免疫学（英文版）：Chapter 10

As indicated in Chapter 2, the thymus occupies a central role in T-cell biology. Aside from being the main source of all T cells, it is where T cells diversify and then are shaped into an effective primary T-cell repertoire by an extraordinary pair of selection processes. One of these, positive selection, permits the survival of only those T cells whose TCRs are capable of recognizing self-MHC molecules. It is thus responsible for the creation of a self-MHC-restricted repertoire of T cells. The other, negative selection, eliminates T cells that react too strongly with self-MHC or with self-MHC plus self- peptides. It is an extremely important factor in generating a primary T-cell repertoire that is self-tolerant. As shown in Figure 10-1, when T-cell precursors arrive at the thymus, they do not express such signature surface mark- ers of T cells as the T-cell receptor, the CD3 complex, or the coreceptors CD4 and CD8. In fact, these progenitor cells have chapter 10 a73 T-Cell Maturation and the Thymus a73 Thymic Selection of the T-Cell Repertoire a73 T H -Cell Activation a73 T-Cell Differentiation a73 Cell Death and T-Cell Populations a73 Peripheral H9253H9254 T-Cells T-Cell Maturation, Activation, and Differentiation T ?? ????????? ???? ????????????? ??????? recognition by most T cells from recognition by B cells is MHC restriction. In most cases, both the maturation of progenitor T cells in the thymus and the acti- vation of mature T cells in the periphery are influenced by the involvement of MHC molecules. The potential antigenic diversity of the T-cell population is reduced during matura- tion by a selection process that allows only MHC-restricted and nonself-reactive T cells to mature. The final stages in the maturation of most T cells proceed along two different de- velopmental pathways, which generate functionally distinct CD4 H11001 and CD8 H11001 subpopulations that exhibit class II and class I MHC restriction, respectively. Activation of mature peripheral T cells begins with the interaction of the T-cell receptor (TCR) with an antigenic peptide displayed in the groove of an MHC molecule. Al- though the specificity of this interaction is governed by the TCR, its low avidity necessitates the involvement of corecep- tors and other accessory membrane molecules that strengthen the TCR-antigen-MHC interaction and trans- duce the activating signal. Activation leads to the prolifera- tion and differentiation of T cells into various types of effector cells and memory T cells. Because the vast majority of thymocytes and peripheral T cells express the H9251H9252 T-cell receptor rather than the H9253H9254 T-cell receptor, all references to the T-cell receptor in this chapter denote the H9251H9252 receptor un- less otherwise indicated. Similarly, unless otherwise indi- cated, all references to T cells denote those H9251H9252 receptor- bearing T cells that undergo MHC restriction. T-Cell Maturation and the Thymus Progenitor T cells from the early sites of hematopoiesis begin to migrate to the thymus at about day 11 of gestation in mice and in the eighth or ninth week of gestation in humans. In a manner similar to B-cell maturation in the bone marrow, T- cell maturation involves rearrangements of the germ-line TCR genes and the expression of various membrane mark- ers. In the thymus, developing T cells, known as thymocytes, proliferate and differentiate along developmental pathways that generate functionally distinct subpopulations of mature T cells. Engagement of TcR by Peptide: MHC Initiates Signal Transduction ζζ γδε 8536d_ch10_221 8/29/02 2:03 PM Page 221 mac83 Mac 83:379_kyw: not yet rearranged their TCR genes and do not express pro- teins, such as RAG-1 and RAG-2, that are required for re- arrangement. After arriving at the thymus, these T-cell precursors enter the outer cortex and slowly proliferate. Dur- ing approximately three weeks of development in the thy- mus, the differentiating T cells progress through a series of stages that are marked by characteristic changes in their cell- surface phenotype. For example, as mentioned previously, thymocytes early in development lack detectable CD4 and CD8. Because these cells are CD4 H11002 CD8 H11002 , they are referred to as double-negative (DN) cells. Even though these coreceptors are not expressed during the DN early stages, the differentiation program is progress- ing and is marked by changes in the expression of such cell surface molecules as c-Kit, CD44, and CD25. The initial thy- mocyte population displays c-Kit, the receptor for stem-cell growth factor, and CD44, an adhesion molecule involved in homing; CD25, the H9252-chain of the IL-2 receptor, also appears 222 PART II Generation of B-Cell and T-Cell Responses on early-stage DN cells. During this period, the cells are pro- liferating but the TCR genes remain unrearranged. Then the cells stop expressing c-Kit, markedly reduce CD44 expres- sion, turn on expression of the recombinase genes RAG-1 and RAG-2 and begin to rearrange their TCR genes. Al- though it is not shown in Figure 10-1, a small percentage (H110215%) of thymocytes productively rearrange the H9253- and H9254-chain genes and develop into double-negative CD3 H11001 H9253H9254 T cells. In mice, this thymocyte subpopulation can be detected by day 14 of gestation, reaches maximal numbers between days 17 and 18, and then declines until birth (Figure 10-2). Most double-negative thymocytes progress down the H9251H9252 developmental pathway. They stop proliferating and begin to rearrange the TCR H9252-chain genes, then express the H9252 chain. Those cells of the H9251H9252 lineage that fail to productively re- arrange and express H9252 chains die. Newly synthesized H9252 chains combine with a 33-kDa glycoprotein known as the pre-TH9251 chain and associate with the CD3 group to form a novel com- VISUALIZING CONCEPTS FIGURE 10-1 Development of H9251H9252 T cells in the mouse. T-cell precursors arrive at the thymus from bone mar- row via the bloodstream, undergo de- velopment to mature T cells, and are exported to the periphery where they can undergo antigen-induced activa- tion and differentiation into effector cells and memory cells. Each stage of development is characterized by stage-specific intracellular events and the display of distinctive cell-surface markers. Hematopoietic stem cell (HSC) Common lymphoid precursor T-cell precursor c-Kit CD3 CD44 Pre-T α TCR β chain TCR α chain CD4 and CD8 CD4 or CD8 CD4+CD8+ CD4+CD8+ CD25 Pro-T cell (double negative, DN) Pre-T cell (double negative, DN) Pro-T cell (double positive, DP) migration migration Surface markers Peripheral tissues Marrow Blood Blood Thymus RAG expression on D β -J β V β -D β -J β V β -D β -J β and V α -J β TCR locus rearrangement T c cell 8536d_ch10_221 8/27/02 1:37 PM Page 222 Mac 109 Mac 109:1254_BJN:Goldsby et al. / Immunology 5e: plex called the pre-T-cell receptor or pre-TCR (Figure 10-3). Some researchers have suggested that the pre-TCR recog- nizes some intra-thymic ligand and transmits a signal through the CD3 complex that activates signal-transduction pathways that have several effects: a73 Indicates that a cell has made a productive TCR H9252-chain rearrangement and signals its further proliferation and maturation. T-Cell Maturation, Activation, and Differentiation CHAPTER 10 223 a73 Suppresses further rearrangement of TCR H9252-chain genes, resulting in allelic exclusion. a73 Renders the cell permissive for rearrangement of the TCR H9251 chain. a73 Induces developmental progression to the CD4 H11001 8 H11001 double-positive state. After advancing to the double-positive (DP) stage, where both CD4 and CD8 coreceptors are expressed, the thymo- cytes begin to proliferate. However, during this proliferative phase, TCR H9251-chain gene rearrangement does not occur; both the RAG-1 and RAG-2 genes are transcriptionally ac- tive, but the RAG-2 protein is rapidly degraded in proliferat- ing cells, so rearrangement of the H9251-chain genes cannot take place. The rearrangement of H9251-chain genes does not begin until the double-positive thymocytes stop proliferating and RAG-2 protein levels increase. The proliferative phase prior to the rearrangement of the H9251-chain increases the diversity of the T-cell repertoire by generating a clone of cells with a sin- gle TCR H9252-chain rearrangement. Each of the cells within this clone can then rearrange a different H9251-chain gene, thereby generating a much more diverse population than if the orig- inal cell had first undergone rearrangement at both the H9252- and H9251-chain loci before it proliferated. In mice, the TCR H9251- chain genes are not expressed until day 16 or 17 of gestation; double-positive cells expressing both CD3 and the H9251H9252 T-cell receptor begin to appear at day 17 and reach maximal levels about the time of birth (see Figure 10-2). The possession of a complete TCR enables DP thymocytes to undergo the rigors of positive and negative selection. T-cell development is an expensive process for the host. An estimated 98% of all thymocytes do not mature—they die by apoptosis within the thymus either because they fail to make a productive TCR-gene rearrangement or because they fail to survive thymic selection. Double-positive thymocytes that express the H9251H9252 TCR-CD3 complex and survive thymic selection develop into immature single-positive CD4 H11545 thymocytes or single-positive CD8 H11545 thymocytes. These single-positive cells undergo additional negative selection and migrate from the cortex to the medula, where they pass from the thymus into the circulatory system. Thymic Selection of the T-Cell Repertoire Random gene rearrangement within TCR germ-line DNA combined with junctional diversity can generate an enor- mous TCR repertoire, with an estimated potential diversity exceeding 10 15 for the H9251H9252 receptor and 10 18 for the H9253H9254 recep- tor. Gene products encoded by the rearranged TCR genes have no inherent affinity for foreign antigen plus a self-MHC mol- ecule; they theoretically should be capable of recognizing sol- uble antigen (either foreign or self), self-MHC molecules, or FIGURE 10-2 Time course of appearance of H9253H9254 thymocytes and H9251H9252 thymocytes during mouse fetal development. The graph shows the percentage of CD3 H11001 cells in the thymus that are double-negative (CD4 H11002 8 H11002 ) and bear the H9253H9254 T-cell receptor (black) or are double- positive (CD4 H11001 8 H11001 ) and bear the H9251H9252 T-cell receptor (blue). FIGURE 10-3 Structure and activity of the pre–T-cell receptor (pre- TCR). Binding of ligands yet to be identified to the pre-TCR generates intracellular signals that induce a variety of processes. 