《免疫学》（英文版） Chapter 14 Cell-mediated Effector Responses

Cell-Mediated chapter 14 Effector responses HE CELL-MEDIATED AND HUMORAL BRANCHES OF the immune system assume different roles tecting the host. The effectors of the humoral branch are secreted antibodies, highly specific molecules that can bind and neutralize antigens on the surface of cells and in the extracellular spaces. The primary domain of anti- body protection lies outside the cell. If antibodies were the Big CtL Attacks Little Tumor Cell only agents of immunity, pathogens that managed to evade them and colonize the intracellular environment would Effector Responses escape the immune system. This is not the case. The princi pal role of cell-mediated immunity is to detect and eliminate a General Properties of Effector T Cells cells that harbor intracellular pathogens. Cell-mediated im a Cytotoxic T Cells munity also can recognize and eliminate cells, such as tumor lls, that have undergone genetic modifications so that they Natural Killer Cells xpress antigens not typical of normal cells Antibody-Dependent Cell-Mediated Cytotoxicity Both antigen-specific and-nonspecific cells can contri bute to the cell-mediated immune response. Specific cells in- Experimental Assessment of Cell-Mediated chude CD8 cytotoxic T lymphocytes(Tc cells or CTls)and Cytotoxicity cytokine-secreting CD4 TH cells that mediate delayed-type ypersensitivity(DTH). The discussion of DTH reactions and the role of CD4 T cells in their orchestration appears in Chapter 16. Nonspecific cells include nK cells and non- lymphoid cell types such as macrophages, neutrophils, and sinophils. The activity of both specific and nonspecific components usually depends on effective local concentra- Effector Responses tions of various cytokines. T cells, NK cells, and macrophages are the most important sources of the cytokines that organize The importance of cell-mediated immunity becomes evident and support cell-mediated immunity. Finally, although hu- when the system is defective. Children with DiGeorge syn noral and cell-mediated immunity have many distinctive drome, who are born without a thymus and therefore lack the features, they are not completely independent. Cells such T-cell component of the cell-mediated immune system, gen- as macrophages, NK cells, neutrophils, and eosinophils can erally are able to cope with infections of extracellular bacteria, use antibodies as receptors to recognize and target cells for but they cannot effectively eliminate intracellular pathogens killing. Also, chemotactic peptides generated by the activa- Their lack of functional cell-mediated immunity results in tion of complement in response to antigen-antibody com- repeated infections with viruses, intracellular bacteria, and plexes can contribute to assembling the cell types required for fungi. The severity of the cell-mediated immunodeficiency in a cell-mediated response these children is such that even the attenuated virus present in In the preceding chapters, various aspects of the humoral a vaccine, capable of only limited growth in normal individu and cell-mediated effector responses have been described. This als, can produce life-threatening infections. chapter addresses cytotoxic effector mechanisms mediated by Cell-mediated immune responses can be divided into two Tc cells, NK cells, antibody-dependent cell-mediated cytoto major categories according to the different effecto city(adcc), and the tions that are mobilized. One group comprises effector cells

■ Effector Responses ■ General Properties of Effector T Cells ■ Cytotoxic T Cells ■ Natural Killer Cells ■ Antibody-Dependent Cell-Mediated Cytotoxicity ■ Experimental Assessment of Cell-Mediated Cytotoxicity Big CTL Attacks Little Tumor Cell Cell-Mediated Effector Responses T -     the immune system assume different roles in pro￾tecting the host. The effectors of the humoral branch are secreted antibodies, highly specific molecules that can bind and neutralize antigens on the surface of cells and in the extracellular spaces. The primary domain of anti￾body protection lies outside the cell. If antibodies were the only agents of immunity, pathogens that managed to evade them and colonize the intracellular environment would escape the immune system. This is not the case. The princi￾pal role of cell-mediated immunity is to detect and eliminate cells that harbor intracellular pathogens. Cell-mediated im￾munity also can recognize and eliminate cells, such as tumor cells, that have undergone genetic modifications so that they express antigens not typical of normal cells. Both antigen-specific and -nonspecific cells can contri￾bute to the cell-mediated immune response. Specific cells in￾clude CD8+ cytotoxic T lymphocytes (TC cells or CTLs) and cytokine-secreting CD4+ TH cells that mediate delayed-type hypersensitivity (DTH). The discussion of DTH reactions and the role of CD4+ T cells in their orchestration appears in Chapter 16. Nonspecific cells include NK cells and non￾lymphoid cell types such as macrophages, neutrophils, and eosinophils. The activity of both specific and nonspecific components usually depends on effective local concentra￾tions of various cytokines. T cells, NK cells, and macrophages are the most important sources of the cytokines that organize and support cell-mediated immunity. Finally, although hu￾moral and cell-mediated immunity have many distinctive features, they are not completely independent. Cells such as macrophages, NK cells, neutrophils, and eosinophils can use antibodies as receptors to recognize and target cells for killing. Also, chemotactic peptides generated by the activa￾tion of complement in response to antigen-antibody com￾plexes can contribute to assembling the cell types required for a cell-mediated response. In the preceding chapters, various aspects of the humoral and cell-mediated effector responses have been described. This chapter addresses cytotoxic effector mechanisms mediated by TC cells, NK cells, antibody-dependent cell-mediated cytotoxi￾city (ADCC), and the experimental assay of cytotoxicity. Effector Responses The importance of cell-mediated immunity becomes evident when the system is defective. Children with DiGeorge syn￾drome, who are born without a thymus and therefore lack the T-cell component of the cell-mediated immune system, gen￾erally are able to cope with infections of extracellular bacteria, but they cannot effectively eliminate intracellular pathogens. Their lack of functional cell-mediated immunity results in repeated infections with viruses, intracellular bacteria, and fungi. The severity of the cell-mediated immunodeficiency in these children is such that even the attenuated virus present in a vaccine, capable of only limited growth in normal individu￾als, can produce life-threatening infections. Cell-mediated immune responses can be divided into two major categories according to the different effector popula￾tions that are mobilized. One group comprises effector cells chapter 14

