrent pulmonary bacterial infections leading to bron-chiectasis. A patient with congenital CD8α de�ciency had adequate titers to cytomegalovirus, rubella, and other viruses, con�rming prior exposure and successful protective immunity.333 However, in later

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CD1 Antigen Presentation and Host Defense against Infectious DiseasesMycobacteriaMycobacterial lipids, speci�cally mycolic acids, were the �rst CD1-presented antigens to be identi�ed.267 Several other mycobacterial lipid antigens are presented by CD1, including lipoarabinomannan268 and glycosylated isoprenoids.343 Because of the noted di�erences between mouse and human CD1 loci, the mouse model has not been informa-tive in de�ning an important role for CD1 in immune defense against mycobacterial infection.344-346 Recent puri�ed protein derivative con-verters have CD1-restricted T cells that react to mycobacterial isopren-oid glycolipids, showing that CD1-restricted antigen presentation does occur in the context of human infection. With the use of CD1b tetramer-based isolation methods (see later section “Primer on Basic Immunologic Techniques: Foundation of Immunologic Models”), a recent study isolated mycolyl lipid-reactive T cells; these CD1b-restricted T cells are present in the native repertoire and appear to expand during tuberculosis.166 In addition, CD1a through CD1c are expressed strongly on DCs in tuberculoid leprosy lesions and reversal reactions, but not in lepromatous lesions, suggesting that CD1 expres-sion correlates with e�ective antimycobacterial immunity.347 Although this system likely does not exist solely for the purpose of defense against mycobacterial infections, it is widely accepted that CD1 mol-ecules likely play an important role in antimycobacterial immunity. Several other studies using mouse models or α-galactosyl ceramide–activated NK cells have implicated CD1 and NK cells in antimicrobial defense against tuberculosis,348 opportunistic fungi,349,350 malaria, try-panosomiasis,351 and others.160

MAJOR HISTOCOMPATIBILITY COMPLEX IMMUNOGENETICS�e MHC, so named because its discovery resulted from studies of tissue transplant rejection, contains many of the genes associated with cell-mediated immune defenses. A map of the human MHC is shown in Figure 6-8. �e MHC complex encodes the α-chains of the MHC class I molecules HLA-A, HLA-B, and HLA-C and the α- and β-chains of the MHC class II molecules HLA-DR, HLA-DP, and HLA-DQ, all of which are expressed in a co-dominant fashion. Genes associated with the antigen-processing pathways are found in the MHC as well. Several proteasome subunits (i.e., LMP-2, LMP-7, and MECL-1); the peptide transporter TAP1 and TAP2 proteins; and the MHC class II processing–associated HLA-DM and HLA-DO proteins are encoded in the MHC.352 TNF and its receptor also are encoded in the MHC.

An important feature of the MHC, which led to its discovery, is the polymorphism of some of its genes, particularly the MHC class I and class II genes.353 For the MHC class I HLA-B gene, there are more than 2000 known alleles. Although some of these alleles bind similar pep-tides, most, by virtue of their morphologically distinct peptide-binding grooves, bind distinct families of peptides. An evolutionary force driving the diversity of MHC alleles comes from the microbial world and its ability to undergo antigenic variation.354-356 It is clear from studies of African populations where malaria is endemic that certain HLA alleles are associated with a greater ability to survive malaria infection.357,358 Conversely, malaria has a confounding ability to alter protein sequences that are bound by HLA molecules, allowing parasite populations to evolve that are invisible to individuals with certain HLA

clinical syndrome has been termed mendelian susceptibility to myco-bacterial disease337 and includes patients with disseminated BCG infec-tion and progressive infection with low-pathogenicity mycobacteria. �ese patients also show increased susceptibility to Salmonella infec-tions. All of the mutations that cause this mendelian susceptibility to mycobacterial disease are in the IFN-γ and IL-12 pathways, including IFN-γ receptor mutations, STAT1 mutations, IL-12 cytokine/receptor, and NF-κB essential modulator (NEMO).15-17,338,339

�e data presented here document the central role of the �1 immunity in protection from and control of phagosomal pathogens. As such, it is logical to assume that pathogens of this type have evolved countermeasures to dampen or subvert e�ective host immunity. M. tuberculosis infection of macrophages renders these cells resistant to activation with IFN-γ,340 limiting the bacteriostatic actions of the host cell that depend on this cytokine. M. tuberculosis also prevents acidi-�cation of vacuoles by excluding the proton-adenosine triphosphatase (ATPase) from the endosomes it occupies. One possible outcome of diminished vacuolar acidi�cation is decreased antigen degradation, resulting in diminished presentation of mycobacterial peptides by MHC class II molecules. Other pathogens, such as L. pneumophila, segregate themselves in an endosomal compartment that does not communicate with the MHC class II antigen-processing pathway.341