100 75 50 25 0 17 18161514 CD 3 + cells, % Days of gestation 19 Birth Adult αβ Thymocytes γδ Thymocytes Pre-TCR TCR β H9280 H9280 δ γ ? ? Pre-Tα Signals Cell becomes permissive for TCR α-chain locus arrangement Stimulates expression of CD4 and CD8 coreceptors Stimulates proliferation Stops additional TCR β-chain locus arrangements (allelic exclusion) SS SS SS SS 8536d_ch10_221-247 8/28/02 3:58 PM Page 223 mac76 mac76:385_reb: antigen plus a nonself-MHC molecule. Nonetheless, the most distinctive property of mature T cells is that they recognize only foreign antigen combined with self-MHC molecules. As noted, thymocytes undergo two selection processes in the thymus: a73 Positive selection for thymocytes bearing receptors capable of binding self-MHC molecules, which results in MHC restriction. Cells that fail positive selection are eliminated within the thymus by apoptosis. a73 Negative selection that eliminates thymocytes bearing high-affinity receptors for self-MHC molecules alone or self-antigen presented by self-MHC, which results in self-tolerance. Both processes are necessary to generate mature T cells that are self-MHC restricted and self-tolerant. As noted already, some 98% or more of all thymocytes die by apoptosis within the thymus. The bulk of this high death rate appears to reflect a weeding out of thymocytes that fail positive selection be- cause their receptors do not specifically recognize foreign antigen plus self-MHC molecules. Early evidence for the role of the thymus in selection of the T-cell repertoire came from chimeric mouse experi- ments by R. M. Zinkernagel and his colleagues (Figure 10-4). These researchers implanted thymectomized and ir- radiated (A H11003 B) F 1 mice with a B-type thymus and then reconstituted the animal’s immune system with an intra- venous infusion of F 1 bone-marrow cells. To be certain that the thymus graft did not contain any mature T cells, it was irradiated before being transplanted. In such an experi- mental system, T-cell progenitors from the (A H11003 B) F 1 bone-marrow transplant mature within a thymus that ex- presses only B-haplotype MHC molecules on its stromal cells. Would these (A H11003 B) F 1 T cells now be MHC- restricted for the haplotype of the thymus? To answer this question, the chimeric mice were infected with LCM virus and the immature T cells were then tested for their ability to kill LCM-infected target cells from the strain A or strain B mice. As shown in Figure 10-4, when T C cells from the chimeric mice were tested on LCM virus infected target cells from strain A or strain B mice, they could only lyse LCM-infected target cells from strain B mice. These mice have the same MHC haplotype, B, as the implanted thymus. Thus, the MHC haplotype of the thymus in which T cells develop determines their MHC restriction. Thymic stromal cells, including epithelial cells, macro- phages, and dendritic cells, play essential roles in positive and negative selection. These cells express class I MHC molecules and can display high levels of class II MHC also. The interac- tion of immature thymocytes that express the TCR-CD3 complex with populations of thymic stromal cells results in positive and negative selection by mechanisms that are under intense investigation. First, we’ll examine the details of each selection process and then study some experiments that pro- vide insights into the operation of these processes. Positive Selection Ensures MHC Restriction Positive selection takes place in the cortical region of the thy- mus and involves the interaction of immature thymocytes with cortical epithelial cells (Figure 10-5). There is evidence that the T-cell receptors on thymocytes tend to cluster with 224 PART II Generation of B-Cell and T-Cell Responses FIGURE 10-4 Experimental demonstration that the thymus selects for maturation only those T cells whose T-cell receptors recognize antigen presented on target cells with the haplotype of the thymus. Thymectomized and lethally irradiated (A H11003 B) F 1 mice were grafted with a strain-B thymus and reconstituted with (A H11003 B) F 1 bone- marrow cells. After infection with the LCM virus, the CTL cells were assayed for their ability to kill 51 Cr-labeled strain-A or strain-B target cells infected with the LCM virus. Only strain-B target cells were lysed, suggesting that the H-2 b grafted thymus had selected for maturation only those T cells that could recognize antigen combined with H-2 b MHC molecules. Lethal x-irradiation Thymectomy EXPERIMENT (A × B)F 1 (H–2 a/b ) Strain-B thymus graft (H–2 b ) (A × B)F 1 hematopoietic stem cells (H–2 a/b ) Infect with LCM virus Spleen cells CONTROL Infect with LCM virus (A × B)F 1 Spleen cells Killing Killing LCM-infected strain-B cells LCM-infected strain-A cells No killing Killing LCM-infected strain-B cells LCM-infected strain-A cells 1 2 8536d_ch10_221-247 8/29/02 10:23 AM Page 224 mac114 Mac 114:2nd shift:1268_tm:8536d: MHC molecules on the cortical cells at sites of cell-cell con- tact. Some researchers have suggested that these interactions allow the immature thymocytes to receive a protective signal that prevents them from undergoing cell death; cells whose receptors are not able to bind MHC molecules would not in- teract with the thymic epithelial cells and consequently would not receive the protective signal, leading to their death by apoptosis. During positive selection, the RAG-1, RAG-2, and TdT proteins required for gene rearrangement and modification continue to be expressed. Thus each of the immature thymo- cytes in a clone expressing a given H9252 chain have an opportu- nity to rearrange different TCR H9251-chain genes, and the resulting TCRs are then selected for self-MHC recognition. Only those cells whose H9251H9252 TCR heterodimer recognizes a self-MHC molecule are selected for survival. Consequently, the presence of more than one combination of H9251H9252 TCR chains among members of the clone is important because it increases the possibility that some members will “pass” the test for positive selection. Any cell that manages to rearrange an H9251chain that allows the resulting H9251H9252 TCR to recognize self- MHC will be spared; all members of the clone that fail to do so will die by apoptosis within 3 to 4 days. Negative Selection Ensures Self-Tolerance The population of MHC-restricted thymocytes that survive positive selection comprises some cells with low-affinity re- ceptors for self-antigen presented by self-MHC molecules and other cells with high-affinity receptors. The latter thy- mocytes undergo negative selection by an interaction with thymic stromal cells. During negative selection, dendritic cells and macrophages bearing class I and class II MHC mol- ecules interact with thymocytes bearing high-affinity recep- tors for self-antigen plus self-MHC molecules or for self-MHC molecules alone (see Figure 10-5). However, the precise details of the process are not yet known. Cells that ex- perience negative selection are observed to undergo death by apoptosis. Tolerance to self-antigens encountered in the thy- mus is thereby achieved by eliminating T cells that are reac- tive to these antigens. Experiments Revealed the Essential Elements of Positive and Negative Selection Direct evidence that binding of thymocytes to class I or class II MHC molecules is required for positive selection in the thymus came from experimental studies with knockout mice incapable of producing functional class I or class II MHC molecules (Table 10-1). Class I–deficient mice were found to have a normal distribution of double-negative, double-posi- tive, and CD4 H11001 thymocytes, but failed to produce CD8 H11001 thy- mocytes. Class II–deficient mice had double-negative, double-positive, and CD8 H11001 thymocytes but lacked CD4 H11001 thymocytes. Not surprisingly, the lymph nodes of these class II–deficient mice lacked CD4 H11001 T cells. Thus, the absence of class I or II MHC molecules prevents positive selection of CD8 H11001 or CD4 H11001 T cells, respectively. Further experiments with transgenic mice provided addi- tional evidence that interaction with MHC molecules plays a role in positive selection. In these experiments, rearranged H9251H9252-TCR genes derived from a CD8 H11001 T-cell clone specific for influenza antigen plus H-2 k class I MHC molecules were in- jected into fertilized eggs from two different mouse strains, T-Cell Maturation, Activation, and Differentiation CHAPTER 10 225 FIGURE 10-5 Positive and negative selection of thymocytes in the thymus. Thymic selection involves thymic stromal cells (epithelial cells, dendritic cells, and macrophages), and results in mature T cells that are both self-MHC restricted and self-tolerant. T-cell receptor Immature thymocyte Positive selection of cells whose receptor binds MHC molecules Death by apoptosis of cells that do not interact with MHC molecules CD8 CD3 CD4 T-cell precursor Class I and/or class II MHC molecules Epithelial cell Rearrangement of TCR genes Negative selection and death of cells with high-affinity receptors for self-MHC or self-MHC + self-antigen CD4 + CD8 + T H cell T C cell Mature CD4 + or CD8 + T lymphocytes Macrophage Dendritic cell 8536d_ch10_221-247 8/28/02 3:58 PM Page 225 mac76 mac76:385_reb: one with the H-2 k haplotype and one with the H-2 d haplo- type (Figure 10-6). Since the receptor transgenes were al- ready rearranged, other TCR-gene rearrangements were suppressed in the transgenic mice; therefore, a high percent- age of the thymocytes in the transgenic mice expressed the T-cell receptor encoded by the transgene. Thymocytes expressing the TCR transgene were found to mature into CD8 H11001 T cells only in the transgenic mice with the H-2 k class I MHC haplotype (i.e., the haplotype for which the transgene receptor was restricted). In transgenic mice with a different MHC haplotype (H-2 d ), immature, double-positive thymo- cytes expressing the transgene were present, but these thy- mocytes failed to mature into CD8 H11001 T cells. These findings confirmed that interaction between T-cell receptors on im- mature thymocytes and self-MHC molecules is required for positive selection. In the absence of self-MHC molecules, as in the H-2 d transgenic mice, positive selection and subse- quent maturation do not occur. Evidence for deletion of thymocytes reactive with self- antigen plus MHC molecules comes from a number of ex- perimental systems. In one system, thymocyte maturation was analyzed in transgenic mice bearing an H9251H9252 TCR trans- gene specific for the class I D b MHC molecule plus H-Y anti- gen, a small protein that is encoded on the Y chromosome and is therefore a self-molecule only in male mice. In this ex- periment, the MHC haplotype of the transgenic mice was H-2 b , the same as the MHC restriction of the transgene- encoded receptor. Therefore any differences in the selection of thymocytes in male and female transgenics would be re- lated to the presence or absence of H-Y antigen. Analysis of thymocytes in the transgenic mice revealed that female mice contained thymocytes expressing the H-Y– specific TCR transgene, but male mice did not (Figure 10-7). In other words, H-Y–reactive thymocytes were self-reactive in the male mice and were eliminated. However, in the female transgenics, which did not express the H-Y antigen, these cells were not self-reactive and thus were not eliminated. When thymocytes from these male transgenic mice were cul- tured in vitro with antigen-presenting cells expressing the H-Y antigen, the thymocytes were observed to undergo apoptosis, providing a striking example of negative selection. Some Central Issues in Thymic Selection Remain Unresolved Although a great deal has been learned about the develop- mental processes that generate mature CD4 H11001 and CD8 H11001 T cells, some mysteries persist. Prominent among them is a seeming paradox: If positive selection allows only thymo- cytes reactive with self-MHC molecules to survive, and nega- tive selection eliminates the self-MHC–reactive thymo- cytes, then no T cells would be allowed to mature. Since this is not the outcome of T-cell development, clearly, other fac- tors operate to prevent these two MHC-dependent processes from eliminating the entire repertoire of MHC-restricted T cells. Experimental evidence from fetal thymic organ culture (FTOC) has been helpful in resolving this puzzle. In this sys- tem, mouse thymic lobes are excised at a gestational age of day 16 and placed in culture. At this time, the lobes consist pre- dominantly of CD4 H11002 8 H11002 thymocytes. Because these immature, double-negative thymocytes continue to develop in the organ culture, thymic selection can be studied under conditions that permit a range of informative experiments. Particular use has 226 PART II Generation of B-Cell and T-Cell Responses TABLE 10-1 Effect of class I or II MHC deficiency on thymocyte populations * KNOCKOUT MICE Control Class I Class II Cell type mice deficient deficient CD4 H11002 CD8 H11002 H11001H11001 H11001 CD4 H11001 CD8 H11001 H11001H11001 H11001 CD4 H11001 H11001H11001 H11002 CD8 H11001 H11001H11002 H11002 * Plus sign indicates normal