320 paRI I Immune Effector mechanisms that have direct cytotoxic activity. These effectors eliminate naive T cells) are able to respond to TCR-mediated signals foreign cells and altered self-cells by mounting a cytotoxic with little, if any co-stimulation. reaction that lyses their target. The various cytotoxic effector The reason for the different activation requirements of cells can be grouped into two general categories: one com- naive and activated t cells is an area of continuing research, prises antigen-specific cytotoxic T lymphocytes( CTls) and but some clues have been found. One is that many popula unspecific cells, such as natural killer(NK) cells and macro- tions of naive and effector T cells express different isoforms phages. The target cells to which these effectors are directed of CD45, designated CD45RA and CD45RO, which are pro include allogeneic cells, malignant cells, virus-infected cells, duced by alternative splicing of the Rna transcript of the and chemically conjugated cells. The other group is a sub- CD45 gene. This membrane molecule mediates TCR signal population of effector CD4* T cells that mediates delayed- transduction by catalyzing dephosphorylation of a tyrosine type hypersensitivity reactions(see Chapter 16). The next residue on the protein tyrosine kinases Lck and Fyn, activat- section reviews the general properties of effector T cells and ing these kinases and triggering the subsequent steps in T-cell how they differ from naive T cells. activation(see figures 10-10 and 10-11). The CD45RO iso- form, which is expressed on effector T cells, associates with the TCR complex and its coreceptors, CD4 and CD8, much General Properties of Effector T Cells better than does the CD45RA isoform, which is expressed by naive T cells. Memory T cells have both isoforms, but the The three types of effectorTcells-CD4*, THl and TH2 cells, CD45RO is predominant. As a result, effector and memory and CD8* CTLs-exhibit several properties that set them T cells are more sensitive to TCR-mediated activation by a art from naive helper and cytotoxic T cells (Table 14-1). In peptide-MHC complex. They also have less stringent re particular, effector cells are characterized by their less strin quirements for co-stimulatory signals and therefore are able gent activation requirements, increased expression of cell- to respond to peptide -MHC complexes displayed on target adhesion molecules, and production of both membrane- cells or antigen-presenting cells that lack the co-stimulatory bound and soluble effector molecules B7 molecules The Activation Requirements Cell-Adhesion molecules facilitate of t cells differ TCR-Mediated Interactions T cells at different stages of differentiation may respond with CD2 and the integrin LFA-1 are cell-adhesion molecules on different efficiencies to signals mediated by the T-cell recep- the surfaces of T cells that bind, respectively, to LFA-3 and tor and may consequently require different levels of a second ICAMs(intracellular cell-adhesion molecules) on antigen set of co-stimulatory signals. As described in Chapter 10, presenting cells and various target cells(see Figure 9-13). The activation of naive T cells and their subsequent proliferation level of LFA-1 and CD2 is twofold to fourfold higher on and differentiation into effector T cells require both a pri- effector T cells than on naive T cells, enabling the effector mary signal, delivered when the TCR complex and CD4 T cells to bind more effectively to antigen-presenting cells or CD8 coreceptor interact with a foreign peptide-MHc and to various target cells that express low levels of ICAMs molecule complex, and a co-stimulatory signal, delivered by LFA-3 nteraction between particular membrane molecules on the As Chapter 9 showed, the initial interaction of an effector T cell and the antigen-presenting cell. In contrast, antigen T cell with an antigen-presenting cell or target cell is weak, experienced effector cells and memory cells(as opposed to allowing the TCr to scan the membrane for specific peptides TABLE 14-1 Comparison of naive and effector T cells Property Naive t cells Effector T cells stimulatory signal (CD28-B7 interaction) Required for activation equired for activation CD45RA CD45RO Cell-adhesion molecules(CD2 and LFA-1) fficking patterns HEVs' in secondary lymphoid tissue Tertiary lymphoid tissue inflammatory sites HEV= high endothelial venules, sites in blood vessel used by lymphocytes for extravasation

that have direct cytotoxic activity. These effectors eliminate foreign cells and altered self-cells by mounting a cytotoxic reaction that lyses their target. The various cytotoxic effector cells can be grouped into two general categories: one com￾prises antigen-specific cytotoxic T lymphocytes (CTLs) and nonspecific cells, such as natural killer (NK) cells and macro￾phages. The target cells to which these effectors are directed include allogeneic cells, malignant cells, virus-infected cells, and chemically conjugated cells. The other group is a sub￾population of effector CD4+ T cells that mediates delayed￾type hypersensitivity reactions (see Chapter 16). The next section reviews the general properties of effector T cells and how they differ from naive T cells. General Properties of Effector T Cells The three types of effector T cells—CD4+ , TH1 and TH2 cells, and CD8+ CTLs—exhibit several properties that set them apart from naive helper and cytotoxic T cells (Table 14-1). In particular, effector cells are characterized by their less strin￾gent activation requirements, increased expression of cell￾adhesion molecules, and production of both membrane￾bound and soluble effector molecules. The Activation Requirements of T Cells Differ T cells at different stages of differentiation may respond with different efficiencies to signals mediated by the T-cell recep￾tor and may consequently require different levels of a second set of co-stimulatory signals. As described in Chapter 10, activation of naive T cells and their subsequent proliferation and differentiation into effector T cells require both a pri￾mary signal, delivered when the TCR complex and CD4 or CD8 coreceptor interact with a foreign peptide–MHC molecule complex, and a co-stimulatory signal, delivered by interaction between particular membrane molecules on the T cell and the antigen-presenting cell. In contrast, antigen￾experienced effector cells and memory cells (as opposed to naive T cells) are able to respond to TCR-mediated signals with little, if any co-stimulation. The reason for the different activation requirements of naive and activated T cells is an area of continuing research, but some clues have been found. One is that many popula￾tions of naive and effector T cells express different isoforms of CD45, designated CD45RA and CD45RO, which are pro￾duced by alternative splicing of the RNA transcript of the CD45 gene. This membrane molecule mediates TCR signal transduction by catalyzing dephosphorylation of a tyrosine residue on the protein tyrosine kinases Lck and Fyn, activat￾ing these kinases and triggering the subsequent steps in T-cell activation (see figures 10-10 and 10-11). The CD45RO iso￾form, which is expressed on effector T cells, associates with the TCR complex and its coreceptors, CD4 and CD8, much better than does the CD45RA isoform, which is expressed by naive T cells. Memory T cells have both isoforms, but the CD45RO is predominant. As a result, effector and memory T cells are more sensitive to TCR-mediated activation by a peptide-MHC complex. They also have less stringent re￾quirements for co-stimulatory signals and therefore are able to respond to peptide-MHC complexes displayed on target cells or antigen-presenting cells that lack the co-stimulatory B7 molecules. Cell-Adhesion Molecules Facilitate TCR-Mediated Interactions CD2 and the integrin LFA-1 are cell-adhesion molecules on the surfaces of T cells that bind, respectively, to LFA-3 and ICAMs (intracellular cell-adhesion molecules) on antigen￾presenting cells and various target cells (see Figure 9-13). The level of LFA-1 and CD2 is twofold to fourfold higher on effector T cells than on naive T cells, enabling the effector T cells to bind more effectively to antigen-presenting cells and to various target cells that express low levels of ICAMs or LFA-3. As Chapter 9 showed, the initial interaction of an effector T cell with an antigen-presenting cell or target cell is weak, allowing the TCR to scan the membrane for specific peptides 320 PART III Immune Effector Mechanisms TABLE 14-1 Comparison of naive and effector T cells Property Naive T cells Effector T cells Co-stimulatory signal (CD28-B7 interaction) Required for activation Not required for activation CD45 isoform CD45RA CD45RO Cell-adhesion molecules (CD2 and LFA-1) Low High Trafficking patterns HEVs* in secondary lymphoid tissue Tertiary lymphoid tissues; inflammatory sites *HEV = high endothelial venules, sites in blood vessel used by lymphocytes for extravasation