Cytoplasmic PathogensSome bacterial pathogens have evolved a di�erent intracellular survival strategy. �ese pathogens, which include L. monocytogenes, Shigella "exneri, and various Rickettsia species, escape the phagocytic vacuole and replicate in the cytoplasm of host cells. �ese pathogens secrete proteins that are essential for virulence and that destroy the vacuolar membrane, providing direct access to the host cell cytosol. In terms of the cellular immune response, these pathogens are similar to viruses because defense against these agents predominantly depends on the MHC class I/CD8+ T-cell axis. Because of their cytoplasmic location, the antimicrobial e�ector mechanisms of phagocytic cells cannot be localized spatially to the cytoplasmic site of infection, necessitating killing of the infected cell by cytolytic T cells to eliminate the infection. Extensive evidence from animal models of L. monocytogenes supports the role of CD8+ T cells in protective immunity against cytoplasmic bacterial pathogens.

Extracellular BacteriaBacteria that replicate extracellularly are accessible to antibody-mediated neutralization or killing by externalized microbial products of phagocytic cells. Defense against pyogenic bacteria, such as Staphy-lococcus aureus and Streptococcus pneumoniae, depends on adequate humoral immunity and intact neutrophil function. To the extent that adequate speci�c and high-a"nity antibody production depends on CD4+ �-cell function, patients with impaired CD4+ T-cell function are susceptible to these pathogens. �17 cells have been implicated in defense against Klebsiella infections in the lung.52,342 A role for CD8+ T-cell responses in defense against extracellular bacteria has been postulated in anaerobic abscesses. In this setting, CD8+ T cells recognize carbohydrate antigens of Bacteroides fragilis. Patients with hyperimmunoglobulin E syndrome have defects in �17 cells and are susceptible to staphylococcal infection due to mutations in TYK2 or STAT3.53

FIGURE 6-8 Genomic organization of major histocompatibility complex (MHC) locus. The MHC locus contains genes for many proteins involved in adaptive immune defense against infectious diseases. In addition to MHC class I and class II genes, the MHC locus also contains genes coding for TNF, for lymphotoxin, and for essential components of the antigen-processing pathway. HLA, human leukocyte antigen; TAP, transporter associated with antigen processing.

HLA-DP

HLA-DM HLA-DQ

TAP1 and TAP2LMP-2 and

LMP-7 HLA-DRTNF

lymphotoxin

HLA-B

HLA-C

HLA-E

HLA-A

HLA-G

HLA-F

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L-selectin) and the chemokine receptor CCR7,380 a combination of adhesion molecule and chemokine receptor that targets cells for the T-cell zone of lymph nodes and spleen. �e diversity of T cells in the peripheral compartment is enormous; it is estimated that naïve T cells of an immunologically normal mouse express approximately 25 million distinct TCRs.381

�ymic selection is crucial for the development of peripheral T lymphocytes. Patients with DiGeorge syndrome have a congenital absence of thymic tissue and a corresponding absence of peripheral T lymphocytes.382 Transplantation of thymic tissue into these patients corrects this problem.383

�ymic function dramatically diminishes in humans on aging. Young children have active thymic function and produce large numbers of naïve T lymphocytes, whereas older adults have a markedly smaller thymus that produces few new T lymphocytes. However, adults in their 50s continue to have active thymic tissue in the mediastinum and recent thymic emigrants in the bloodstream suggest the thymus con-tinues to function despite its relatively small size.384 Many endogenous and exogenous factors can injure thymic function. Administration of corticosteroids results in thymic involution and decreased output of naïve T lymphocytes. Many forms of chemotherapy also are toxic to the thymus and result in thymic atrophy and diminished output of T lymphocytes. �is issue is of particular importance in allogeneic hema-topoietic cell transplantation, where long-term survival depends on reconstitution of the peripheral T-cell compartment. Studies that measure TCR excision circles, an indication of recent emigration from the thymus, and the kinetics of TCR repertoire reconstitution have shown that thymic function returns and TCR repertoire diversity increases in the post-transplant setting.385,386 �ymic function also is relevant in HIV infection,387 particularly in patients with advanced HIV disease on highly active antiretroviral therapy. In this setting, viral replication o�en is halted, but increases in the peripheral CD4+ T-cell count depend on de novo thymic production of naïve T cells or expan-sion of the residual peripheral T-cell compartment or both.388,389

Lymphoid AnatomyMost studies of the human immune system focus on circulating blood cells and, less frequently, on lymphocytes derived from lymph nodes, spleen, or other tissues. Studies in mice have focused on lymphocytes derived from spleen and lymph nodes and, recently, on mucosal tissues such as the intestines. In aggregate, studies with humans and mice provide a fairly detailed picture of immune system compartmentaliza-tion. Understanding of immune cell populations in various lymphoid and nonlymphoid tissues has increased dramatically,390 as has under-standing of the signals that direct the tra"cking of lymphocytes in vivo.391,392 Although exceptions to this rule may exist, T-cell–mediated immune responses generally are initiated in secondary lymphoid tissues, speci�cally in lymph nodes and in the spleen.393 Subsequently, activated lymphocytes tra"c to nonlymphoid sites of infection. Even a�er infection with microbial pathogens that do not infect lymphoid tissues directly, naïve antigen-speci�c T cells are activated in secondary lymphoid tissues and then tra"c to the actual site of infection. Because lymphoid tissues play this vital role in adaptive immunity, understand-ing the anatomy of the lymph node and spleen is essential to under-standing the co-mingling of antigen with its cognate immune cell.