distribution of indicated cell types in thymus. Minus sign indicates absence of cell type. FIGURE 10-6 Effect of host haplotype on T-cell maturation in mice carrying transgenes encoding an H-2 b class I–restricted T-cell recep- tor specific for influenza virus. The presence of the rearranged TCR transgenes suppressed other gene rearrangements in the transgen- ics; therefore, most of the thymocytes in the transgenics expressed the H9251H9252 T-cell receptor encoded by the transgene. Immature double- positive thymocytes matured into CD8 H11001 T cells only in transgenics with the haplotype (H-2 k ) corresponding to the MHC restriction of the TCR transgene. Thymocytes in transgenics TCR + /CD4 + 8 + TCR + /CD8 + H–2 k transgenic + + + ? Influenza- infected target cell T C -cell clone (H-2 k ) CD8 Class I MHC (H-2 k ) αβ-TCR genes H–2 d transgenic 8536d_ch10_221-247 8/28/02 3:58 PM Page 226 mac76 mac76:385_reb: been made of mice in which the peptide transporter, TAP-1, has been knocked out. In the absence of TAP-1, only low levels of MHC class I are expressed on thymic cells, and the develop- ment of CD8 H11001 thymocytes is blocked. However, when exoge- nous peptides are added to these organ cultures, then peptide-bearing class I MHC molecules appear on the surface of the thymic cells, and development of CD8 H11001 T cells is re- stored. Significantly, when a diverse peptide mixture is added, the extent of CD8 H11001 T-cell restoration is greater than when a single peptide is added. This indicates that the role of peptide is not simply to support stable MHC expression but also to be recognized itself in the selection process. Two competing hypotheses attempt to explain the para- dox of MHC-dependent positive and negative selection. The avidity hypothesis asserts that differences in the strength of the signals received by thymocytes undergoing positive and negative selection determine the outcome, with signal strength dictated by the avidity of the TCR-MHC-peptide in- teraction. The differential-signaling hypothesis holds that the outcomes of selection are dictated by different signals, rather than different strengths of the same signal. The avidity hypothesis was tested with TAP-1 knockout mice transgenic for an H9251H9252TCR that recognized an LCM virus peptide-MHC complex. These mice were used to prepare fe- tal thymic organ cultures (Figure 10-8). The avidity of the TCR-MHC interaction was varied by the use of different concentrations of peptide. At low peptide concentrations, few MHC molecules bound peptide and the avidity of the TCR-MHC interaction was low. As peptide concentrations were raised, the number of peptide-MHC complexes dis- played increased and so did the avidity of the interaction. In this experiment, very few CD8 H11001 cells appeared when peptide was not added, but even low concentrations of the relevant peptide resulted in the appearance of significant numbers of CD8 H11001 T cells bearing the transgenic TCR receptor. Increas- ing the peptide concentrations to an optimum range yielded the highest number of CD8 H11001 T cells. However, at higher con- centrations of peptide, the numbers of CD8 H11001 T cells pro- duced declined steeply. The results of these experiments show that positive and negative selection can be achieved with signals generated by the same peptide-MHC combina- tion. No signal (no peptide) fails to support positive selec- tion. A weak signal (low peptide level) induces positive selection. However, too strong a signal (high peptide level) results in negative selection. The differential-signaling model provides an alternative explanation for determining whether a T cell undergoes posi- tive or negative selection. This model is a qualitative rather than a quantitative one, and it emphasizes the nature of the signal delivered by the TCR rather than its strength. At the core of this model is the observation that some MHC-peptide complexes can deliver only a weak or partly activating signal T-Cell Maturation, Activation, and Differentiation CHAPTER 10 227 FIGURE 10-7 Experimental demonstration that negative selection of thymocytes requires self-anti- gen plus self-MHC. In this experiment, H-2 b male and female transgenics were prepared carrying TCR transgenes specific for H-Y antigen plus the D b mol- ecule. This antigen is expressed only in males. FACS analysis of thymocytes from the transgenics showed that mature CD8 H11001 T cells expressing the transgene were absent in the male mice but present in the fe- male mice, suggesting that thymocytes reactive with a self-antigen (in this case, H-Y antigen in the male mice) are deleted during thymic selection. [Adapted from H. von Boehmer and P. Kisielow, 1990, Science 248:1370.] Use to make α H-Y TCR transgenic mice Male H-2D b Female H-2D b H-Y expression Thymocytes CD4 ? 8 ? CD4 + 8 + CD4 + CD8 + + ++ + + ? ? + ++ + ++ Clone TCR α and β genes Female cell (H-2D b )Male cell (H-2D b ) CTL H-Y specific H-2D b restricted H-Y peptide αβ × 8536d_ch10_221-247 8/28/02 3:58 PM Page 227 mac76 mac76:385_reb: while others can deliver a complete signal. In this model, pos- itive selection takes place when the TCRs of developing thy- mocytes encounter MHC-peptide complexes that deliver weak or partial signals to their receptors, and negative selec- tion results when the signal is complete. At this point it is not possible to decide between the avidity model and the differen- tial-signaling model; both have experimental support. It may be that in some cases, one of these mechanisms operates to the complete exclusion of the other. It is also possible that no sin- gle mechanism accounts for all the outcomes in the cellular interactions that take place in the thymus and more than one mechanism may play a significant role. Further work is re- quired to complete our understanding of this matter. The differential expression of the coreceptor CD8 also can affect thymic selection. In an experiment in which CD8 ex- pression was artificially raised to twice its normal level, the concentration of mature CD8 H11001 cells in the thymus was one- thirteenth of the concentration in control mice bearing nor- mal levels of CD8 on their surface. Since the interaction of T cells with class I MHC molecules is strengthened by partici- pation of CD8, perhaps the increased expression of CD8 would increase the avidity of thymocytes for class I mole- cules, possibly making their negative selection more likely. Another important open question in thymic selection is how double-positive thymocytes are directed to become ei- ther CD4 H11001 8 H11002 or CD4 H11002 8 H11001 T cells. Selection of CD4 H11001 8 H11001 thy- mocytes gives rise to class I MHC–restricted CD8 H11001 T cells and class II–restricted CD4 H11001 T cells. Two models have been proposed to explain the transformation of a double-positive precursor into one of two different single-positive lineages 228 PART II Generation of B-Cell and T-Cell Responses FIGURE 10-8 Role of peptides in selection. Thymuses harvested before their thymocyte populations have undergone positive and negative selection allow study of the develop- ment and selection of single positive (CD4 H11001 CD8 H11002 and CD4 H11002 CD8 H11001 ) T cells. (a) Outline of the experimental procedure for in vitro fetal thymic organ culture (FTOC). (b) The development and selection of CD8 H11001 CD4 H11002 class I–restricted T cells depends on TCR peptide-MHC I interactions. TAP -1 knockout mice are unable to form peptide- MHC complexes unless peptide is added. The mice used in this study were transgenic for the H9251 and H9252 chains of a TCR that recog- nizes the added peptide bound to MHC I molecules of the TAP -1 knockout/TCR trans- genic mice. Varying the amount of added pep- tide revealed that low concentrations of peptide, producing low avidity of binding, re- sulted in positive selection and nearly normal levels of CD4 H11002 CD8 H11001 cells. High concentra- tions of peptide, producing high avidity of binding to the TCR, caused negative selection, and few CD4 H11002 CD8 H11001 T cells appeared. [Adapted from Ashton Rickardt et al. (1994) Cell 25:651.] (a) Experimental procedure—fetal thymic organ culture (FTOC) (b) Development of CD8 + CD4 ? MHC I–restricted cells Thymus donor Amount of peptide added Thymocyte Thymic stromal cell Degree of CD8 + T-cell development None Peptide Normal None Minimal Optimal Approaches normal High Minimal Remove thymus Place in FTOC Porous membrane Growth medium Normal TCR-transgenic TAP-1 deficient Weak signal No signal Weak signal Strong signal 8536d_ch10_221-247 8/28/02 3:58 PM Page 228 mac76 mac76:385_reb: (Figure 10-9). The instructional model postulates that the multiple interactions between the TCR, CD8 H11001 or CD4 H11001 coreceptors, and class I or class II MHC molecules instruct the cells to differentiate into either CD8 H11001 or CD4 H11001 single- positive cells, respectively. This model would predict that a class I MHC–specific TCR together with the CD8 coreceptor would generate a signal that is different from the signal in- duced by a class II MHC–specific TCR together with the CD4 coreceptor. The stochastic model suggests that CD4 or CD8 expression is switched off randomly with no relation to the specificity of the TCR. Only those thymocytes whose TCR and remaining coreceptor recognize the same class of MHC molecule will mature. At present, it is not possible to choose one model over the other. T H -Cell Activation The central event in the generation of both humoral and cell- mediated immune responses is the activation and clonal ex- pansion of T H cells. Activation of T C cells, which is generally similar to T H -cell activation, is described in Chapter 14. T H - cell activation is initiated by interaction of the TCR-CD3 complex with a processed antigenic peptide bound to a class II MHC molecule on the surface of an antigen-presenting cell. This interaction and the resulting activating signals also involve various accessory membrane molecules on the T H cell and the antigen-presenting cell. Interaction of a T H cell with antigen initiates a cascade of biochemical events that in- duces the resting T H cell to enter the cell cycle, proliferating and differentiating into memory cells or effector cells. Many of the gene products that appear upon interaction with anti- gen can be grouped into one of three categories depending on how early they can be detected after antigen recognition (Table 10-2): a73 Immediate genes, expressed within half an hour of antigen recognition, encode a number of transcription factors, including c-Fos, c-Myc, c-Jun, NFAT, and NF-H9260B a73 Early genes, expressed within 1–2 h of antigen recognition, encode IL-2, IL-2R (IL-2 receptor), IL-3, IL-6, IFN-H9253, and numerous other proteins a73 Late genes, expressed more than 2 days after antigen recognition, encode various adhesion molecules These profound changes are the result of signal-transduction pathways that are activated by the encounter between the TCR and MHC-peptide complexes. An overview of some of the basic strategies of cellular signaling will be useful back- ground for appreciating the specific signaling pathways used by T cells. Signal-Transduction Pathways Have Several Features in Common The detection and interpretation of signals from the environ- ment is an indispensable feature of all cells, including those of the immune system. Although there are an enormous number of different signal-transduction pathways, some common themes are typical of these crucial integrative processes: T-Cell Maturation, Activation, and Differentiation CHAPTER 10 229 FIGURE 10-9 Proposed models for the role of the CD4 and CD8 coreceptors in thymic se- lection of double positive thymocytes leading to single positive T cells. According to the in- structive model, interaction of one coreceptor with MHC molecules on stromal cells results in down-regulation of the other coreceptor. According to the stochastic model, down- regulation of CD4 or CD8 is a random process. INSTRUCTIVE MODEL CD8 engagement signal CD4 engagement signal STOCHASTIC MODEL CD4 lo 8 hi CD4 hi 8 lo CD4 + 8 + CD4 + 8 + CD4 lo 8 hi CD4 hi 8 lo Random CD4 Random CD8 CD4 + 8 + CD4 + 8 + CD4 ? 