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 kidney, 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 targetcell 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 dissociates from the target cell and goes on to bind to another target 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 target 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 HIVderived peptide, for example) is conjugated to biotin and mixed with fluorescently labeled Streptavidin. Four biotinylated MHC-peptide complexes 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 specific for the target antigen, making them readily detectable by flow cytometry. [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 during 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 conjugates only with appropriate target cells that display antigenic peptides associated with class I MHC molecules. LFA-1 persists in the high-avidity state for only 5–10 min after antigenmediated activation, and then it returns to the low-avidity state. This downshift in LFA-1 avidity may facilitate dissociation 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 protein called perforin and several serine proteases called granzymes (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 conjugate, 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 membrane; 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 apoptosis. [Adapted from P. A. Henkart, 1985, Annu. Rev. Immunol. 3:31.]