Lymph NodesLymph nodes serve as a nexus for lymphocyte interaction with antigen-presenting cells, overcoming the di"cult problem of bringing together rare, antigen-speci�c lymphocytes with small quantities of antigen.394 Located at the interface between the blood and lymphatic systems, lymph nodes receive lymphatic drainage from peripheral tissues as well as blood !ow through arteries that enter the hilum of the node. �e mechanics of !uid, cellular, and particulate movement through lymph nodes have been elucidated only recently.395,396 �e !ow of cells, pro-teins, in!ammatory mediators, and, in some instances, microbial pathogens, to the lymph node from distant sites of infection via a�erent lymphatic channels facilitates the activation of protective cell-mediated immunity (Fig. 6-9).

At peripheral sites of infection, microbial antigens are taken up by migratory DCs that, a�er receiving signals through innate immune

molecules.359 �is type of escape strategy also has been described for viral pathogens,360,361 including HIV.362 �e sequencing of the human genome, the advent of microarray-based, high-throughput genotyping technology, and the completion of the international HapMap project have facilitated numerous genome-wide association studies for human susceptibility to infectious agents.18 Genome-wide association studies have identi�ed signi�cant associations of polymorphisms in MHC class I genes with HIV control, HIV viral load at setpoint, and long-term nonprogression,363 and in MHC class II genes with susceptibility to leprosy364 and hepatitis B.365

�e MHC complex also encodes proteins for MHC class I mole-cules that are not highly polymorphic. �ese molecules also are called MHC class Ib molecules and in humans consist of HLA-E, HLA-F, and HLA-G. HLA-G is highly expressed in the placenta and may protect cells from NK cell–mediated lysis.366 HLA-E binds the signal sequences of conventional MHC class I molecules, providing a readout of cellular synthesis of MHC class I molecules. HLA-E also binds to NK cell receptors CD94/NKG2B and CD94/NKG2C, thus inhibiting NK cell–mediated cytolysis.367 �e role of these proteins in antimicrobial immu-nity is unclear, although there is evidence that HLA-E can present antigens derived from M. tuberculosis to T lymphocytes.368 Because MHC class Ib molecules generally are highly conserved among indi-viduals (i.e., unlike HLA-A, HLA-B, and HLA-C, they are not highly polymorphic), there has been interest in exploiting them for pathogen-speci�c vaccine development.368

Thymic Selection of CD4+ and CD8+ T CellsMature CD4+ and CD8+ T cells are generated in the thymus by a complex process of positive and negative selection.369,370 As mentioned earlier, T cells detect peptide antigen in the context of MHC molecules and express receptors that bind the foreign antigen and the MHC molecule. �ymic selection has evolved to ensure that peripheral T cells are able to interact with self-MHC molecules, through the process of positive selection, but not to respond to self-peptides, through the process of negative selection. T-lymphocyte progenitors originate in the bone marrow and travel to the thymus, where they enter the cortex at the double-negative stage, de�ned by the absence of the CD4 and CD8 co-receptors on the cell surface. In the thymic cortex, the TCR β-chain gene undergoes VDJ (i.e., variable, diverse, and joining) recombination, which, if successful, gives rise to the TCR β-chain protein. �is gene recombination event is catalyzed by the RAG1 and RAG2 genes.371 �e TCR β-chain �rst complexes with the invariant pre-TCR α-chain and travels to the cell surface.372,373 If this process is successful, the endogenous α-chain locus undergoes VJ recombina-tion and replaces the pre-TCR-α in subsequent TCR complexes. At this stage, the cell upregulates the expression of CD4 and CD8 on its cell surface and is referred to as a double-positive thymocyte. Positive selection occurs �rst through a process in which the double-positive thymocyte interacts with a thymic epithelial cell. �e consensus model is that low-avidity interactions between the TCR on the double-positive thymocyte and MHC/self-peptide complexes on the thymic epithelial cell drive cell proliferation and survival, whereas high-a"nity interactions or no interaction results in cell death.374,375 On completion of positive selection, the double-positive thymocyte down-regulates either CD4+ or CD8+, depending on whether positive selec-tion occurred on MHC class II or class I. At this stage, thymocytes enter the thymic medulla, where they come into contact with bone marrow–derived DCs and medullary epithelial cells expressing a range of self-antigens. High-a"nity interaction with self-antigens results in deletion of the single positive thymocyte, providing a second opportu-nity to eliminate potentially autoreactive T lymphocytes. Studies have identi�ed a transcription factor, AIRE, which promotes the ectopic expression of peripheral antigens in the thymus.376 Loss of function mutations in the human AIRE gene result in systemic autoimmune disease.