Cell-Mediated Effector Responses CHAPTER 14 TABLE 14-2 Effector molecules produced by effector T cells Cell type Soluble effectors Membrane-bound effectors Cytotoxins(perforins and granzymes), IFN-Y, TNF-B Fas ligand( FASL) IL-2, IL-3, TNF-B, IFN-Y, GM-CSF(high) Tumor necrosis factorβ(TNFβ) IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, GM-CSF (low) CD40 ligand presented by self-MHC molecules. If no peptide-MHC com- tumor cells)and in graft-rejection reactions. In general, plex is recognized by the effector cell, it will disengage from CTLs are CD8* and are therefore class I mhc restricted, al the aPC or target cell Recognition of a peptide-MHC com- though in rare instances CD4 class Il-restricted T cells have plex by the TCR, however, produces a signal that increases the been shown to function as CTls. since virtually all nucleated affinity of LFA-1 for ICAMs on the APC or target-cell cells in the body express class I MHC molecules, CTLs can membrane, prolonging the interaction between the cells. recognize and eliminate almost any altered body cell For example, THl effector cells remain bound to macro The CTL-mediated immune response can be divided into phages that display peptide-class II MHC complexes; TH2 ef- two phases, reflecting different aspects of the response. The fector cells remain bound to B cells that display peptide-class first phase activates and differentiates naive Tc cells into II MHC complexes; and CTl effector cells bind tightly to functional effector CTLs In the second phase, effector CTls virus-infected target cells that display peptide-class I MHc recognize antigen-class I MHC complexes on specific target com plexes cells, which leads them to destroy the target cells Effector T Cells Express a Variety Effector CTLs Are generated of Effector molecules from ctl Precursors Unlike naive T cells, effector T cells express certain effector Naive Tc cells are incapable of killing target cells and are there molecules, which may be membrane bound or soluble Table fore referred to as Ctl precursors( CTL-Ps)to denote their 14-2). The membrane-bound molecules belong to the tumor functionally immature state. Only after a CTL-P has been acti- necrosis factor (TNF) family of membrane proteins and vated will the cell differentiate into a functional CTl with include the Fas ligand( FASl)on CD8 CTLs, TNF-Bon THl cytotoxic activity. Generation of CTls from CTL-Ps appears to cells, and the CD40 ligand on TH2 cells. Each of the effector require at least three sequential signals(Figure 14-1 T-cell populations also secretes distinct panels of soluble effector molecules. CTLs secrete cytotoxins(perforins and An antigen-specific signal 1 transmitted by the TCR granzymes)as well as two cytokines, IFN-y and TNF-B As complex upon recognition of a peptide-class I MHC described in Chapter 12, the THl and TH2 subsets secrete largely nonoverlapping sets of cytokines Each of these membrane-bound and secreted molecules A co-stimulatory signal transmitted by the CD28-B7 plays an important role in various T-cell effector functions interaction of the CTL-P and the antigen-presenting The Fas ligand, perforins, and granzymes, for example, medi- Sle target-cell destruction by the CTL; membrane-bound. A signal induced by the interaction of IL-2 with the F-B and soluble IFN-y and GM-csF promote macro- h-affinity IL-2 receptor, resulting in proliferation and phage activation by the THl cell; and the membrane-bound differentiation of the antigen-activated CTl-P into CD40 ligand and soluble IL-4, IL-5, and IL-6 all play a role in effector Ctls B-cell activation by the TH2 cell Unactivated CTL-Ps do not express IL-2 or IL-2 receptors do not proliferate, and do not display cytotoxic activity. Anti Cytotoxic T Cells gen activation induces a CTL-P to begin expressing the IL-2 receptor and to a lesser extent IL-2, the principal cytokine CytotoxicT lymphocytes, or CTls, are generated by immune required for proliferation and differentiation of activated activation of T cytotoxic tc) cells. These effector cells have CTL-Ps into effector CTLs. In some cases, the amount of lytic capability and are critical in the recognition and elimi- IL-2 secreted by an antigen-activated CTL-P may be suffi nation of altered self-cells (e.g, virus-infected cells and cient to induce its own proliferation and differentiation; this

presented by self-MHC molecules. If no peptide-MHC com￾plex is recognized by the effector cell, it will disengage from the APC or target cell. Recognition of a peptide-MHC com￾plex by the TCR, however, produces a signal that increases the affinity of LFA-1 for ICAMs on the APC or target-cell membrane, prolonging the interaction between the cells. For example, TH1 effector cells remain bound to macro￾phages that display peptide–class II MHC complexes; TH2 ef￾fector cells remain bound to B cells that display peptide–class II MHC complexes; and CTL effector cells bind tightly to virus-infected target cells that display peptide–class I MHC complexes. Effector T Cells Express a Variety of Effector Molecules Unlike naive T cells, effector T cells express certain effector molecules, which may be membrane bound or soluble (Table 14-2). The membrane-bound molecules belong to the tumor necrosis factor (TNF) family of membrane proteins and include the Fas ligand (FASL) on CD8+ CTLs, TNF- on TH1 cells, and the CD40 ligand on TH2 cells. Each of the effector T-cell populations also secretes distinct panels of soluble effector molecules. CTLs secrete cytotoxins (perforins and granzymes) as well as two cytokines, IFN- and TNF-. As described in Chapter 12, the TH1 and TH2 subsets secrete largely nonoverlapping sets of cytokines. Each of these membrane-bound and secreted molecules plays an important role in various T-cell effector functions. The Fas ligand, perforins, and granzymes, for example, medi￾ate target-cell destruction by the CTL; membrane-bound TNF- and soluble IFN- and GM-CSF promote macro￾phage activation by the TH1 cell; and the membrane-bound CD40 ligand and soluble IL-4, IL-5, and IL-6 all play a role in B-cell activation by the TH2 cell. Cytotoxic T Cells Cytotoxic T lymphocytes, or CTLs, are generated by immune activation of T cytotoxic (TC) cells. These effector cells have lytic capability and are critical in the recognition and elimi￾nation of altered self-cells (e.g., virus-infected cells and tumor cells) and in graft-rejection reactions. In general, CTLs are CD8+ and are therefore class I MHC restricted, al￾though in rare instances CD4+ class II–restricted T cells have been shown to function as CTLs. Since virtually all nucleated cells in the body express class I MHC molecules, CTLs can recognize and eliminate almost any altered body cell. The CTL-mediated immune response can be divided into two phases, reflecting different aspects of the response. The first phase activates and differentiates naive TC cells into functional effector CTLs. In the second phase, effector CTLs recognize antigen–class I MHC complexes on specific target cells, which leads them to destroy the target cells. Effector CTLs Are Generated from CTL Precursors Naive TC cells are incapable of killing target cells and are there￾fore referred to as CTL precursors (CTL-Ps) to denote their functionally immature state. Only after a CTL-P has been acti￾vated will the cell differentiate into a functional CTL with cytotoxic activity. Generation of CTLs from CTL-Ps appears to require at least three sequential signals (Figure 14-1): ■ An antigen-specific signal 1 transmitted by the TCR complex upon recognition of a peptide–class I MHC molecule complex ■ A co-stimulatory signal transmitted by the CD28-B7 interaction of the CTL-P and the antigen-presenting cell ■ A signal induced by the interaction of IL-2 with the high-affinity IL-2 receptor, resulting in proliferation and differentiation of the antigen-activated CTL-P into effector CTLs Unactivated CTL-Ps do not express IL-2 or IL-2 receptors, do not proliferate, and do not display cytotoxic activity. Anti￾gen activation induces a CTL-P to begin expressing the IL-2 receptor and to a lesser extent IL-2, the principal cytokine required for proliferation and differentiation of activated CTL-Ps into effector CTLs. In some cases, the amount of IL-2 secreted by an antigen-activated CTL-P may be suffi￾cient to induce its own proliferation and differentiation; this Cell-Mediated Effector Responses CHAPTER 14 321 TABLE 14-2 Effector molecules produced by effector T cells Cell type Soluble effectors Membrane-bound effectors CTL Cytotoxins (perforins and granzymes), IFN-, TNF- Fas ligand (FASL) TH1 IL-2, IL-3, TNF-, IFN-, GM-CSF (high) Tumor necrosis factor (TNF-) TH2 IL-3, IL-4, IL-5,IL-6, IL-10,IL-13,GM-CSF (low) CD40 ligand