8 + T cell CD4 + 8 ? T cell CD4 ? 8 + T cell Able to bind Ag + class I MHC Able to bind Ag + class II MHC Not able to bind Ag + class II MHC Not able to bind Ag + class I MHC Apoptosis CD4 + 8 ? T cell Apoptosis 8536d_ch10_221-247 8/28/02 3:58 PM Page 229 mac76 mac76:385_reb: a73 Signal transduction begins with the interaction between a signal and its receptor. Signals that cannot penetrate the cell membrane bind to receptors on the surface of the cell membrane. This group includes water-soluble signaling molecules and membrane-bound ligands (MHC-peptide complexes, for example). Hydrophobic signals, such as steroids, that can diffuse through the cell membrane are bound by intracellular receptors. a73 Signals are often transduced through G proteins, membrane-linked macromolecules whose activities are controlled by binding of the guanosine nucleotides GTP and GDP, which act as molecular switches. Bound GTP turns on the signaling capacities of the G protein; hydrolysis of GTP or exchange for GDP turns off the signal by returning the G protein to an inactive form. There are two major categories of G proteins. Small G proteins consist of a single polypeptide chain of about 21 kDa. An important small G protein, known as Ras, is a key participant in the activation of an important proliferation-inducing signal-transduction cascade triggered by binding of ligands to their receptor tyrosine kinases. Large G proteins are composed of H9251, H9252, and H9253 subunits and are critically involved in many processes, including vision, olfaction, glucose metabolism, and phenomena of immunological interest such as leukocyte chemotaxis. 230 PART II Generation of B-Cell and T-Cell Responses TABLE 10-2 Time course of gene expression by T H cells following interaction with antigen Time mRNA Ratio of activated to Gene product Function expression begins Location nonactivated cells IMMEDIATE c-Fos Protooncogene; 15 min Nucleus H11022 100 nuclear-binding protein c-Jun Cellular oncogene; 15–20 min Nucleus ? transcription factor NFAT Transcription factor 20 min Nucleus 50 c-Myc Cellular oncogene 30 min Nucleus 20 NF-H9260B Transcription factor 30 min Nucleus H11022 10 EARLY IFN-H9253 Cytokine 30 min Secreted H11022 100 IL-2 Cytokine 45 min Secreted H11022 1000 Insulin receptor Hormone receptor 1 h Cell membrane 3 IL-3 Cytokine 1–2 h Secreted H11022 100 TGF-H9252 Cytokine H11021 2 h Secreted H11022 10 IL-2 receptor (p55) Cytokine receptor 2 h Cell membrane H11022 50 TNF-H9252 Cytokine 1–3 h Secreted H11022 100 Cyclin Cell-cycle protein 4–6 h Cytoplasmic H11022 10 IL-4 Cytokine H11021 6 h Secreted H11022 100 IL-5 Cytokine H11021 6 h Secreted H11022 100 IL-6 Cytokine H11021 6 h Secreted H11022 100 c-Myb Protooncogene 16 h Nucleus 100 GM-CSF Cytokine 20 h Secreted ? LATE HLA-DR Class II MHC molecule 3–5 days Cell membrane 10 VLA-4 Adhesion molecule 4 days Cell membrane H11022 100 VLA-1, VLA-2, VLA-3, VLA-5 Adhesion molecules 7–14 days Cell membrane H11022 100, ?, ?, ? SOURCE: Adapted from G. Crabtree, Science 243:357. 8536d_ch10_221 8/27/02 1:37 PM Page 230 Mac 109 Mac 109:1254_BJN:Goldsby et al. / Immunology 5e: a73 Signal reception often leads to the generation within the cell of a “second messenger,” a molecule or ion that can diffuse to other sites in the cell and evoke changes. Examples are cyclic nucleotides (cAMP, cGMP), calcium ion (Ca 2H11001 ), and membrane phospholipid derivatives such as diacylglycerol (DAG) and inositol triphosphate (IP 3 ). a73 Protein kinases and protein phosphatases are activated or inhibited. Kinases catalyze the phosphorylation of target residues (tyrosine, serine, or threonine) of key elements in many signal-transduction pathways. Phosphatases catalyze dephosphorylation, reversing the effect of kinases. These enzymes play essential roles in many signal-transduction pathways of immunological interest. a73 Many signal transduction pathways involve the signal- induced assembly of some components of the pathway. Molecules known as adaptor proteins bind specifically and simultaneously to two or more different molecules with signaling roles, bringing them together and promoting their combined activity. a73 Signals are amplified by enzyme cascades. Each enzyme in the cascade catalyzes the activation of many copies of the next enzyme in the sequence, greatly amplifying the signal at each step and offering many opportunities to modulate the intensity of a signal along the way. a73 The default setting for signal-transduction pathways is OFF. In the absence of an appropriately presented signal, transmission through the pathway does not take place. Multiple Signaling Pathways Are Initiated by TCR Engagement The events that link antigen recognition by the T-cell recep- tor to gene activation echo many of the themes just reviewed. The key element in the initiation of T-cell activation is the recognition by the TCR of MHC-peptide complexes on antigen-presenting cells. As described in Chapter 9, the TCR consists of a mostly extracellular ligand-binding unit, a predominantly intracel- lular signaling unit, the CD3 complex, and the homodimer of H9256 (zeta) chains. Experiments with knockout mice have shown that all of these components are essential for signal transduc- tion. Two phases can be recognized in the antigen-mediated induction of T-cell responses: a73 Initiation. The engagement of MHC-peptide by the TCR leads to clustering with CD4 or CD8 coreceptors as these coreceptors bind to invariant regions of the MHC molecule (Figure 10-10). Lck, a protein tyrosine kinase associated with the cytoplasmic tails of the coreceptors, is brought close to the cytoplasmic tails of the TCR complex and phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs, described in Chapter 9). The phosphorylated tyrosines in the ITAMs of the zeta chain provide docking sites to which a protein tyrosine kinase called ZAP-70 attaches (step 2 in Figure 10-10) and becomes active. ZAP-70 then catalyzes the phosphorylation of a number of membrane-associated adaptor molecules (step 3), which act as anchor points for the recruitment of several intracellular signal transduction pathways. One set of pathways involves a form of the enzyme phospholipase C (PLC), which anchors to an adaptor molecule, is activated by phosphorylation and cleaves a membrane phospholipid to generate second messengers. Another set activates small G proteins. a73 Generation of multiple intracellular signals.Many signaling pathways are activated as a consequence of the steps that occur in the initiation phase, as shown to the right in Figure 10-10, and described below. We shall consider several of the signaling pathways re- cruited by T-cell activation, but the overall process is quite complex and many of the details will not be presented here. The review articles suggested at the end of this chapter pro- vide extensive coverage of this very active research area. Phospholipase CH9253 (PLCH9253): PLCH9253 is activated by phosphoryla- tion and gains access to its substrate by binding to a mem- brane-associated adaptor protein (Figure 10-11a). PLCH9253 hydrolyzes a phospholipid component of the membrane to generate inositol 1,4,5-triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 causes a rapid release of Ca 2H11001 from the endoplas- mic reticulum and opens Ca 2H11001 channels in the cell mem- brane (Figure 10-11b). DAG activates protein kinase C, a multifunctional kinase that phosphorylates many different targets (Figure 10-11c). Ca 2H11001 : Calcium ion is involved in an unusually broad range of processes, including vision, muscle contraction, and many others. It is an essential element in many T-cell responses, in- cluding a pathway that leads to the movement of a major transcription factor, NFAT, from the cytoplasm into the nu- cleus (Figure 10-11b). In the nucleus, NFAT supports the transcription of genes required for the expression of the T- cell growth-promoting cytokines IL-2, IL-4, and others. Protein kinase C (PKC): This enzyme, which affects many pathways, causes the release of an inhibitory molecule from the transcription factor NF-H9260B, allowing NF-H9260B to enter the nucleus, where it promotes the expression of genes required for T-cell activation (Figure 10-11c). NF-H9260B is essential for a variety of T-cell responses and provides survival signals that protect T cells from apoptotic death. The Ras/MAP kinase pathway: Ras is a pivotal component of a signal-transduction pathway that is found in many cell types and is evolutionarily conserved across a spectrum of eukaryotes from yeasts to humans. Ras is a small G protein whose activation by GTP initiates a cascade of protein ki- nases known as the mitogen-activated protein kinase (MAP kinase) pathway. As shown in Figure 10-12, phosphorylation of the end product of this cascade, MAP kinase (also called T-Cell Maturation, Activation, and Differentiation CHAPTER 10 231 8536d_ch10_221 8/27/02 1:37 PM Page 231 Mac 109 Mac 109:1254_BJN:Goldsby et al. / Immunology 5e: VISUALIZING CONCEPTS FIGURE 10-10 Overview of TCR-mediated signaling. TCR en- gagement by peptide-MHC complexes initiates the assembly of a signaling complex. An early step is the Lck-mediated phosphory- lation of ITAMs on the zeta (H9256) chains of the TCR complex, creat- ing docking sites to which the protein kinase ZAP-70 attaches and becomes activated by phosphorylation. A series of ZAP-70- catalyzed protein phosphorylations enable the generation of a variety of signals. (Abbreviations: DAG = diacylglycerol; GADS = Grb2-like adaptor downstream of Shc; GEF = guanine nucleotide exchange factor; ITAM = immunoreceptor tyrosine-based activa- tion motif; Itk = inducible T cell kinase; IP3 = inositol 1,4,5 triphosphate; LAT = linker of activated T cells; PIP 2 = phospho- inositol biphosphage; PLCH9253 = phospholipase C gamma; Lck = lymphocyte kinase; SLP-76 = SH2-containing leukocyte-specific protein of 76 kDa; ZAP-70 = zeta associated protein of 70 kDa.) 1 Engagement of MHC-peptide initiates processes that lead to assembly of signaling complex. CD4/8-associated Lck phosphorylates ITAMs of coreceptors, creates docking site for ZAP-70 3 ZAP-70 phosphorylates adaptor molecules that recruit components of several signaling pathways ζζ γ H9280δ Class II MHC Peptide CD4/8 TCR P P P P P P P PP Lck ZAP-70 SLP-76 LAT LATGADS SLP67 ItK PLCγ GEF DAG PIP 2 IP 3 PkC-mediated pathways CA 2+ -mediated pathways Small G-protein— mediated pathways ? Ras Pathway ? Rac Pathway ? Changes in gene expression ? Functional changes ? Differentiation ? Activation 2 8536d_ch10_221-247 8/29/02 2:31 PM Page 232 mac114 Mac 114:2nd shift:1268_tm:8536d: ERK), allows it to activate Elk, a transcription factor neces- sary for the expression of Fos. Phosphorylation of Fos by MAP kinase allows it to associate with Jun to form AP-1, which is an essential transcription factor for T-cell activation. Co-Stimulatory Signals Are Required for Full T-Cell Activation T-cell activation requires the dynamic interaction of multiple membrane molecules described above, but this interaction, by itself, is not sufficient to fully activate naive T cells. Naive T cells require more than one signal for activation and subse- quent proliferation into effector cells: a73 Signal 1, the initial signal, is generated by interaction of an antigenic peptide with the TCR-CD3 complex. T-Cell Maturation, Activation, and Differentiation CHAPTER 10 233 a73 A subsequent antigen-nonspecific co-stimulatory signal, signal 2, is provided primarily by interactions between CD28 on the T cell and members of the B7 family on the APC. There are two related forms of B7, B7-1 and B7-2 (Figure 10-13). These molecules are members of the