On completion of positive and negative selection, thymocytes exit the thymus in a process that is regulated by sphingosine-1- phosphate.377,378 �e recent thymic emigrants undergo post-thymic maturation and become part of the peripheral T-cell compartment.379 Naïve T cells generally express high levels of CD62L (also called

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FIGURE 6-9 Anatomy of lymph nodes. A, Humans typically contain 500 to 600 bean-shaped lymph nodes. Lymph nodes filter lymphatic fluid that is draining from peripheral tissues to the bloodstream via lymphatic vessels. Lymph nodes are encapsulated by dense collagen-rich fibers that extend trabeculae into the lymph node substance. Afferent lymph fluid enters lymph nodes through channels that drain into subcapsular sinuses. Lymph fluid percolates through cortical and medullary sinuses before exiting the lymph node via efferent lymph channels. The outer region of the lymph node, termed the cortex, consists primarily of B-cell–rich lymphoid follicles and T-cell–rich paracortical aggregates. The inner region of the lymph node, termed the medulla, is far less cellular than the cortex, and this region contains coalescing lymph-filled sinuses. The blood supply to the lymph node enters and exits via the hilum. High endothelial venules represent the site of circulating leukocyte entry into the lymph node. Leukocytes exit the lymph node via the efferent lymphatic vessels and rejoin the circulation. B, Lymph node microanatomy and network of conduits. This panel provides an outline of lymph node anatomy at a level that cannot be visualized by light microscopy. The anatomic relationships were deduced by introducing low molecular weight markers into lymphatic fluid and into blood that circulate into intact lymph nodes and analyzing trafficking patterns. Antigen-bearing migratory dendritic cells (DCs) can traverse the cellular lining of subcapsular and cortical sinuses to enter the paracortical cords to initiate antigen presentation to naïve T cells. Soluble inflammatory mediators and antigens are funneled into fibrous conduits that link lymphatic channels with high endothelial venules (HEV) and lymphoid follicles. Resident dendritic cells (rDC) can sample the antigenic content of fibrous conduits and present antigens to T cells that enter the paracortex through the high endothelial venules. Low-molecular-weight proteins, such as chemokines, can flow from the lymph node sinus through the conduit system to the high endothelial venules, where they can influence the recruitment of circulating lymphocytes. CF, collagen fibers that provide structural support to the conduits and to the lymph node. FRC, fibroblastic reticular cells that surround the conduits; JC, junctional complexes that provide a tight barrier to the contents of the conduit; SLCs, sinus-lining cells.

Subcapsular Sinus

Afferent LymphChannel

Vein

High EndothelialVenule

Efferent LymphChannel

B-CellFollicle

Para-cortex

Medulla

Arteriole

Subcapsular Sinus

Migratory DCSLCs

Fibrous Conduit

FRC

CF

JC

HEV

rDC

Trabecula

CorticalSinus

MedullarySinus

Low MW Lymph Constituents

ParacorticalCords (T Cells)

Direction ofLymph Flow

B-Cell

Follicle

A

B

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level, they also di�er in some important respects (Fig. 6-10). �e spleen is not a site for lymphatic drainage. Instead, antigens, pathogens, and circulating cells enter the spleen through the splenic artery. �e splenic artery branches into central arterioles, which course through the splenic cortex and are surrounded by lymphocytes in a structure called the periarteriolar sheath.408 T lymphocytes are most proximal to the central arteriole and are surrounded by aggregates of B lymphocytes in regions termed B-cell follicles (see Fig. 6-10B). �e densely packed B and T lymphocytes constitute the splenic white pulp and are sur-rounded by the marginal zone (see Fig. 6-10C), which separates white pulp from red pulp.409 �e splenic red pulp is rich in macrophages and contains many red blood cells, resulting from the percolating blood !ow delivered by the termini of the splenic arterioles. �e blood !ow to the spleen predominantly terminates in the red pulp and the mar-ginal zone. Most pathogens, on clearance from the bloodstream, �rst are localized to the marginal zone and red pulp of the spleen.410 With respect to cellular tra"cking into the spleen, it seems that the major entry point into the white pulp is from the marginal zone.411 From here, activated antigen-presenting cells can enter the white pulp areas, most likely by traversing the marginal zone sinus and the metallophilic macrophage layer that forms a boundary between white pulp and marginal zone. Access of protein antigens and other molecules into the splenic white pulp is highly restricted and, in many ways, similar to the system identi�ed in lymph nodes. Speci�cally, �broblastic reticular cells form small channels that surround collagen �bers that enter the T-cell and the B-cell zones of the spleen and deliver small molecules (i.e., generally 60 kDa) into the white pulp. �e channels found in B-cell follicles bind chemokines associated with B-cell recruitment, whereas the channels identi�ed in T-cell zones bind chemokines asso-ciated with T-cell recruitment.409