322 PART I Immune Effector Mechanisms Class II mhc CD4 THl cell Co-stimulatory signal IL-2 HI cell Class I mhc IL-2R. Activation arget cell Ta CTL-P g-activated CTL-P Proliferation Effector cytotoxic function FIGURE 14-1 Generation of effector CTLS. Upon interaction with additional IL-2 secreted by TH1 cells resulting from antigen activation antigen-class I MHC complexes on appropriate target cells, CTL-Ps be- and proliferation of CD4"' T cells. In the subsequent effector phase, CTLS gin to express IL-2 receptors (IL-2R) and lesser amounts of IL-2 Prolif- destroy specific target cells. eration and differentiation of antigen-activated CTL-Ps generally require is particularly true of memory CTL-Ps, which have lower the immune response is rapidly terminated, lessening the activation requirements than naive cells do( figure 14-2a). likelihood of nonspecific tissue damage from the inflamma In general, though, most activated CTL-Ps require addi- tory response tional IL-2 produced by proliferating THl cells to proliferate The role of THl cells in the generation of CTls from naive and differentiate into effector CTLs. The fact that the IL-2 CTL-Ps is not completely understood, and it is unlikely that a receptor is not expressed until after a CTL-P has been acti- THl cell and CTl-P interact directly. However, IL-2 and vated by antigen plus a class I MHc molecule favors the stimulation are important in the transformation of naive clonal expansion and acquisition of cytotoxicity by only the CTL-Ps into effector cells, and THl cells can be mediators antigen-specific CTL-Ps in the provision of these essential requirements. As shown The proliferation and differentiation of both antigen- in Figure 14-2b, the interaction of helper cells with antigen activated THl cells and CTL-Ps depend on IL-2. In IL-2 presenting cells can result in production of IL-2 by the TH1 knockout mice, the absence of IL-2 has been shown to abol- cell. The paracrine action of this cytokine on nearby naive ish CTL-mediated cytotoxicity. After clearance of antigen, CTL-Ps whose TCRs are engaged can cause them to prolifer- the level of IL-2 declines, which induces THl cells and Ctls ate and differentiate into active Ctls. Additionally, thl can to undergo programmed cell death by apoptosis. In this way, induce the up-regulation of co-stimulatory molecules on the

is particularly true of memory CTL-Ps, which have lower activation requirements than naive cells do (Figure 14-2a). In general, though, most activated CTL-Ps require addi￾tional IL-2 produced by proliferating TH1 cells to proliferate and differentiate into effector CTLs. The fact that the IL-2 receptor is not expressed until after a CTL-P has been acti￾vated by antigen plus a class I MHC molecule favors the clonal expansion and acquisition of cytotoxicity by only the antigen-specific CTL-Ps. The proliferation and differentiation of both antigen￾activated TH1 cells and CTL-Ps depend on IL-2. In IL-2 knockout mice, the absence of IL-2 has been shown to abol￾ish CTL-mediated cytotoxicity. After clearance of antigen, the level of IL-2 declines, which induces TH1 cells and CTLs to undergo programmed cell death by apoptosis. In this way, the immune response is rapidly terminated, lessening the likelihood of nonspecific tissue damage from the inflamma￾tory response. The role of TH1 cells in the generation of CTLs from naive CTL-Ps is not completely understood, and it is unlikely that a TH1 cell and CTL-P interact directly. However, IL-2 and co￾stimulation are important in the transformation of naive CTL-Ps into effector cells, and TH1 cells can be mediators in the provision of these essential requirements. As shown in Figure 14-2b, the interaction of helper cells with antigen￾presenting cells can result in production of IL-2 by the TH1 cell. The paracrine action of this cytokine on nearby naive CTL-Ps whose TCRs are engaged can cause them to prolifer￾ate and differentiate into active CTLs. Additionally, TH1 can induce the up-regulation of co-stimulatory molecules on the 322 PART III Immune Effector Mechanisms IL-2 APC Class II MHC + antigen CD4 IL-2R TH1 cell TH1 cell TH1 cell IL-2 Co-stimulatory signal Activation Proliferation, differentiation Target cell CTL-P CD8 CTL Class I MHC + antigen IL-2R CD3 Ag-activated CTL-P Co-stimulatory signal IL-2R expression Proliferation Effector cytotoxic function – + – – + IL-2 expression – + + – – ± ± FIGURE 14-1 Generation of effector CTLs. Upon interaction with antigen–class I MHC complexes on appropriate target cells, CTL-Ps be￾gin to express IL-2 receptors (IL-2R) and lesser amounts of IL-2. Prolif￾eration and differentiation of antigen-activated CTL-Ps generally require additional IL-2 secreted by TH1 cells resulting from antigen activation and proliferation of CD4+ T cells. In the subsequent effector phase, CTLs destroy specific target cells

Cell-Mediated Effector Responses cHAPTER 14 32 Y Virus-infected dendritic cell FIGURE 14-2 Proliferation of memory CTL-Ps may not require help activation. (b)A TH cell may provide the IL-2 necessary for proliferation from TH cells.(a)Antigen-activated memory CTL-Ps appear to secrete suf- of an antigen-activated naive CTL-P when it binds to the same APC as the ficient IL-2 to stimulate their own proliferation and differentiation into effec CTL-P. Also, TH cells may alter the behavior of APCs in a number of way tor CTLs. They also may not require the CD28-B7 co-stimulatory signal for such as increasing the display of co-stimulatory molecules by the APC. surface of antigen-presenting cells. In this manner, THl cells the tetramer become fluorescently labeled(Figure 14-3) help CTL-P division and differentiation by causing the gen- Using flow cytometry, it is then possible to determine the eration of adequate levels of co-stimulation. proportion of cells in a population that have TCRs specific for a particular antigen by counting the number of fluores- CD8 CTLs Can be tracked with mHc cently labeled cells in a cell population. This very sensitive Tetramer Technology approach can detect antigen-specific T cells even when their frequency in the CD8t population is as low as 0.1%. Further MHC tetramers are laboratory-generated complexes of four more one can directly measure the increase in antigen MHC class I molecules bound to a specific peptide and specific CD8* T cells in response to exposure to pathogens linked to a fluorescent molecule. a given MHC-tetramer- such as viruses or cancer-associated antigens. In a related peptide complex binds only CD8 T cells that have TCRs application, researchers infected mice with vesicular stomati- specific for the particular peptide-MHC complex that makes tis virus(VSv) and systematically examined the distribution up the tetramer. Thus, when a particular tetramer is added to of CD8* cells specific for a VSV-derived peptide-MHC com a cell population containing T cells(spleen cells or lymph- plex throughout the entire body. This study demonstrated ode cells, for example), cells that bear TCRs specific for that during acute infection with VSV, the distribution of

surface of antigen-presenting cells. In this manner, TH1 cells help CTL-P division and differentiation by causing the gen￾eration of adequate levels of co-stimulation. CD8+ CTLs Can Be Tracked with MHC Tetramer Technology MHC tetramers are laboratory-generated complexes of four MHC class I molecules bound to a specific peptide and linked to a fluorescent molecule. A given MHC-tetramer– peptide complex binds only CD8+ T cells that have TCRs specific for the particular peptide-MHC complex that makes up the tetramer. Thus, when a particular tetramer is added to a cell population containing T cells (spleen cells or lymph￾node cells, for example), cells that bear TCRs specific for the tetramer become fluorescently labeled (Figure 14-3). Using flow cytometry, it is then possible to determine the proportion of cells in a population that have TCRs specific for a particular antigen by counting the number of fluores￾cently labeled cells in a cell population. This very sensitive approach can detect antigen-specific T cells even when their frequency in the CD8+ population is as low as 0.1%. Further￾more, one can directly measure the increase in antigen￾specific CD8+ T cells in response to exposure to pathogens such as viruses or cancer-associated antigens. In a related application, researchers infected mice with vesicular stomati￾tis virus (VSV) and systematically examined the distribution of CD8+ cells specific for a VSV-derived peptide-MHC com￾plex throughout the entire body. This study demonstrated that during acute infection with VSV, the distribution of Cell-Mediated Effector Responses CHAPTER 14 323 (a) IL-2 Memory CTL-P B7 CD28 CTL (b) Virus-infected dendritic cell Naive CTL-P TH1 IL-2 CTL Virus-infected dendritic cell 1 2 1 2 1 2 FIGURE 14-2 Proliferation of memory CTL-Ps may not require help from TH cells. (a) Antigen-activated memory CTL-Ps appear to secrete suf￾ficient IL-2 to stimulate their own proliferation and differentiation into effec￾tor CTLs. They also may not require the CD28-B7 co-stimulatory signal for activation. (b) A TH cell may provide the IL-2 necessary for proliferation of an antigen-activated naive CTL-P when it binds to the same APC as the CTL-P. Also, TH cells may alter the behavior of APCs in a number of ways, such as increasing the display of co-stimulatory molecules by the APC