immunoglobu- lin superfamily and have a similar organization of extracel- lular domains but markedly different cytosolic domains. Both B7 molecules are constitutively expressed on dendritic cells and induced on activated macrophages and activated B cells. The ligands for B7 are CD28 and CTLA-4 (also known as CD152), both of which are expressed on the T-cell mem- brane as disulfide-linked homodimers; like B7, they are members of the immunoglobulin superfamily (Figure 10-13). Although CD28 and CTLA-4 are structurally similar glycoproteins, they act antagonistically. Signaling through FIGURE 10-11 Signal-transduction path- ways associated with T-cell activation. (a) Phospholipase CH9253 (PLC) is activated by phosphorylation. Active PLC hydrolyzes a phospholipid component of the plasma membrane to generate the second mes- sengers, DAG and IP 3 . (b) Protein kinase C (PKC) is activated by DAG and Ca 2H11001 . Among the numerous effects of PKC is phosphorylation of IkB, a cytoplasmic pro- tein that binds the transcription factor NF- H9260B and prevents it from entering the nucleus. Phosphorylation of IkB releases NF-H9260B, which then translocates into the nucleus. (c) Ca 2H11001 -dependent activation of calcineurin. Calcineurin is a Ca 2H11001 /calmod- ulin dependent phosphatase. IP 3 mediates the release of Ca 2H11001 from the endoplasmic reticulum. Ca 2H11001 binds the protein calmod- ulin, which then associates with and acti- vates the Ca 2H11001 /calmodulin-dependent phosphatase calcineurin. Active calcine- urin removes a phosphate group from NFAT, which allows this transcription fac- tor to translocate into the nucleus. DAG Cytoplasm Nucleus IP 3 Intracellular Ca 2+ stores IκB/NF-κB NF-κB NF-κB IκB Phospho- lipase Cγ (inactive) PKC (active) ATP ADP ATP ADP ZAP-70 + + + Ca 2+ Calmodulin-Ca 2+ Calcineurin- calmodulin-Ca 2+ (active) Calmodulin Calcineurin (inactive) NFAT NFAT Transcriptional activation of several genes NFAT P P P P (c) (a) (b) 8536d_ch10_221-247 8/28/02 3:58 PM Page 233 mac76 mac76:385_reb: CD28 delivers a positive co-stimulatory signal to the T cell; signaling through CTLA-4 is inhibitory and down-regulates the activation of the T cell. CD28 is expressed by both resting and activated T cells, but CTLA-4 is virtually undetectable on resting cells. Typically, engagement of the TCR causes the in- duction of CTLA-4 expression, and CTLA-4 is readily de- 234 PART II Generation of B-Cell and T-Cell Responses FIGURE 10-13 T H -cell activation requires a co-stimulatory signal provided by antigen-presenting cells (APCs). Interaction of B7 family members on APCs with CD28 delivers the co-stimulatory signal. En- gagement of the closely related CTLA-4 molecule with B7 produces an inhibitory signal. All of these molecules contain at least one im- munoglobulin-liké domain and thus belong to the immunoglobulin superfamily. [Adapted from P. S. Linsley and J. A. Ledbetter, 1993, Annu. Rev. Immunol. 11:191.] FIGURE 10-12 Activation of the small G protein, Ras. Signals from the T-cell receptor result in activation of Ras via the action of specific guanine nucleotide exchange factors (GEFs) that catalyze the ex- change of GDP for GTP. Active Ras causes a cascade of reactions that result in the increased production of the transcription factor Fos. Following their phosphorylation, Fos and Jun dimerize to yield the transcription factor AP-1. Note that all these pathways have impor- tant effects other than the specific examples shown in the figure. Cytoplasm MAP kinase pathway Nucleus + + P i Ras-GDP (inactive) Ras-GDP (active) P P P P P GTP GDP TCR-mediated signals Raf MEK MAP kinase GEFs Elk Elk Jun AP-1 Jun Fos Fos Fos Transcriptional activation of several genes S S S S S S S S S S S S SSSS B7 SSSS B7 T H cell CD28 is expressed by both resting and activated T cells CTLA-4 APC Both B7 molecules are expressed on dendritic cells, activated macrophages, and activated B cells CD28 CTLA-4 is expressed on activated T cells tectable within 24 hours of stimulation, with maximal ex- pression within 2 or 3 days post-stimulation. Even though the peak surface levels of CTLA-4 are lower than those of CD28, it still competes favorably for B7 molecules because it has a significantly higher avidity for these molecules than CD28 does. Interestingly, the level of CTLA-4 expression is increased by CD28-generated co-stimulatory signals. This provides regulatory braking via CTLA-4 in proportion to the acceleration received from CD28. Some of the importance of CTLA-4 in the regulation of lymphocyte activation and pro- liferation is revealed by experiments with CTLA-4 knockout mice. T cells in these mice proliferate massively, which leads to lymphadenopathy (greatly enlarged lymph nodes), splenomegaly (enlarged spleen), and death at 3 to 4 weeks after birth. Clearly, the production of inhibitory signals by engagement of CTLA-4 is important in lymphocyte home- ostasis. CTLA-4 can effectively block CD28 co-stimulation by competitive inhibition at the B7 binding site, an ability that holds promise for clinical use in autoimmune diseases and transplantation. As shown in Figure 10-14, an ingeniously engineered chimeric molecule has been designed to explore the therapeutic potential of CTLA-4. The Fc portion of human IgG has been fused to the B7-binding domain of CTLA-4 to produce a chimeric molecule called CTLA-4Ig. The human Fc region endows the molecule with a longer half-life in the body and the presence of B7 binding domains 8536d_ch10_221-247 8/28/02 3:58 PM Page 234 mac76 mac76:385_reb: allows it to bind to B7. A promising clinical trial of CTLA-4 has been conducted in patients with psoriasis vulgaris, a T-cell–mediated autoimmune inflammatory skin disease. During the course of this trial, forty-three patients received four doses of CTLA-4Ig, and 46% of this group experienced a 50% or greater sustained improvement in their skin condi- tion. Further studies of the utility of CTLA-4Ig are also being pursued in other areas. Clonal Anergy Ensues If a Co-Stimulatory Signal Is Absent T H -cell recognition of an antigenic peptide–MHC complex sometimes results in a state of nonresponsiveness called clonal anergy, marked by the inability of cells to proliferate in response to a peptide-MHC complex. Whether clonal ex- pansion or clonal anergy ensues is determined by the pres- ence or absence of a co-stimulatory signal (signal 2), such as that produced by interaction of CD28 on T H cells with B7 on antigen-presenting cells. Experiments with cultured cells show that, if a resting T H cell receives the TCR-mediated sig- nal (signal 1) in the absence of a suitable co-stimulatory sig- nal, then the T H cell will become anergic. Specifically, if resting T H cells are incubated with glutaraldehyde-fixed APCs, which do not express B7 (Figure 10-15a), the fixed APCs are able to present peptides together with class II MHC molecules, thereby providing signal 1, but they are unable to provide the necessary co-stimulatory signal 2. In the absence of a co-stimulatory signal, there is minimal production of cy- T-Cell Maturation, Activation, and Differentiation CHAPTER 10 235 FIGURE 10-14 CTLA-4Ig, a chimeric suppressor of co-stimulation. (a) CTLA-4Ig, a genetically engineered molecule in which the Fc por- tion of human IgG is joined to the B7-binding domain of CTLA-4. (b) CTLA-4Ig blocks costimulation by binding to B7 on antigen pre- senting cells and preventing the binding of CD28, a major co-stimula- tory molecule of T cells. (a) CTLA-4Ig (b) B7 blockade by CTLA-4Ig CTLA-4 binding domain SS IgG F c CD4 B7 CD28 T cell APC TCR tokines, especially of IL-2. Anergy can also be induced by in- cubating T H cells with normal APCs in the presence of the Fab portion of anti-CD28, which blocks the interaction of CD28 with B7 (Figure 10-15b). Two different control experiments demonstrate that fixed APCs bearing appropriate peptide-MHC complexes can de- liver an effective signal mediated by T-cell receptors. In one experiment, T cells are incubated both with fixed APCs bear- ing peptide-MHC complexes recognized by the TCR of the T cells and with normal APCs, which express B7 (Figure 10-15d). The fixed APCs engage the TCRs of the T cells, and the B7 molecules on the surface of the normal APCs cross- link the CD28 of the T cell. These T cells thus receive both signals and undergo activation. The addition of bivalent anti-CD28 to mixtures of fixed APCs and T cells also pro- vides effective co-stimulation by crosslinking CD28 (Figure 10-15e). Well-controlled systems for studying anergy in vitro have stimulated considerable interest in this phenome- non. However, more work is needed to develop good animal systems for establishing anergy and studying its role in vivo. Superantigens Induce T-Cell Activation by Binding the TCR and MHC II Simultaneously Superantigens are viral or bacterial proteins that bind simul- taneously to the V H9252 domain of a T-cell receptor and to the H9251 chain of a class II MHC molecule. Both exogenous and en- dogenous superantigens have been identified. Crosslinkage of a T-cell receptor and class II MHC molecule by either type of superantigen produces an activating signal that induces T-cell activation and proliferation (Figure 10-16). Exogenous superantigens are soluble proteins secreted by bacteria. Among them are a variety of exotoxins secreted by gram-positive bacteria, such as staphylococcal enterotoxins, toxic-shock-syndrome toxin, and exfoliative-dermatitis toxin. Each of these exogenous superantigens binds particu- lar V H9252 sequences in T-cell receptors (Table 10-3) and crosslinks the TCR to a class II MHC molecule. Endogenous superantigens are cell-membrane proteins encoded by certain viruses that infect mammalian cells. One group, encoded by mouse mammary tumor virus (MTV), can integrate into the DNA of certain inbred mouse strains; after integration, retroviral proteins are expressed on the membrane of the infected cells. These viral proteins, called minor lymphocyte stimulating (Mls) determinants, bind particular V H9252 sequences in T-cell receptors and crosslink the TCR to a class II MHC molecule. Four Mls superantigens, originating in different MTV strains, have been identified. Because superantigens bind outside of the TCR antigen- binding cleft, any T cell expressing a particular V H9252 sequence will be activated by a corresponding superantigen. Hence, the activation is polyclonal and can affect a significant per- centage (5% is not unusual) of the total T H population. The massive activations that follow crosslinkage by a superanti- gen results in overproduction of T H -cell cytokines, leading to systemic toxicity. The food poisoning induced by staphy- 8536d_ch10_221-247 8/28/02 3:58 PM Page 235 mac76 mac76:385_reb: lococcal enterotoxins and the toxic shock induced by toxic- shock-syndrome toxin are two examples of the consequences of cytokine overproduction induced by superantigens. Superantigens can also influence T-cell maturation in the thymus. A superantigen present in the thymus during thymic processing will induce the negative selection of all thymo- cytes bearing a TCR V H9252 domain corresponding to the super- antigen specificity. Such massive deletion can be caused by exogeneous or endogenous superantigens and is character- ized by the absence of all T cells whose receptors possess V H9252 domains targeted by the superantigen. T-Cell Differentiation CD4 H11001 and CD8 H11001 T cells leave the thymus and enter the cir- culation as resting cells in the G 0 stage of the cell cycle. There are about twice as many CD4 H11001 T cells as CD8 H11001 T cells in the periphery. T cells that have not yet encountered antigen (naive T cells) are characterized by condensed chromatin, very little cytoplasm, and little transcriptional activity. Naive T cells continually recirculate between the blood and lymph systems. During recirculation, naive T cells reside in sec- ondary lymphoid tissues such