Chemokine-Mediated and Integrin-Mediated Trafficking to Lymphoid Tissues�e mechanism of entry of T and B lymphocytes into lymph nodes has been investigated in detail, and the sequence of events is well characterized. First, lymphocytes passing through high endothelial venules roll along the surface of the endothelium in a process that involves selectins on lymphocytes and counterreceptors on the endo-thelial surface.412-414 �e next step involves recognition of chemokines on the endothelial surface by chemokine receptors on the lymphocyte surface, which triggers a G1-protein–mediated activation process that induces integrin-mediated adhesion and movement across the endo-thelium.391,415 In recent years, many of the components of this process have been delineated. �e chemokine receptors CXCR4 and CCR7 are activated by the chemokine CXCL12, promoting the entry of B lym-phocytes into peripheral lymph nodes. B-cell entry into Peyer’s patches is mediated by CXCL13 and CXCR5.416 LFA-1/ICAM-1 and α4β1/VCAM (vascular cell adhesion molecule) interactions are required for B-cell retention in the marginal zone of the spleen.417 Antibody-blocking studies showed that B-cell entry into follicles requires interac-tion between LFA-1 on lymphocytes and ICAM-1 on spleen cells. �e interaction between α4β1 integrin molecules and VCAM also contrib-utes to B-cell entry into splenic follicles.418 When lymphocytes have entered the splenic white pulp, their movement between T-cell and B-cell zones is dictated in large part by chemokines and their respective receptors. B lymphocytes, on activation by antigen, upregulate the expression of CCR7, a chemokine receptor that responds to CCL19 and CCL21 chemokines.419 Naïve T lymphocytes express CCR7, allow-ing them to respond to CCL19 and CCL21 and directing them to T-cell zones of lymph nodes. When activated by antigen, the upregulation of CCR7 on B cells directs them to the T-cell zone, enabling their direct interaction with T cells and enabling B-cell antigen presentation to T cells and T-cell–mediated maturation of B cells.419

Immune Tissues Associated with Mucosal SurfacesOne of the challenges faced by the immune system is to distinguish pathogenic from innocuous microbes.420 �is issue is of particular relevance in the intestine, a location that is teeming with microbes that, under most circumstances, pose little threat to healthy individu-als. �e role of endogenous commensal microbial !ora in tuning

receptors, mature and travel in the a�erent lymph !uid to a draining lymph node.325 In!ammatory cytokines and chemokines produced at the site of infection travel in lymph !uid to the draining lymph node concurrently.397 �is cellular and molecular cocktail enters the subcap-sular space of the draining lymph node. Here the cellular and molecu-lar immigrants follow di�erent paths.

A�erent lymph draining from peripheral tissues enters the subcap-sular space of a lymph node, then is �ltered slowly through the capsular sinus and the cortical sinus, and converges on the medullary sinus in the hilum before exiting via an e�erent lymph vessel.398 Lymph-borne DCs enter the subcapsular sinus, actively traverse an endothelial and �broblastic layer of cells that forms the !oor of the subcapsular sinus, and reach the lymph node cortex, directly moving into the lymph node parenchyma and the collection of lymphocytes in the paracortical cords.399 Small protein molecules, such as cytokines and chemokines, and small microbe-derived molecules, do not traverse this barrier and fail to enter lymph node parenchyma.398,400 Instead, these molecules are channeled into de�ned �brous conduits that are both produced and enclosed by a tight network of �broblastic reticular cells and contain an internal matrix consisting of collagen bundles. �e collagen �bers provide structural support to the lymph node and de�ne its architec-ture. Despite the enclosed nature of these conduits, leukocytes within the lymph node are able to sample soluble material, including antigens, from the lymphatic content.401 Fibrous lymphatic conduits provide connections from the subcapsular and cortical sinuses to the high endothelial venules, enabling !uid !ow and substrate transport from peripheral tissues, via the subcapsular sinuses and �brous conduits, to the high endothelial venules, the site of cellular entry into the lymph node from the bloodstream.398 Fibrous conduits channel soluble, lymph-borne antigens and chemoattractants from the subcapsular sinus to B-cell follicles as well.402

Blood !ow to the lymph node, a�er entering the lymph node hilum, is channeled through the high endothelial venules, where intravascular lymphocytes can adhere to the endothelial barrier and traverse into the paracortical cords of the lymph node. Endothelial cells in the high endothelial venules can produce chemokines on their luminal surface and, because many �brous conduits end in the high endothelial venules, intravascular lymphocytes can respond to lymph-borne che-mokines, both of which trigger lymphocyte extravasation into the lymph node parenchyma.400 On traversing the high endothelial venules, lymphocytes enter the perivenular channel, a narrow space bounded by �broblastic reticular cells, that provides entry into the corridors of the paracortical cord. �e paracortical cord is a labyrinthine structure that accommodates many T cells that are presumed to roam the cor-ridors, eventually exiting by moving either toward a B-cell follicle or toward a cortical or medullary sinus.403 As lymphocytes move, side by side, through this labyrinth, they have extensive opportunities to contact antigen-bearing DCs that have entered the cord from the sub-capsular sinus. If lymphocytes recognize an antigen presented by a DC, the T-cell activation process is initiated.