324 paRT I Immune Effector Mechanisms Streptavidin mouse strains carrying mutations that affect the ability of Purified CTls to induce death have led to the identification of the molecules class I mhc The primary events in CTL-mediated death are conjugate formation, membrane attack, CTL dissociation, and target- Biotin fluorescently cell destruction(Figure 14-6). When antigen-specific CTLs Streptavidin are incubated with appropriate target cells, the two cell types interact and undergo conjugate formation. Formation of a CTL-target cell conjugate is followed within several minutes MHC tetramer by a Ca-dependent, energy-requiring step in which the CTL programs the target cell for death. The Ctl then disso ciates from the target cell and goes on to bind to another tar get cell. Within a variable period of time(up to a few hours) Tetramer binds after CTL dissociation, the target cell dies by apoptosis. Each of the steps in this process has been studied in detail with loned Ctls complementary CD8 s to selected The TCR-CD3 membrane complex on a CTl recognizes peptide -MHC antigen in association with class I MHC molecules on a tar get cell. After this antigen-specific recognition, the integri Signal measured by flow cytometer FIGURE 14-3 MHC tetramers. a homogeneous population of peptide-bound class I MHC molecules(HLA-Al bound to an HIv derived peptide, for example) is conjugated to biotin and mixed with fluorescently labeled Streptavidin. Four biotinylated MHC-peptide com- Lung 30% lexes bind to the high affinity binding sites of Streptavidin to form a lymph node 0.6% tetramer. Addition of the tetramer to a population of T cells results in Periphera lood 19% Liver 30% exclusive binding of the fluorescent tetramer to those CD8*T cells with TCRs complementary to the peptide-MHC complexes of the tetramer. This results in the labeling of the subpopulation of T cells that are spe- Kidney 41% cific for the target antigen, making them readily detectable by flow cy- Mesenteric tometry. /Adapted in part from P. Klenerman, V. Cerundolo, and P.R. lymph node Dunbar, 2002, Nature Reviews/Immunology 2: 264.] Gut 28% marrow 7%E VSV-specific CD8 cells is far from uniform(Figure 14-4) large populations of antigen-specific cells are not limited to the lymphoid system, but can be found in the liver and kid nev too FIGURE 14.4 Localizing antigen specific CD8* T-cell populations in CTLs Kill Cells in Two Ways vivo Mice were infected with vesicular stomatitis virus (VSv)and dur- ing the course of the acute stage of the infection, cell populations were The effector phase of a CTl-mediated response involves a isolated from the tissues indicated in the figure and incubated with carefully orchestrated sequence of events that begin with the tetramers containing VSV-peptide/MHC complexes. Flow cytometric embrace of the target cell by the attacking cell(Figure 14-5). analysis allowed determination of the percentages of CD8* T cells that Long-term cultures of Ctl clones have been used to identify were VSv-specific in each of the populations examined (Adapted from many of the membrane molecules and membrane events P. Klenerman, V. Cerundolo, and P. R. Dunbar, 2002, Nature Reviews/ involved in this process. As described below, studies with Immunology 2: 269.1 Gotowww.whfreeman.com/immunology Animation Cell Death

VSV-specific CD8+ cells is far from uniform (Figure 14-4); large populations of antigen-specific cells are not limited to the lymphoid system, but can be found in the liver and kid￾ney, too. CTLs Kill Cells in Two Ways The effector phase of a CTL-mediated response involves a carefully orchestrated sequence of events that begin with the embrace of the target cell by the attacking cell (Figure 14-5). Long-term cultures of CTL clones have been used to identify many of the membrane molecules and membrane events involved in this process. As described below, studies with mouse strains carrying mutations that affect the ability of CTLs to induce death have led to the identification of the necessary molecules. The primary events in CTL-mediated death are conjugate formation, membrane attack, CTL dissociation, and target￾cell destruction (Figure 14-6). When antigen-specific CTLs are incubated with appropriate target cells, the two cell types interact and undergo conjugate formation. Formation of a CTL–target cell conjugate is followed within several minutes by a Ca2+-dependent, energy-requiring step in which the CTL programs the target cell for death. The CTL then disso￾ciates from the target cell and goes on to bind to another tar￾get cell. Within a variable period of time (up to a few hours) after CTL dissociation, the target cell dies by apoptosis. Each of the steps in this process has been studied in detail with cloned CTLs. The TCR-CD3 membrane complex on a CTL recognizes antigen in association with class I MHC molecules on a tar￾get cell. After this antigen-specific recognition, the integrin 324 PART III Immune Effector Mechanisms MHC tetramer Signal measured by flow cytometer Purified biotinylated class I MHC Fluorescently labeled Streptavidin Streptavidin Tetramer binds exclusively to TCR complementary to selected peptide-MHC complex Peptide Biotin CD8 Peripheral lymph node 0.6% Spleen 11% Lung 30% Blood and organs Lymphoid tissues and organs Peripheral blood 19% Liver 30% Kidney 41% Gut 28% Mesenteric lymph node 2.5% Bone marrow 7% FIGURE 14-3 MHC tetramers. A homogeneous population of peptide-bound class I MHC molecules (HLA-A1 bound to an HIV￾derived peptide, for example) is conjugated to biotin and mixed with fluorescently labeled Streptavidin. Four biotinylated MHC-peptide com￾plexes bind to the high affinity binding sites of Streptavidin to form a tetramer. Addition of the tetramer to a population of T cells results in exclusive binding of the fluorescent tetramer to those CD8+ T cells with TCRs complementary to the peptide-MHC complexes of the tetramer. This results in the labeling of the subpopulation of T cells that are spe￾cific for the target antigen, making them readily detectable by flow cy￾tometry. [Adapted in part from P. Klenerman, V. Cerundolo, and P. R. Dunbar, 2002, Nature Reviews/Immunology 2:264.] FIGURE 14-4 Localizing antigen specific CD8+ T-cell populations in vivo. Mice were infected with vesicular stomatitis virus (VSV) and dur￾ing the course of the acute stage of the infection, cell populations were isolated from the tissues indicated in the figure and incubated with tetramers containing VSV-peptide/MHC complexes. Flow cytometric analysis allowed determination of the percentages of CD8+ T cells that were VSV-specific in each of the populations examined. [Adapted from P. Klenerman, V. Cerundolo, and P. R. Dunbar, 2002, Nature Reviews/ Immunology 2:269.] Go to www.whfreeman.com/immunology Animation Cell Death

Cell-Mediated Effector Responses CHAPTER 14 325 peptides associated with class I MHC molecules. LFA-1 per sists in the high-avidity state for only 5-10 min after antigen tion of the CTL from the target celi rns to the low-avidity mediated activation and then it ret state. This downshift in LFA-1 avidity may facilitate dissocia- ectron microscopy of cultured Ctl clones reveals the presence of intracellular electron-dense storage granules. These granules have been isolated by fractionation and shown to mediate target-cell damage by themselves. Analysis of their contents revealed 65-kDa monomers of a pore-forming pro- tein called perforin and several serine proteases called gran zymes(or fragmentis). CTL-Ps lack cytoplasmic granules FIGURE 14-5 Scanning electron micrograph of tumor-cell attack and perforin; upon activation, cytoplasmic granules appear, by a ctl. the Ctl makes contact with a smaller tumor cell. From bearing newly expressed perforin monomers. J.D. E. Young and Z. A Cohn, 1988, Sci. Am. 258(1): 38.1 Immediately after formation of a CTL-target cell conju gate, the Golgi stacks and storage granules reorient within the cytoplasm of the Ctl to concentrate near the junction with the target cell(Figure 14-8). Evidence suggests that perforin receptor LFA-1 on the CTl membrane binds to ICAMs on monomers and the granzyme proteases are then released from the target-cell membrane, resulting in the formation of a the granules by exocytosis into the junctional space between conjugate. Antigen-mediated CTl activation converts LFA-1 the two cells. As the perforin monomers contact the target-cell from a low-avidity state to a high-avidity state(Figure 14-7). membrane they undergo a conformational change, exposing Because of this phenomenon, CTLs adhere to and form conju- an amphipathic domain that inserts into the target-cell mem- gates only with appropriate target cells that display antigenic brane; the monomers then polymerize(in the presence of Granule conjugate cytoplasmic rearrangement Target cell Ctl granule exocytosis 一( FIGURE 14-6 Stages in CTL-mediated killing of target cells. T-cell reorient towards the point of contact with the target cell, and the receptors on a CTL interact with processed antigen-class I MHc granule,'s contents are released by exocytosis. After dissociation of complexes on an appropriate target cell, leading to formation of a the conjugate, the CTL is recycled and the target cell dies by apopto. TL/target-cell conjugate. The Golgi stacks and granules in the CTL sis. /Adapted from P. A. Henkart, 1985, Annu. Rev. Immunol. 3: 31