as lymph nodes. If a naive cell does not encounter antigen in a lymph node, it exits through the efferent lymphatics, ultimately draining into the thoracic duct and rejoining the blood. It is estimated that each naive T cell recirculates from the blood to the lymph nodes and back again every 12–24 hours. Because only about 1 in 10 5 naive T cells is specific for any given antigen, this large-scale recircu- lation increases the chances that a naive T cell will encounter appropriate antigen. Activated T Cells Generate Effector and Memory T Cells If a naive T cell recognizes an antigen-MHC complex on an appropriate antigen-presenting cell or target cell, it will be activated, initiating a primary response. About 48 hours after activation, the naive T cell enlarges into a blast cell and begins undergoing repeated rounds of cell division. As described earlier, activation depends on a signal induced by engage- ment of the TCR complex and a co-stimulatory signal in- duced by the CD28-B7 interaction (see Figure 10-13). These signals trigger entry of the T cell into the G 1 phase of the cell 236 PART II Generation of B-Cell and T-Cell Responses FIGURE 10-15 Experimental demonstration of clonal anergy ver- sus clonal expansion. (a,b) Only signal 1 is generated when resting T H cells are incubated with glutaraldehyde-fixed antigen-presenting cells (APCs) or with normal APCs in the presence of the Fab portion of anti-CD28. (c) The resulting anergic T cells cannot respond to nor- mal APCs. (d,e) In the presence of normal allogeneic APCs or anti- CD28, both of which produce the co-stimulatory signal 2, T cells are activated by fixed APCs. (a) (b) Normal APC Fab anti-CD28 B7 (c) Normal APC No response (d) Fixed APC 1 Anergic genes 1 Anergic genes Anergic T cell 1 IL-2 gene 2 Normal allogeneic APC IL-2 (e) Fixed APC 1 IL-2 gene 2 IL-2 Anti-CD28 Fixed APC (No B7) 8536d_ch10_221-247 8/28/02 3:58 PM Page 236 mac76 mac76:385_reb: cycle and, at the same time, induce transcription of the gene for IL-2 and the H9251 chain of the high-affinity IL-2 receptor. In addition, the co-stimulatory signal increases the half-life of the IL-2 mRNA. The increase in IL-2 transcription, together with stabilization of the IL-2 mRNA, increases IL-2 produc- tion by 100-fold in the activated T cell. Secretion of IL-2 and its subsequent binding to the high-affinity IL-2 receptor in- duces the activated naive T cell to proliferate and differenti- ate (Figure 10-17). T cells activated in this way divide 2–3 times per day for 4–5 days, generating a large clone of prog- eny cells, which differentiate into memory or effector T-cell populations. The various effector T cells carry out specialized functions such as cytokine secretion and B-cell help (activated CD4 H11001 T H cells) and cytotoxic killing activity (CD8 H11001 CTLs). The genera- tion and activity of CTL cells are described in detail in Chapter 14. Effector cells are derived from both naive and memory cells after antigen activation. Effector cells are short-lived cells, whose life spans range from a few days to a few weeks. The ef- fector and naive populations express different cell-membrane molecules, which contribute to different recirculation patterns. As described in more detail in Chapter 12, CD4 H11001 effector T cells form two subpopulations distinguished by the differ- ent panels of cytokines they secrete. One population, called the T H 1 subset, secretes IL-2, IFN-H9253, and TNF-H9252. The T H 1 subset is responsible for classic cell-mediated functions, such as delayed-type hypersensitivity and the activation of cyto- toxic T lymphocytes. The other subset, called the T H 2 subset, secretes IL-4, IL-5, IL-6, and IL-10. This subset functions more effectively as a helper for B-cell activation. The memory T-cell population is derived from both naive T cells and from effector cells after they have encountered antigen. Memory T cells are antigen-generated, generally T-Cell Maturation, Activation, and Differentiation CHAPTER 10 237 FIGURE 10-16 Superantigen-mediated crosslinkage of T-cell re- ceptor and class II MHC molecules. A superantigen binds to all TCRs bearing a particular V H9252 sequence regardless of their antigenic speci- ficity. Exogenous superantigens are soluble secreted bacterial pro- teins, including various exotoxins. Endogenous superantigens are membrane-embedded proteins produced by certain viruses; they in- clude Mls antigens encoded by mouse mammary tumor virus. APC TCR Peptide for which TCR is not specific Class II MHC T H cell V β Superantigen Endogenous superantigen is membrane-bound αβ βα TABLE 10-3 Exogenous superantigens and their V H9252 specificity V H9252 SPECIFICITY Superantigen Disease ? Mouse Human Staphylococcal enterotoxins SEA Food poisoning 1, 3, 10, 11, 12, 17 nd SEB Food poisoning 3, 8.1, 8.2, 8.33, 12, 14, 15, 17, 20 SEC1 Food poisoning 7, 8.2, 8.3, 11 12 SEC2 Food poisoning 8.2, 10 12, 13, 14, 15, 17, 20 SEC3 Food poisoning 7, 8.25, 12 SED Food poisoning 3, 7, 8.3, 11, 17 5, 12 SEE Food poisoning 11, 15, 17 5.1, 6.1–6.3, 8, 18 Toxic-shock-syndrome toxin (TSST1) Toxic-shock syndrome 15, 16 2 Exfoliative-dermatitis toxin (ExFT) Scalded-skin syndrome 10, 11, 15 2 Mycoplasma-arthritidis supernatant Arthritis, shock 6, 8.1–8.3 nd (MAS) Streptococcal pyrogenic exotoxins Rheumatic fever, shock nd nd (SPE-A, B, C, D) ? Disease results from infection by bacteria that produce the indicated superantigens. 8536d_ch10_221-247 8/28/02 3:58 PM Page 237 mac76 mac76:385_reb: long-lived, quiescent cells that respond with heightened reac- tivity to a subsequent challenge with the same antigen, gen- erating a secondary response. An expanded population of memory T cells appears to remain long after the population of effector T cells has declined. In general, memory T cells express many of the same cell-surface markers as effector T cells; no cell-surface markers definitively identify them as memory cells. Like naive T cells, most memory T cells are resting cells in the G 0 stage of the cell cycle, but they appear to have less stringent requirements for activation than naive T cells do. For example, naive T H cells are activated only by dendritic cells, whereas memory T H cells can be activated by macrophages, dendritic cells, and B cells. It is thought that the expression of high levels of numerous adhesion mole- cules by memory T H cells enables these cells to adhere to a broad spectrum of antigen-presenting cells. Memory cells also display recirculation patterns that differ from those of naive or effector T cells. A CD4 + CD25 + Subpopulation of T cells Negatively Regulates Immune Responses Investigators first described T cell populations that could sup- press immune responses during the early 1970s. These cells were called suppressor T cells (T s ) and were believed to be CD8 + T cells. However, the cellular and molecular basis of the observed suppression remained obscure, and eventually great doubt was cast on the existence of CD8 + suppressor T cells. Recent research has shown that there are indeed T cells that suppress immune responses. Unexpectedly, these cells have turned out to be CD4 + rather than CD8 + T cells. Within the population of CD4 + CD25 + T cells, there are regulatory T cells that can inhibit the proliferation of other T cell populations in vitro. Animal studies show that members of the CD4 + CD25 + population inhibit the development of autoimmune diseases such as experimentally induced inflammatory bowel disease, experimental allergic encephalitis, and autoimmune diabetes. The suppression by these regulatory cells is antigen specific because it depends upon activation through the T cell recep- tor. Cell contact between the suppressing cells and their tar- gets is required. If the regulatory cells are activated by antigen but separated from their targets by a permeable barrier, no suppression occurs. The existence of regulatory T cells that specifically suppress immune responses has clinical implica- tions. The depletion or inhibition of regulatory T cells fol- lowed by immunization may enhance the immune responses to conventional vaccines. In this regard, some have suggested that elimination of T cells that suppress responses to tumor antigens may facilitate the development of anti-tumor immu- nity. Conversely, increasing the suppressive activity of regula- tory T cell populations could be useful in the treatment of allergic or autoimmune diseases. The ability to increase the activity of regulatory T cell populations might also be useful in suppressing organ and tissue rejection. Future work on this regulatory cell population will seek deeper insights into the mechanisms by which members of CD4 + CD25 + T cell popu- lations regulate immune responses. There will also be deter- mined efforts to discover ways in which the activities of these populations can be increased to diminish unwanted immune responses and decreased to promote desirable ones. Antigen-Presenting Cells Have Characteristic Co-Stimulatory Properties Only professional antigen-presenting cells (dendritic cells, macrophages, and B cells) are able to present antigen to- gether with class II MHC molecules and deliver the co-stim- ulatory signal necessary for complete T-cell activation that leads to proliferation and differentiation. The principal co- stimulatory molecules expressed on antigen-presenting cells are the glycoproteins B7-1 and B7-2 (see Figure 10-13). The professional antigen-presenting cells differ in their ability to display antigen and also differ in their ability to deliver the co-stimulatory signal (Figure 10-18). 238 PART II Generation of B-Cell and T-Cell Responses FIGURE 10-17 Activation of a T H cell by both signal 1 and co- stimulatory signal 2 up-regulates expression of IL-2 and the high- affinity IL-2 receptor, leading to the entry of the T cell into the cell cycle and several rounds of proliferation. Some of the cells differenti- ate into effector cells, others into memory cells. CD28 B7 IL-2 IL-2 gene IL-2R gene Normal APC 1 2 IL-2 IL-2 receptor Activation Population of memory and effector cells Several divisions G 1 G 2 MS MMEEEE 8536d_ch10_221-247 8/28/02 3:58 PM Page 238 mac76 mac76:385_reb: Dendritic cells constitutively express high levels of class I and class II MHC molecules as well as high levels of B7-1 and B7-2. For this reason, dendritic cells are very potent activa- tors of naive, memory, and effector T cells. In contrast, all other professional APCs require activation for expression of co-stimulatory B7 molecules on their membranes; conse- quently, resting macrophages are not able to activate naive T cells and are poor activators of memory and effector T cells. Macrophages can be activated by phagocytosis of bacteria or by bacterial products such as LPS or by IFN-H9253,a T H 1-derived cytokine. Activated macrophages up-regulate their expres- sion of class II MHC molecules and co-stimulatory B7 mole- cules. Thus, activated macrophages are common activators of memory and effector T cells, but their effectiveness in acti- vating naive T cells is considered minimal. B cells also serve as antigen-presenting cells in T-cell acti- vation. Resting B cells express class II MHC molecules but fail to express co-stimulatory B7 molecules. Consequently, rest- ing B cells cannot activate naive T cells, although they can ac- tivate the effector and memory T-cell populations. Upon activation, B cells up-regulate their expression of class II MHC molecules and begin expressing B7. These activated B cells can now activate naive T cells as well as the memory and effector populations. Cell Death and T-Cell Populations Cell death is an important feature of development in all multicellular organisms. During fetal life it is used to mold and sculpt, removing unnecessary cells to provide shape and form. It also is an important feature of lymphocyte homeostasis, returning T- and B-cell populations to their ap- propriate levels after