In!ammatory cytokines and chemokines enter the �brous conduits that lead to the high endothelial venules, where they relay the message to circulating lymphocytes to adhere and traverse the endothelium. �e recruitment of lymphocytes into the paracortical cords is rapid and e"cient, and the labyrinths can become engorged with lymphocytes within a few hours of an in!ammatory stimulus. Beyond their trans-port function for cytokines and chemokines, resident DCs within the T-cell–rich paracortical zones in the lymph node can access and process soluble antigens sequestered in the conduits, linking the conduit system to the process of antigen presentation. Recent advances in microscopy404 have enabled investigators to visualize interactions between T lymphocytes and DCs in lymph nodes over the course of several hours405 and have demonstrated, for example, that CD4+ T-cell interactions with DCs enhance recruitment and activation of naïve CD8+ T cells.406

Spleen�e spleen is a large secondary lymphoid organ that also serves as an important �ltration site for clearance of microbial pathogens from the bloodstream.407 Although spleen and lymph nodes share many T-cell, B-cell, and DC subsets and are similarly complex at the microanatomic

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functions, such as cytolytic activity and cytokine secretion. �e intestinal lamina propria also is populated with T lymphocytes, but conventional CD8α+β+ T cells with αβ TCRs predominate at this site.430,431

Innate lymphoid cells, a heterogenous group of innate immune cells that are classi�ed in the lymphoid lineage in humans and in mice,167 are abundant within the intestinal lamina propria and cooperate with T cells to regulate barrier functions and to balance immune tone within intestinal tissues. For example, a recent study demonstrated that murine RORγt-expressing innate lymphoid cells express MHC class II molecules and present microbial-derived antigen to limit CD4+ T-cell responses to commensal bacteria, a mechanism that may dampen intestinal in!ammatory tone under homeostatic conditions.432 A�er challenge with the enteropathogenic bacterium Citrobacter rodentium, IL-22 production by innate lymphoid cells and CD4+ T cells appears to be critical for the induction of antimicrobial peptides by intestinal epithelial cells and for host protection.433,434

Although intestinal epithelial cells are postulated to present antigen to T lymphocytes,435-437 DCs are present in intestinal tissues as well. Two reports have shown that DCs sample intestinal contents by tran-siently traversing the tight junctions of the intestinal epithelial cell layer.438,439 A�er engul�ng bacterial pathogens, such as S. typhimurium, DCs migrate to secondary lymphoid tissues to activate naïve T lymphocytes.

Studies in mouse models have shown that intestinal infection with viral or bacterial pathogens induces robust expansion of

immunologic homeostasis and in limiting host tissue damage on encounter of exogenous pathogens (i.e., tolerance to potentially bene�-cial microbes and the induction of in!ammation to resist infection by pathogenic microbes) has been the focus of intense investigation.421,422 Aberrant in!ammatory and T-cell–mediated responses to commensal microbes are postulated to underlie the pathogenesis of various in!am-matory bowel syndromes.423 �e intestine also is one of the most important portals of entry for viral, bacterial, protozoal, and helminth pathogens.424

T lymphocytes are prevalent in the intestinal mucosa and can be found within the innermost epithelial layer, in the adjacent lamina propria, and within secondary lymphoid tissues such as Peyer’s patches (lymphoid aggregates scattered in the small intestinal mucosa) and mesenteric lymph nodes.425 A series of tertiary lymphoid tissues can develop in postnatal life under the in!uence of lymphoid tissue inducer cells; these include cryptopatches, isolated lymphoid follicles, and colonic patches in the intestines as well as inducible bronchus-associated lymphoid tissue in the lung.

T lymphocytes in the epithelial layer are a diverse population that in aggregate are termed intraepithelial lymphocytes. Some intraepithe-lial lymphocytes express the α- and β-chains of the co-receptor CD8 (CD8α+β+), whereas others express exclusively α-chains (CD8αα+) and others do not express CD8 at all.426 With respect to TCRs, some express αβ TCRs whereas others express γδ TCRs.427-429 Nearly all intraepithelial lymphocytes express adhesion molecules associated with an activated or memory phenotype and readily express e�ector

FIGURE 6-10 Anatomy of the spleen. The spleen is a complex secondary lymphoid organ with compartmentalized cell populations. A, Hematoxylin-eosin–stained spleen section with areas of densely packed cells, referred to as the white pulp (WP), separated by areas with more dispersed cell popula-tions referred to as the red pulp (RP). B, Section of spleen stained with fluorescently labeled antibodies specific for B cells (orange) and T cells (green), demonstrating the distinct localization of B cells and T cells within the white pulp. C, Staining for macrophages (orange) and the splenic marginal zone (green), demonstrating the density of macrophages and phagocytic cells in the red pulp and marginal zone.