receptor LFA-1 on the CTL membrane binds to ICAMs on the target-cell membrane, resulting in the formation of a conjugate. Antigen-mediated CTL activation converts LFA-1 from a low-avidity state to a high-avidity state (Figure 14-7). Because of this phenomenon, CTLs adhere to and form conju￾gates only with appropriate target cells that display antigenic peptides associated with class I MHC molecules. LFA-1 per￾sists in the high-avidity state for only 5–10 min after antigen￾mediated activation, and then it returns to the low-avidity state. This downshift in LFA-1 avidity may facilitate dissocia￾tion of the CTL from the target cell. Electron microscopy of cultured CTL clones reveals the presence of intracellular electron-dense storage granules. These granules have been isolated by fractionation and shown to mediate target-cell damage by themselves. Analysis of their contents revealed 65-kDa monomers of a pore-forming pro￾tein called perforin and several serine proteases called gran￾zymes (or fragmentins). CTL-Ps lack cytoplasmic granules and perforin; upon activation, cytoplasmic granules appear, bearing newly expressed perforin monomers. Immediately after formation of a CTL–target cell conju￾gate, the Golgi stacks and storage granules reorient within the cytoplasm of the CTL to concentrate near the junction with the target cell (Figure 14-8). Evidence suggests that perforin monomers and the granzyme proteases are then released from the granules by exocytosis into the junctional space between the two cells. As the perforin monomers contact the target-cell membrane, they undergo a conformational change, exposing an amphipathic domain that inserts into the target-cell mem￾brane; the monomers then polymerize (in the presence of Cell-Mediated Effector Responses CHAPTER 14 325 FIGURE 14-5 Scanning electron micrograph of tumor-cell attack by a CTL. The CTL makes contact with a smaller tumor cell. [From J. D. E . Young and Z. A. Cohn, 1988, Sci. Am. 258(1):38.] CTL CTL granule exocytosis Target cell Conjugate formation CTL-target cell conjugate CTL cytoplasmic rearrangement CTL recycling Dissociation Granule FIGURE 14-6 Stages in CTL-mediated killing of target cells. T-cell receptors on a CTL interact with processed antigen-class I MHC complexes on an appropriate target cell, leading to formation of a CTL/target-cell conjugate. The Golgi stacks and granules in the CTL reorient towards the point of contact with the target cell, and the granule’s contents are released by exocytosis. After dissociation of the conjugate, the CTL is recycled and the target cell dies by apopto￾sis. [Adapted from P. A. Henkart, 1985, Annu. Rev. Immunol. 3:31.]

PaRt I Immune Effector Mechanisms tion of the target-cell DNA into oligomers of 200 bp: this gen-activated Ctls ype of DNA fragmentation is typical of apoptosis. Since granzymes are proteases, they cannot directly mediate dNA fragmentation. Rather, they activate an apoptotic pathway within the target cell. This apoptotic process does not require mRNA or protein synthesis in either the Ctl or the target ell. Within 5 min of CTL contact, target cells begin to exhibit DNA fragmentation. Interestingly, viral DNA within infected target cells has also been shown to be fragmented during thi process. This observation shows that CTL-mediated killing Resting CTLs not only kills virus-infected cells but can also destroy the viral DNA in those cells. It has been suggested that the rapid onset of dNA fragmentation after CTl contact may prevent continued viral replication and assembly in the period before the target cell is destroyed FIGURE.7 Effect of antigen activation on the ability of CTLs to bind to the intercellular cell-adhesion molecule ICAM-1. Restin mouse ctls were first incubated with anti-CD3 antibodies crosslink- age of CD3 molecules on the CTL membrane by anti-CD3 has the same activating effect as interaction with antigen-class I MHC com- plexes on a target cell Adhesion was assayed by binding radiolabeled CTLs to microwells coated with ICAM-1. Antigen activation increased CTL binding to ICAM-1 more than 10-fold. The presence of excess monoclonal antibody to LFA-1 or ICAM-1 in the microwell abolished binding, demonstrating that both molecules are necessary for adhe TC sion. Based on M. L. Dustin and T. A. Springer, 1989, Nature 341: 619. b Caz*)to form cylindrical pores with an internal diameter of 5-20 nm(Figure 14-9a). A large number of perforin pores are visible on the target-cell membrane in the region of conju- gate formation(Figure 14-9b) Interestingly, perforin exhibits some sequence homology of the complement system, and the membrane pores formed by perforin are similar to those observed in complement mediated lysis. The importance of perforin to CTL-mediated ling is demonstrated by perforin-deficient knockout mice, which are unable to eliminate lymphocytic choriomeningitis virus(LCMv) even though they mount a significant CD8+ Immu ne response to the virus. d Pore formation in the cell membrane of the target is one way that perforin mediates granzyme entry; another is the FIGURE 14-8 Formation of a conjugate between a CTL and target perforin-assisted pathway Many target cells have a molecule cell and reorientation of CTL cytoplasmic granules as recorded by known as the mannose 6-phosphate receptor on their sur- time-lapse cinematography.(a)A motile mouse CTL( thin arrow)ap- face that also binds to granzyme B. Granzyme B/mannose proaches an appropriate target cell (TC). The thick arrow indicates di 6-phosphate receptor complexes are internalized and appear rection of movement.(b)Initial contact of the CTL and target cell has inside vesicles. In this case, perforin is necessary for releas- occurred. (c)Within 2 min of initial contact, the membrane-contact ing granzyme B from the vesicle into the cytoplasm of the region has broadened and the rearrangement of dark cytoplasmic granules within the CTL(thin arrow) is under way.(d)Further move- Once it enters the cytoplasm of the target cell, granzyme b ment of dark granules toward the target cell is evident 10 min after initiates a cascade of reactions that result in the fragmenta- initial contact /From 1. R. Yanelli et al, 1986, J Immunol. 136: 377)