bursts of antigen-induced proliferation. Apoptosis also plays a crucial role in the deletion of poten- tially autoreactive thymocytes during negative selection and in the removal of developing T cells unable to recognize self (failure to undergo positive selection). Although the induction of apoptosis involves different signals depending on the cell types involved, the actual death of the cell is a highly conserved process amongst vertebrates and invertebrates. For example, T cells may be induced to die by many different signals, including the withdrawal of growth factor, treatment with glucocorticoids, or TCR sig- naling. Each of these signals engages unique signaling path- ways, but in all cases, the actual execution of the cell involves the activation of a specialized set of proteases known as cas- pases. The role of these proteases was first revealed by studies of developmentally programmed cell deaths in the nematode T-Cell Maturation, Activation, and Differentiation CHAPTER 10 239 FIGURE 10-18 Differences in the properties of professional antigen-presenting cells affect their ability to present antigen and induce T-cell activation. Note that activation of effector and memory T cells does not require the co-stimulatory B7 molecule. Antigen uptake Class II MHC expression Co-stimulatory activity T-cell activation Endocytosis phagocytosis (by Langerhans cells) Phagocytosis Inducible (?) Inducible B7 (?) Inducible (++) Inducible B7 (++) Effector T cells Memory T cells Phagocytosis Receptor-mediated endocytosis Receptor-mediated endocytosis Constitutive (++) Constitutive (+++) Constitutive B7 (+++) Naive T cells Effector T cells Memory T cells Constitutive (+++) Inducible B7 (?) Inducible B7 (++) Effector T cells Memory T cells Naive T cells Effector T cells Memory T cells Resting Resting Class II MHC Class II MHC Class II MHC Class I MHC Class I MHC Class I MHC Class I MHC B7 Activated B Lymphocyte Activated Dendritic cell B7 Class II MHC Class I MHC Macrophage LPS INF-γ B7 (?) 8536d_ch10_221-247 8/28/02 3:58 PM Page 239 mac76 mac76:385_reb: C. elegans, where the death of cells was shown to be totally dependent upon the activity of a gene that encoded a cysteine protease with specificity for aspartic acid residues. We now know that in mammals there are at least 14 cysteine proteases or caspases, and all cell deaths require the activity of at least a subset of these molecules. We also know that essentially every cell in the body produces caspase proteins, suggesting that every cell has the potential to initiate its own death. Cells protect themselves from apoptotic death under nor- mal circumstances by keeping caspases in an inactive form within a cell. Upon reception of the appropriate death signal, certain caspases are activated by proteolytic cleavage and then activate other caspases in turn, leading to the activation of effec- tor caspases.This catalytic cascade culminates in cell death. Al- though it is not well understood how caspase activation directly results in apoptotic death of the cell, presumably it is through the cleavage of critical targets necessary for cell survival. T cells use two different pathways to activate caspases (Figure 10-19). In peripheral T cells, antigen stimulation re- sults in proliferation of the stimulated T cell and production of several cytokines including IL-2. Upon activation, T cells increase the expression of two key cell-surface proteins in- volved in T-cell death, Fas and Fas ligand (FasL). When Fas binds its ligand, FasL, FADD (Fas-associated protein with death domain) is recruited and binds to Fas, followed by the recruitment of procaspase 8, an inactive form of caspase 8. The association of FADD with procaspase 8 results in the proteolytic cleavage of procaspase 8 to its active form; cas- pase 8 then initiates a proteolytic cascade that leads to the death of the cell. Outside of the thymus, most of the TCR-mediated apop- tosis of mature T cells is mediated by the Fas pathway. Repeated or persistent stimulation of peripheral T cells re- sults in the coexpression of both Fas and Fas ligand, followed by the apoptotic death of the cell. The Fas/FasL mediated death of T cells as a consequence of activation is called acti- vation-induced cell death (AICD) and is a major homeostatic mechanism, regulating the size of the pool of T cells and re- moving T cells that repeatedly encounter self antigens. The importance of Fas and FasL in the removal of acti- vated T cells is underscored by lpr/lpr mice, a naturally occur- ring mutation that results in non-functional Fas. When T cells become activated in these mice, the Fas/FasL pathway is not operative; the T cells continue to proliferate, producing IL-2 and maintaining an activated state. These mice spontaneously develop autoimmune disease, have excessive numbers of T cells, and clearly demonstrate the consequences of a failure to delete activated T cells. An additional mutation, gld/gld, is also informative. These mice lack functional FasL and display much the same abnormalities found in the lpr/lpr mice. Re- cently, humans with defects in Fas have been reported. As expected, these individuals display characteristics of autoim- mune disease. (See the Clinical Focus box.) Fas and FasL are members of a family of related recep- tor/ligands including tumor necrosis factor (TNF) and its 240 PART II Generation of B-Cell and T-Cell Responses FIGURE 10-19 Two pathways to apoptosis in T cells. (a) Acti- vated peripheral T cells are induced to express high levels of Fas and FasL. FasL induces the trimerization of Fas on a neighboring cell. FasL can also engage Fas on the same cell, resulting in a self- induced death signal. Trimerization of Fas leads to the recruitment of FADD, which leads in turn to the cleavage of associated mole- cules of procaspase 8 to form active caspase 8. Caspase 8 cleaves procaspase 3, producing active caspase 3, which results in the death of the cell. Caspase 8 can also cleave Bid to a truncated form that can activate the mitochondrial death pathway. (b) Other signals, such as the engagement of the TCR by peptide-MHC complexes on an APC, result in the activation of the mitochondrial death pathway. A key feature of this pathway is the release of AIF (apoptosis induc- ing factor) and cytochrome c from the inner mitochondrial mem- brane into the cytosol. Cytochrome c interacts with Apaf-1 and subsequently with procaspase 9 to form the active apoptosome. The apoptosome initiates the cleavage of procaspase 3, producing active caspase 3, which initiates the execution phase of apoptosis by proteolysis of substances whose cleavage commits the cell to apop- tosis. [Adapted in part from S. H. Kaufmann and M. O. Hengartner, 2001. Trends Cell Biol. 11:526.] (a) (b) T cell Fas TCR MHC FADD Procaspase-8 (inactive) Caspase-8 (active) FasL Mitochondrion Procaspase-3 (inactive) Caspase-3 (active) Apoptosis substrates Apoptosis Active apoptotic effectors Caspase 9 Procaspase-9 Apoptosome Released cytochrome c Apaf-1 Promotion of apoptosis AIF Bid Truncated Bid 8536d_ch10_221-247 8/28/02 3:58 PM Page 240 mac76 mac76:385_reb: ligand, TNFR (tumor necrosis factor receptor). Like Fas and FasL, membrane-bound TNFR interacts with TNF to induce apoptosis. Also similar to Fas/FasL-induced apoptosis, TNF/TNFR-induced death is the result of the activation of caspase 8 followed by the activation of effector caspases such as caspase 3. In addition to the activation of apoptosis through death receptor proteins like Fas and TNFR, T cells can die through other pathways that do not activate procaspase 8. For exam- ple, negative selection in the thymus induces the apoptotic death of developing T cells via a signaling pathway that orig- inates at the TCR. We still do not completely understand why some signals through the TCR induce positive selection and others induce negative selection, but we know that the strength of the signal plays a critical role. A strong, negatively selecting signal induces a route to apoptosis in which mitochondria play a central role. In mitochondrially depen- dent apoptotic pathways, cytochrome c, which normally resides in the inner mitochondrial membrane, leaks into the cytosol. Cytochrome c binds to a protein known as Apaf-1 (apoptotic protease-activating factor-1) and undergoes an ATP-dependent conformational change and oligomeriza- tion. Binding of the oligomeric form of Apaf-1 to procaspase 9 results in its transformation to active caspase 9. The com- plex of cytochrome c/Apaf-1/caspase 9, called the apopto- some, proteolytically cleaves procaspase 3 generating active caspase 3, which initiates a cascade of reactions that kills the cell (Figure 10-19). Finally, mitochondria also release an- other molecule, AIF (apoptosis inducing factor), which plays a role in the induction of cell death. Cell death induced by Fas/FasL is swift, with rapid activa- tion of the caspase cascade leading to cell death in 2–4 hours. On the other hand, TCR-induced negative selection appears to be a more circuitous process, requiring the activation of sev- eral processes including mitochondrial membrane failure, the release of cytochrome c, and the formation of the apopto- some before caspases become involved. Consequently, TCR- mediated negative selection can take as long as 8–10 hours. An important feature in the mitochondrially induced cell death pathway is the regulatory role played by Bcl-2 family members. Bcl-2 and Bcl-XL both reside in the mitochondr- ial membrane. These proteins are strong inhibitors of apop- tosis, and while it is not clear how they inhibit cell death, one hypothesis is that they somehow regulate the release of cy- tochrome c from the mitochondria. There are at least three groups of Bcl-2 family members. Group I members are anti- apoptotic and include Bcl-2 and Bcl-xL. Group II and Group III members are pro-apoptotic and include Bax and Bak in Group II and Bid and Bim in Group III. There is clear evi- dence that levels of anti-apoptotic Bcl-2 family members play an important role in regulating apoptosis in lymphocytes. Bcl-2 family members dimerize, and the anti-apoptotic group members may control apoptosis by dimerizing with pro-apoptotic members, blocking their activity. As indicated in Figure 10-19, cleavage of Bid, catalyzed by caspase 8 gen- erated by the Fas pathway, can turn on the mitochondrial pathway. Thus signals initiated through Fas can also involve the mitochondrial death pathway. While it is apparent there are several ways a lymphocyte can be signaled to die, all of these pathways to cell death con- verge upon the activation of caspases. This part of the cell- death pathway, the execution phase, is common to almost all death pathways known in both vertebrates and invertebrates, demonstrating that apoptosis is an ancient process that has been conserved throughout evolution. Peripheral H9253H9254 T Cells In 1986, a small population of peripheral-blood T cells was discovered that expressed CD3 but failed to stain with mon- oclonal antibody specific for the H9251H9252 T-cell receptor, indicat- ing an absence of the H9251H9252 heterodimer. Many of these cells eventually were found to express the H9253H9254 receptor. gd T Cells Are Far Less Pervasive Than ab T Cells In humans, less than 5% of T cells bear the H9253H9254 heterodimer, and the percentage of H9253H9254 T cells in the lymphoid organs of mice has been reported to range from 1% to 3%. In addition to their presence in blood and lymphoid tissues, they also ap- pear in the skin, intestinal epithelium, and pulmonary epithe- lium. Up to 1% of the epidermal cells in the skin of mice are H9253H9254 T cells. In general, H9253H9254 T cells are not MHC-restricted, and most do not express the coreceptors CD4 and CD8 present on populations of H9251H9252 T cells. Although the potential of the H9253 and H9254 TCR loci to generate diversity is great, very little diversity is found in this type of T cell. In fact, as