A B

C

RP

RPWP

WP

B Cells

B Cells

Marginal Zone

Red Pulp Macrophages

T Cells

T Cells

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populations. Flow cytometry is used routinely in the clinical arena for CD4+ and CD8+ T-cell quantitation in HIV-infected and other immu-nocompromised patients.

More recent technical innovations that have a�ected cellular immu-nology studies involve the precise quantitation of antigen-speci�c T lymphocytes. �ree methods—ELISPOT assays, intracellular cytokine staining, and MHC tetramers—have revolutionized the study of pathogen-speci�c T-cell responses. �e ELISPOT assay is relatively simple and does not require a !ow cytometer. ELISPOT assays provide accurate, quantitative data and can be performed with complex mix-tures of cells, such as peripheral blood mononuclear cells or lymph node or spleen cells.444 To perform this assay, complex mixtures of cells are stimulated with antigen on a membrane coated with a monoclonal antibody speci�c for a cytokine, such as IFN-γ, TNF, or IL-4. Antigen-speci�c T cells in the mixture, on stimulation, release cytokines that are captured by membrane-bound antibodies directly adjacent to the stimulated cell. Bound cytokines are detected with a secondary, enzyme-conjugated monoclonal antibody in a fashion identical to a standard sandwich enzyme-linked immunosorbent assay. Each stimu-lated cell leaves a “spot” on the membrane, and the number of spots can be quanti�ed (Fig. 6-12).

pathogen-speci�c T lymphocytes in the small intestinal epithelium and lamina propria.430,431,440 �e presence of speci�c bacterial populations is a requirement for the development of �17 cell populations, exem-pli�ed by their absence in mice that lack microbial !ora.66 Bacteria that typically belong to commensal communities (e.g., segmented �lamen-tous bacteria) or that are considered pathogenic (e.g., S. typhimurium) induce mucosal �17 cells that protect against pathogenic microbes, in part via IL-17–, IL-23–, and IL-22–dependent pathways. Systemic infection with viruses, such as vesicular stomatitis virus, or the intra-cellular bacterium L. monocytogenes also induces marked increases in the frequency of pathogen-speci�c CD8+ T cells in the gut, suggesting that these T cells tra"c to the intestine during systemic immune responses. �e concept that immune responses to infectious pathogens result in the distribution of pathogen-speci�c T lymphocytes through-out the body is supported by two studies that measured whole-animal immunity.441,442 In both studies, although T-cell priming occurred in secondary lymphoid tissues, a�er priming, antigen-speci�c T lympho-cytes were found at higher frequencies in nonlymphoid tissues, such as liver, lamina propria, and adipose tissues.

PRIMER ON BASIC IMMUNOLOGIC TECHNIQUES: FOUNDATION OF IMMUNOLOGIC MODELSRapid evolution of immunologic techniques has facilitated the increas-ingly sophisticated view of the mammalian immune system. Under-standing current immunologic techniques is important for the practicing infectious diseases specialist for two reasons. First, these techniques form the basis on which we formulate our understanding of protective immunity. Second, immunologic techniques, such as !ow cytometry, intracellular cytokine and MHC tetramer staining, and enzyme-linked immunospot (ELISPOT) assays increasingly are used in the clinical setting to evaluate immunologic function. Some of the more recently developed immunologic techniques are reviewed brie!y here.

Characterizing and Measuring Pathogen-Specific ImmunityFlow cytometry has transformed immunologic analysis by allowing rapid and e"cient analysis of complex lymphoid cell populations.443 A !ow cytometer analyzes single cells for the presence of multiple, !uo-rescently labeled monoclonal antibodies or dyes and allows each cell in a complex population to be scored for multiple cellular markers. �is powerful technique provides a detailed picture of mixed cell pop-ulations, such as lymph node, spleen, or peripheral blood cells (Fig. 6-11). With the steady introduction of new monoclonal antibodies speci�c for novel surface or intracellular proteins, !ow cytometry con-tinues to uncover increasingly greater complexity among cell popula-tions that previously were assumed to be homogeneous. A recurring theme in immunologic studies is the discovery that a cell subset, on the basis of a new marker, can be divided into two or three distinct cell

FIGURE 6-11 Flow cytometry can be used to identify distinct immune cell populations within a complex mixture of cells. A, Plotting of the forward light scatter versus the side scatter of individual cells, which is a measure of the cells’ relative size and granularity. The cells within the enclosed box are selected (gated) and consist of monocytes and lymphocytes. B, The gated cells are further divided into B cells (vertical axis) and T cells (horizontal axis) on the basis of staining with specific monoclonal antibodies with different fluorescent emissions. The box surrounds the T-cell population. C, The gated T-cell population is analyzed for surface expression of CD4 or CD8 using monoclonal antibodies that fluoresce at two additional wavelengths.