Ca2+) to form cylindrical pores with an internal diameter of 5–20 nm (Figure 14-9a). A large number of perforin pores are visible on the target-cell membrane in the region of conju￾gate formation (Figure 14-9b). Interestingly, perforin exhibits some sequence homology with the terminal C9 component of the complement system, and the membrane pores formed by perforin are similar to those observed in complement￾mediated lysis. The importance of perforin to CTL-mediated killing is demonstrated by perforin-deficient knockout mice, which are unable to eliminate lymphocytic choriomeningitis virus (LCMV) even though they mount a significant CD8+ immune response to the virus. Pore formation in the cell membrane of the target is one way that perforin mediates granzyme entry; another is the perforin-assisted pathway. Many target cells have a molecule known as the mannose 6-phosphate receptor on their sur￾face that also binds to granzyme B. Granzyme B/mannose 6-phosphate receptor complexes are internalized and appear inside vesicles. In this case, perforin is necessary for releas￾ing granzyme B from the vesicle into the cytoplasm of the target cell. Once it enters the cytoplasm of the target cell, granzyme B initiates a cascade of reactions that result in the fragmenta￾tion of the target-cell DNA into oligomers of 200 bp; this type of DNA fragmentation is typical of apoptosis. Since granzymes are proteases, they cannot directly mediate DNA fragmentation. Rather, they activate an apoptotic pathway within the target cell. This apoptotic process does not require mRNA or protein synthesis in either the CTL or the target cell. Within 5 min of CTL contact, target cells begin to exhibit DNA fragmentation. Interestingly, viral DNA within infected target cells has also been shown to be fragmented during this process. This observation shows that CTL-mediated killing not only kills virus-infected cells but can also destroy the viral DNA in those cells. It has been suggested that the rapid onset of DNA fragmentation after CTL contact may prevent continued viral replication and assembly in the period before the target cell is destroyed. 326 PART III Immune Effector Mechanisms 50 CTLs bound to ICAM-1 coated wells, %40 30 20 10 Antigen-activated CTLs +Ab to LFA-1 Resting CTLs +Ab to ICAM-1 FIGURE 14-7 Effect of antigen activation on the ability of CTLs to bind to the intercellular cell-adhesion molecule ICAM-1. Resting mouse CTLs were first incubated with anti-CD3 antibodies. Crosslink￾age of CD3 molecules on the CTL membrane by anti-CD3 has the same activating effect as interaction with antigen–class I MHC com￾plexes on a target cell. Adhesion was assayed by binding radiolabeled CTLs to microwells coated with ICAM-1. Antigen activation increased CTL binding to ICAM-1 more than 10-fold. The presence of excess monoclonal antibody to LFA-1 or ICAM-1 in the microwell abolished binding, demonstrating that both molecules are necessary for adhe￾sion. [Based on M. L. Dustin and T. A. Springer, 1989, Nature 341:619.] FIGURE 14-8 Formation of a conjugate between a CTL and target cell and reorientation of CTL cytoplasmic granules as recorded by time-lapse cinematography. (a) A motile mouse CTL (thin arrow) ap￾proaches an appropriate target cell (TC). The thick arrow indicates di￾rection of movement. (b) Initial contact of the CTL and target cell has occurred. (c) Within 2 min of initial contact, the membrane-contact region has broadened and the rearrangement of dark cytoplasmic granules within the CTL (thin arrow) is under way. (d) Further move￾ment of dark granules toward the target cell is evident 10 min after initial contact. [From J. R. Yanelli et al., 1986, J. Immunol. 136:377]

Cell-Mediated Effector Responses CHAPTER 14 327 Perforin Nucleus IiiI T Completed P FIGURE. CTL-mediated pore formation in target-cell mem- lows them to insert into the target-cell membrane(4). In the pres- brane. (a)In this model, a rise in intracellular Ca* triggered by CTL- ence of Ca the monomers polymerize within the membrane(5) target cell interaction (1)induces exocytosis, in which the granules forming cylindrical pores( 6).(b)Electron micrograph of perforin fuse with the CTL cell membrane(2)and release monomeric perforin pores on the surface of a rabbit erythrocyte target cell. /Part (a) into the small space between the two cells(3). The released perforin adapted from/ D E. Young and Z A Cohn, 1988, Sci. Am. 258(1): 38 monomers undergo a Ca2-induced conformational change that al- (b)from E. R. Podack and G. Denner, 1983, Nature 301: 442. Some potent CTL lines have been shown to lack perforin pretation CTLs raised from normal mice can kill target cells nd granzymes. In these cases, cytotoxicity is mediated by by a perforin-mediated mechanism, by a mechanism involv Fas As described in Chapter 10, this transmembrane protein, ing engagement of target-cell Fas with Fas ligand displayed which is a member of the TNF-receptor family, can deliver a on the CTl membrane, or, in some cases perhaps, by a com death signal when crosslinked by its natural ligand, a mem- bination of both mechanisms. Such CTls can kill target cells ber of the tumor necrosis family called Fas ligand (see Figure that lack membrane Fas by using the perforin mechanism 10-19). Fas ligand(FasL)is found on the membrane of CTLs, alone. On the other hand, CTls from perforin-knockout and the interaction of FasL with Fas on a target cell triggers mice can kill only by the Fas-FasL mechanism. Consequently o% key insight into the role of perforin and the Fas-FasL sys- mal target cells but not lpr cells, which lack Fas. These work- CTLS from perforin-knockout mice can kill Fas-bearing nor tem in CTL-mediated cytolysis came from experiments with ers also concluded that all of the CTl-mediated killing they mutant mice. These experiments used two types of mutant observed could be traced to the action of perforin-dependent mice, the perforin knockout mice mentioned above and a killing, Fas-mediated killing, or a combination of the two. No strain of mice known as lpr(Figure 14-10). Mice that are other mechanism was detected homozygous for the lpr mutation express little or no Fas and, This experiment taken together with a number of other consequently, cells from these mice cannot be killed by inter- studies shows that two mechanisms are responsible for initi- action with Fas ligand. If lymphocytes from normal H-2 ating all CTL-mediated apoptotic death of target cells mice are incubated with killed cells from h-2k mice. anti H-2 CTLs are generated. These H-2b CTLs will kill target Directional delivery of cytotoxic proteins(perforin cells from normal H-2 mice or from H-2k animals that are and granzymes) that are released from CTLs and enter homozygous for the lpr mutation. Incubation of H-2cells of perforin knockout mice with killed cells from H-2 mice resulted in CTls that killed wild-type target cells but failed to Interaction of the membrane-bound Fas ligand on induce lysis in target cells from H-2 mice homozygous for CTLs with the Fas receptor on the surface of target the lpr mutation. The results of these experiments taken together with other Either of these initiating events results in the activation of a studies allowed the investigators to make the following inter- signaling pathway that culminates in the death of the target cell

Some potent CTL lines have been shown to lack perforin and granzymes. In these cases, cytotoxicity is mediated by Fas. As described in Chapter 10, this transmembrane protein, which is a member of the TNF-receptor family, can deliver a death signal when crosslinked by its natural ligand, a mem￾ber of the tumor necrosis family called Fas ligand (see Figure 10-19). Fas ligand (FasL) is found on the membrane of CTLs, and the interaction of FasL with Fas on a target cell triggers apoptosis. Key insight into the role of perforin and the Fas-FasL sys￾tem in CTL-mediated cytolysis came from experiments with mutant mice. These experiments used two types of mutant mice, the perforin knockout mice mentioned above and a strain of mice known as lpr (Figure 14-10). Mice that are homozygous for the lpr mutation express little or no Fas and, consequently, cells from these mice cannot be killed by inter￾action with Fas ligand. If lymphocytes from normal H-2b mice are incubated with killed cells from H-2k mice, anti￾H-2k CTLs are generated. These H-2b CTLs will kill target cells from normal H-2k mice or from H-2k animals that are homozygous for the lpr mutation. Incubation of H-2b cells of perforin knockout mice with killed cells from H-2k mice resulted in CTLs that killed wild-type target cells but failed to induce lysis in target cells from H-2k mice homozygous for the lpr mutation. The results of these experiments taken together with other studies allowed the investigators to make the following inter￾pretation. CTLs raised from normal mice can kill target cells by a perforin-mediated mechanism, by a mechanism involv￾ing engagement of target-cell Fas with Fas ligand displayed on the CTL membrane, or, in some cases perhaps, by a com￾bination of both mechanisms. Such CTLs can kill target cells that lack membrane Fas by using the perforin mechanism alone. On the other hand, CTLs from perforin-knockout mice can kill only by the Fas-FasL mechanism. Consequently, CTLs from perforin-knockout mice can kill Fas-bearing nor￾mal target cells but not lpr cells, which lack Fas. These work￾ers also concluded that all of the CTL-mediated killing they observed could be traced to the action of perforin-dependent killing, Fas-mediated killing, or a combination of the two. No other mechanism was detected. This experiment taken together with a number of other studies shows that two mechanisms are responsible for initi￾ating all CTL-mediated apoptotic death of target cells: ■ Directional delivery of cytotoxic proteins (perforin and granzymes) that are released from CTLs and enter target cells ■ Interaction of the membrane-bound Fas ligand on CTLs with the Fas receptor on the surface of target cells Either of these initiating events results in the activation of a signaling pathway that culminates in the death of the target cell Cell-Mediated Effector Responses CHAPTER 14 327 (a) (b) Nucleus Granule 2 6 5 4 3 1 Perforin monomers CTL Target cell Polymerized perforin Completed pore Ca2 FIGURE 14-9 CTL-mediated pore formation in target-cell mem￾brane. (a) In this model, a rise in intracellular Ca2+ triggered by CTL￾target cell interaction (1) induces exocytosis, in which the granules fuse with the CTL cell membrane (2) and release monomeric perforin into the small space between the two cells (3). The released perforin monomers undergo a Ca2+ -induced conformational change that al￾lows them to insert into the target-cell membrane (4). In the pres￾ence of Ca2+ , the monomers polymerize within the membrane (5), forming cylindrical pores (6). (b) Electron micrograph of perforin pores on the surface of a rabbit erythrocyte target cell. [Part (a) adapted from J. D. E. Young and Z. A. Cohn, 1988, Sci. Am. 258(1):38; part (b) from E. R. Podack and G. Dennert, 1983, Nature 301:442.]

328 PART I Immune Effector Mechanisms (a) Generation of CTls Normal H-2b Normal h-2k Perforin knockout h-2b Normal h-2k Lymphocyt Lymphocytes Lymphocytes Lymphocytes Mitomycin C Killed lymphocytes Killed lymphocytes bk④b(kb ④b(kb(k(b Normal h-2b anti-H-2k ctls Perforin knockout H-2b anti-H-2k ctls (b)Interaction of CTls with Fast and Fas" targets Normal lpr mutant H-2k CTIs (no Fas) Normal H-2b anti-H-2k Killed Killed Perforin knockout H-2b anti-H-2kKilled urvive FIGURE 14-10 Experimental demonstration that CTLS use Fas by stimulation of lymphocytes from perforin knockout(KO)mice. and perforin pathways. (a) Generation of CTLS. Lymphocytes were they expressed Fas ligand but not perforin. (b)Interaction of CTLS harvested from mice of H-2and H-2 MHC haplotypes H-2 haplo- with Fas* and Fas" targets. Normal H-2 anti-H-2 CTLs that express type cells were killed by treatment with mitomycin C and co-cultured both Fas ligand and perforin kill normal H-2 target cells and H-2"lpr with H-2 haplotype cells to stimulate the generation of H-2 CTLS. If mutant cells, which do not express Fas. In contrast, H-2 anti-H-2k the H-2 lymphocytes were derived from normal mice, they gave rise CTLs from perforin KO mice kill Fas* normal cells by engagement of to CTLs that had both perforin and Fas ligand. If the CTLs were raised Fas with Fas ligand but are unable to kill the lpr cells, which lack Fas by apoptosis(Figure 14-11). A feature of cell death by apopto- get cell by Fas ligand on the ctl causes the activation of an ini sis is the involvement of the caspase family of cysteine pro- tiator caspase in the target cell. Fas is associated with a protein teases, which cleave after an aspartic acid residue. The name known as FADD(Fas-associated protein with death domain) caspase incorporates all of these elements(cysteine, aspartate which in turn associates with a procaspase form of caspase 8 protease). Normally, caspases are present in the cell as inactive Upon Fas crosslinking, procaspase 8 is converted to caspase 8 proenzymes-procaspases-that require proteolytic cleavage and initiates an apoptotic caspase cascade. The end result of for conversion to the active forms. More than a dozen different both the perforin/granzyme and Fas-mediated pathways is the caspases have been found, each with its own specificity Cleav- activation of dormant death pathways that are present in the age of a procaspase produces an active initiator caspase, which target cell. As one immunologist has so aptly put it, CTls dont cleaves other procaspases, thereby activating their proteolytic so much kill target cells as persuade them to commit suicide activity. The end result is the systematic and orderly disassem bly of the cell that is the hallmark of apoptosis. CTLs use granzymes and Fas ligand to initiate caspase cas. Natural Killer Cells cades in their targets. The granzymes introduced into the tar- t cell from the Ctl mediate proteolytic events that activate Natural killer cells were discovered quite by accident when an initiator caspase. Similarly, the engagement of Fas on a tar- immunologists were measuring in vitro activity of tumor-

by apoptosis (Figure 14-11). A feature of cell death by apopto￾sis is the involvement of the caspase family of cysteine pro￾teases, which cleave after an aspartic acid residue. The name caspase incorporates all of these elements (cysteine, aspartate protease). Normally, caspases are present in the cell as inactive proenzymes—procaspases—that require proteolytic cleavage for conversion to the active forms. More than a dozen different caspases have been found, each with its own specificity. Cleav￾age of a procaspase produces an active initiator caspase, which cleaves other procaspases, thereby activating their proteolytic activity. The end result is the systematic and orderly disassem￾bly of the cell that is the hallmark of apoptosis. CTLs use granzymes and Fas ligand to initiate caspase cas￾cades in their targets. The granzymes introduced into the tar￾get cell from the CTL mediate proteolytic events that activate an initiator caspase. Similarly, the engagement of Fas on a tar￾get cell by Fas ligand on the CTL causes the activation of an ini￾tiator caspase in the target cell. Fas is associated with a protein known as FADD (Fas-associated protein with death domain), which in turn associates with a procaspase form of caspase 8. Upon Fas crosslinking, procaspase 8 is converted to caspase 8 and initiates an apoptotic caspase cascade. The end result of both the perforin/granzyme and Fas-mediated pathways is the activation of dormant death pathways that are present in the target cell. As one immunologist has so aptly put it, CTLs don’t so much kill target cells as persuade them to commit suicide. Natural Killer Cells Natural killer cells were discovered quite by accident when immunologists were measuring in vitro activity of tumor- 328 PART III Immune Effector Mechanisms Normal H-2b Normal H-2b anti-H-2k CTLs Normal H-2b anti-H-2k Perforin knockout H-2b anti-H-2k Normal H-2k Killed Killed Killed Survive CTLs lpr mutant H-2k (no Fas) Lymphocytes Normal H-2k Lymphocytes Mitomycin C Killed lymphocytes Killed lymphocytes Culture (a) Generation of CTLs (b) Interaction of CTLs with Fas+ and Fas– targets b k b k b Perforin knockout H-2b Perforin knockout H-2b anti-H-2k CTLs Lymphocytes Normal H-2k Lymphocytes Mitomycin C Culture b k b k b Target cells FIGURE 14-10 Experimental demonstration that CTLs use Fas and perforin pathways. (a) Generation of CTLs. Lymphocytes were harvested from mice of H-2b and H-2k MHC haplotypes. H-2k haplo￾type cells were killed by treatment with mitomycin C and co-cultured with H-2b haplotype cells to stimulate the generation of H-2k CTLs. If the H-2b lymphocytes were derived from normal mice, they gave rise to CTLs that had both perforin and Fas ligand. If the CTLs were raised by stimulation of lymphocytes from perforin knockout (KO) mice, they expressed Fas ligand but not perforin. (b) Interaction of CTLs with Fas+ and Fas– targets. Normal H-2b anti-H-2k CTLs that express both Fas ligand and perforin kill normal H-2k target cells and H-2k lpr mutant cells, which do not express Fas. In contrast, H-2b anti-H-2k CTLs from perforin KO mice kill Fas+ normal cells by engagement of Fas with Fas ligand but are unable to kill the lpr cells, which lack Fas