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FIGURE 6-12 The enzyme-linked immunospot (ELISPOT) assay quantifies T-cell responses to distinct peptides after infection. The four panels show interferon-γ production by spleen cells derived from mice infected with Listeria monocytogenes on stimulation with either no peptide or three defined MHC class I–restricted peptide epitopes. Each spot rep-resents the activation of an individual cell by a specific peptide. The fre-quency of antigen-specific T cells can be calculated by counting the number of spots on the filter and dividing by the number of cells placed in the well. (Redrawn from Vijh S, Pamer EG. Immunodominant and sub-dominant CTL responses to Listeria monocytogenes infection. J Immunol. 1997;158:3366.)

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ACKNOWLEDGMENTEric Pamer and Michael Glickman kindly provided permission to update this chapter based on their text published in the seventh edition of Principles and Practices of Infectious Diseases.

Intracellular cytokine staining is similar to the ELISPOT technique, in that complex cell populations are stimulated with an antigen, but in the presence of either Brefeldin A or monensin, which are drugs that inhibit cellular secretion of cytokines.445 During this incubation, cytokines are produced by antigen-speci!c T cells but instead of being secreted they accumulate within the cell. A"er stimulation, cells are !xed and permeabilized, then stained with cytokine-speci!c, #uorescently tagged antibodies. Permeabilization with a dilute deter-gent is necessary to provide antibody access to the accumulated intra-cellular cytokine. Stained cells are examined by #ow cytometry (Fig. 6-13), and antigen-speci!c T cells are identi!ed and quanti!ed on the basis of cytokine production but also can be scored for other charac-teristics (size, granularity, activation and di$erentiation markers, antigen receptor expression). Although technically more demanding, intracellular cytokine staining is more informative than the ELISPOT assay.

Another direct method for quantifying antigen-speci!c T cells involves the use of MHC tetramers.446 Because the interaction between TCRs and MHC peptide complexes is of low a%nity, attempts to iden-tify antigen-speci!c T cells with soluble MHC-peptide complexes was not possible. Generation of tetrameric forms of MHC-peptide com-plexed with a #uorophore readily enabled antigen-speci!c T cells to be stained and identi!ed by #ow cytometry (Fig. 6-14). In addition, tet-ramer staining can be used to isolate viable, pathogen-speci!c T lym-phocytes.166,447 An advantage of MHC tetramer staining over either intracellular cytokine staining or ELISPOT assays is that T-cell detec-tion does not depend on cytokine production, which in turn depends on the T-cell phenotype.

In many cases, use of the just-described quantitative assays radically revised prior estimates of pathogen-speci!c T-cell frequencies.109,110,448 In some infections, such as primary Epstein-Barr virus infection, the frequency of virus-speci!c CD8+ T cells approaches 70%.449,450 Although Epstein-Barr virus is arguably an extreme example, in other infections, such as those caused by HIV, herpes simplex virus, in#uenza virus, and L. monocytogenes, pathogen-speci!c T-cell frequencies are astonish-ingly large, generally ranging from 2% to 25%.110,448,451,452

FIGURE 6-13 Intracellular cytokine staining is another method for quantifying antigen-specific T lymphocytes in complex mixtures of cells. Single cell suspensions from the spleen, peripheral blood, or organ of interest are stimulated transiently with antigenic peptides and then stained with monoclonal antibodies for the production of cytokines. The dot plot shows production of interferon-γ (INF-γ) and tumor necrosis factor-α (TNF-α) by peripheral blood lymphocytes on stimulation with an antigenic bacterial peptide. The peptide-specific T-cell population demon-strates concurrent production of both cytokines, a common finding for pathogen-specific CD8+ T lymphocytes. The percentage of cells that produce either TNF-α, IFN-γ, or both is indicated in the plot quadrants.

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FIGURE 6-14 Major histocompatibility complex (MHC) tetramers can identify antigen-specific T cells. MHC tetramers are able to stain antigen-specific T lymphocytes for detection by flow cytometry because the fluorochrome-conjugated tetramer can simultaneously interact with multiple antigen-specific T-cell receptors (TCRs). Although the association of individual TCRs with MHC complexes is transient, binding becomes stable in the setting of multiple simultaneous interactions. In most studies, MHC tetramers are generated by biotinylation of the carboxyl terminus of soluble MHC molecules that contain a specific antigenic peptide, followed by complex formation with a fluorescently labeled avidin molecule, which contains four biotin-binding sites. A, MHC tetramer complex associating with an antigen-specific T lymphocyte. B, MHC tetramer staining of human peripheral blood mononuclear cells using tetramers complexed with three different Epstein-Barr virus–derived peptides. The percentage of CD8+ T cells specific for each of these peptides is indicated in the plots. HLA, human leukocyte antigen. (A courtesy Dirk H. Busch, Technical University, Munich.)

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