pointed out in Chapter 9, most of the H9253H9254 T cells in humans have an identical combi- nation of H9253H9254 chains (H92539 and H92542). H9253H9254 T Cells Recognize Nonpeptide Ligands Not all T cells are self-MHC restricted and recognize only peptide antigens displayed in the cleft of the self-MHC mol- ecule. Indeed, Chapters 2 and 8 describe H9251H9252 TCR-bearing T cells (NK1-T cells and CD1-restricted T cells) that are not re- stricted by conventional MHC molecules. In one study, a H9253H9254 T-cell clone was found to bind directly to a herpes-virus pro- tein without requiring antigen processing and presentation together with MHC. Human H9253H9254 T cells have been reported that display MHC-independent binding of a phospholipid derived from M. tuberculosis, the organism responsible for tuberculosis (see Chapter 9). This finding suggests that in many cases the TCR receptors of H9253H9254 T cells bind to epitopes in much the same way that the immunoglobulin receptors of B cells do. The fact that most human H9253H9254 T cells all have the same specificity suggests that like other components of the innate immune system, they recognize and respond to T-Cell Maturation, Activation, and Differentiation CHAPTER 10 241 8536d_ch10_221-247 8/29/02 10:23 AM Page 241 mac114 Mac 114:2nd shift:1268_tm:8536d: 242 PART II Generation of B-Cell and T-Cell Responses amination of these cells by flow cytome- try and fluorescent antibody staining re- vealed an excess of double-negative T cells (see illustration below). Also, like many patients with Canale-Smith syn- drome, she has had cancer, breast can- cer at age 22 and skin cancer at ages 22 and 41. Patient B: A man who was eventually di- agnosed with Canale-Smith syndrome had severe lymphadenopathy and spleno- megaly as an infant and child. He was treated from age 4 to age 12 with corti- costeroids and the immunosuppressive drug mercaptopurine. These appeared to help, and the swelling of lymphoid tis- sues became milder during adolescence and adulthood. At age 42, he died of liver cancer. Patient C: An 8-year-old boy, the son of patient B, was also afflicted with Canale- Smith syndrome and showed elevated T- cell counts and severe lymphadenopathy at the age of seven months. At age 2 his spleen became so enlarged that it had to be removed. He also developed he- molytic anemia and thrombocytopenia. However, although he continued to have elevated T-cell counts, the severity of his hemolytic anemia and thrombocytope- nia have so far been controlled by treat- ment with methotrexate, a DNA- synthe- sis-inhibiting drug used for immunosup- pression and cancer chemotherapy. Recognition of the serious conse- quences of a failure to regulate the num- ber of lymphocytes, as exemplified by these case histories, emerged from de- tailed study of several children whose enlarged lymphoid tissues attracted medical attention. In each of these cases of Canale-Smith syndrome, examination revealed grossly enlarged lymph nodes that were 1–2 cm in girth and some- times large enough to distort the local anatomy. In four of a group of five chil- dren who were studied intensively, the The maintenance of appropriate numbers of various types of lymphocytes is extremely important to an effective immune system. One of the most im- portant elements in this regulation is apoptosis mediated by the Fas/FasL ligand system. The following excerpts from medical histories show what can happen when this key regulatory mecha- nism fails. Patient A: A woman, now 43, has had a long history of immunologic imbalances and other medical problems. By age 2, she was diagnosed with the Canale- Smith syndrome (CSS), a severe enlarge- ment of such lymphoid tissues as lymph nodes (lymphadenopathy) and spleen (splenomegaly). Biopsy of lymph nodes showed that, in common with many other CSS patients, she had greatly in- creased numbers of lymphocytes. She had reduced numbers of platelets (thrombocytopenia) and, because her red blood cells were being lysed, she was anemic (hemolytic anemia). The reduc- tion in numbers of platelets and the lysis of red blood cells could be traced to the action of circulating antibodies that re- acted with these host components. At age 21, she was diagnosed with grossly enlarged pelvic lymph nodes that had to be removed. Ten years later, she was again found to have an enlarged abdom- inal mass, which on surgical removal turned out to be a half-pound lymph- node aggregate. She has continued to have mild lymphadenopathy and, typical of these patients, the lymphocyte popu- lations of enlarged nodes had elevated numbers of T cells (87% as opposed to a normal range of 48%–67% T cells). Ex- CLINICAL FOCUS Failure of Apoptosis Causes Defective Lymphocyte Homeostasis CD4 CD4 CD8 10 3 10 4 10 2 10 1 10 0 10 1 Normal control Patient A 10 0 10 0 10 2 10 3 10 4 20% CD4 – /CD8 + 1% CD4 + /CD8 + 4% CD4 – /CD8 – 75% CD4 + /CD8 – 10 1 10 2 10 3 10 4 24% CD4 – /CD8 + 1% CD4 + /CD8 + 43% CD4 – /CD8 – 32% CD4 + /CD8 – Flow-cytometric analysis of T cells in the blood of Patient A and a control subject. The relative staining by an anti-CD8 antibody is shown on the y axis and the relative staining by an anti-CD4 antibody appears on the x axis. Mature T cells are either CD4 H11001 or CD8 H11001 . While almost all of the T cells in the control subject are CD4 H11001 or CD8 H11001 , the CSS patient shows high numbers of double-negative T cells (43%), which express neither CD4 nor CD8. The percentage of each category of T cells is indicated in the quadrants. [Adapted from Drappa et al., 1996, New England Journal of Medicine 335:1643.] 8536d_ch10_221 8/27/02 1:37 PM Page 242 Mac 109 Mac 109:1254_BJN:Goldsby et al. / Immunology 5e: T-Cell Maturation, Activation, and Differentiation CHAPTER 10 243 below). Such mutations result in the pro- duction of Fas protein that lacks biologi- cal activity but still competes with normal Fas molecules for interactions with essential components of the Fas- mediated death pathway. Other muta- tions have been found in the extracellular domain of Fas, often associated with milder forms of CSS or no disease at all. A number of research groups have conducted detailed clinical studies of CSS patients, and the following general observations have been made: a73 The cell populations of the blood and lymphoid tissues of CSS patients show dramatic elevations (5-fold to as much as 20-fold) in the numbers of lymphocytes of all sorts, including T cells, B cells, and NK cells. a73 Most of the patients have elevated levels of one or more classes of im- munoglobulin (hyper-gammaglobulin- emia). a73 Immune hyperactivity is responsible for such autoimmune phenomena as the production of autoantibodies against red blood cells, resulting in he- molytic anemia, and a depression in platelet counts due to the activity of anti-platelet auto-antibodies. These observations establish the impor- tance of the death-mediated regulation of lymphocyte populations in lympho- cyte homeostasis. Such death is neces- sary because the immune response to antigen results in a sudden and dramatic increase in the populations of respond- ing clones of lymphocytes and temporar- ily distorts the representation of these clones in the repertoire. In the absence of cell death, the periodic stimulation of lymphocytes that occurs in the normal course of life would result in progres- sively increasing, and ultimately unsus- tainable, lymphocyte levels. As the Canale-Smith syndrome demonstrates, without the essential culling of lympho- cytes by apoptosis, severe and life-threat- ening disease can result. spleens were so massive that they had to be removed. Even though the clinical picture in Canale-Smith syndrome can vary from person to person, with some individuals suffering severe chronic affliction and others only sporadic episodes of illness, there is a common feature, a failure of activated lymphocytes to undergo Fas- mediated apoptosis. Isolation and se- quencing of Fas genes from a number of patients and more than 100 controls re- veals that CSS patients are heterozygous ( fas H11001/H11002 ) at the fas locus and thus carry one copy of a defective fas gene. A com- parison of Fas-mediated cell death in T cells from normal controls who do not carry mutant Fas genes with death in- duced in T cells from CSS patients, shows a marked defect in Fas-induced death (see illustration above). Character- ization of the Fas genes so far seen in CSS patients reveals that they have mu- tations in or around the region encoding the death-inducing domain (the “death domain”) of this protein (see illustration Anti-Fas antibody (ng/ml) Percentage of T cells killed 20 40 60 Normal controls Patient A Patient B 16 80 400 Fas-mediated killing takes place when Fas is crosslinked by FasL, its normal ligand, or by treatment with anti-Fas antibody, which artificially crosslinks Fas molecules. This experiment shows the reduction in numbers of T cells after induction of apoptosis in T cells from patients and controls by crosslinking Fas with increasing amounts of an anti- Fas monoclonal antibody. T cells from the Canale-Smith patients (A and B) are resistant to Fas-mediated death. [Adapted from Drappa et al., 1996, New England Journal of Medicine 335:1643.] Map of fas locus. The fas gene is composed of 9 exons separated by 8 introns. Exons 1–5 encode the extracellular part of the protein, exon 6 encodes the transmembrane region, and exons 7–9 encode the intracellular region of the molecule. Much of exon 9 is responsible for encoding the critical death domain. [Adapted from G. H. Fisher et al., 1995, Cell 81:935.] Death domain Exon 1 2 3 4 5 6 7 8 9 Extracellular region Intracellular region Transmembrane region 8536d_ch10_221 8/27/02 1:37 PM Page 243 Mac 109 Mac 109:1254_BJN:Goldsby et al. / Immunology 5e: molecular patterns that are found in certain pathogens but not in humans. Thus they may play a role as first lines of de- fense against certain pathogens, expressing effector functions that help control infection and secreting cytokines that pro- mote an adaptive immune response by H9251H9252 T cells and B cells. SUMMARY a73 Progenitor T cells from the bone marrow enter the thymus and rearrange their TCR genes. In most cases these thymo- cytes rearrange H9251H9252TCR genes and become H9251H9252T cells.A small minority rearrange H9253H9254 TCR genes and become H9253H9254 T cells. a73 The earliest thymocytes lack detectable CD4 and CD8 and are referred to as double-negative cells. During develop- ment, the majority of double-negative thymocytes develop into CD4 H11001 CD8 H11002 H9251H9252 T cells or CD4 H11002 CD8 H11001 H9251H9252 T cells. a73 Positive selection in the thymus eliminates T cells unable to recognize self-MHC and is the basis of MHC restriction. Negative selection eliminates thymocytes bearing high- affinity receptors for self-MHC molecules alone or self- antigen plus self-MHC and produces self-tolerance. a73 T H -cell activation is initiated by interaction of the TCR- CD3 complex with a peptide-MHC complex on an anti- gen-presenting cell. Activation also requires the activity of accessory molecules, including the coreceptors CD4 and CD8. Many different intracellular signal-transduction pathways are activated by the engagement of the TCR. a73 T-cells that express CD4 recognize antigen combined with a class II MHC molecule and generally function as T H cells; T cells that express CD8 recognize antigen combined with a class I MHC molecule and generally function as T C cells. a73 In addition to the signals mediated by the T-cell receptor and its associated accessory molecules (signal 1), activa- tion of the T H cell requires a co-stimulatory signal (signal 2) provided by the antigen-presenting cell. The co-stimu- latory signal is commonly induced by interaction between molecules of the B7 family on the membrane of the APC with CD28 on the T H cell. Engagement of CTLA-4, a close relative of CD28, by B7 inhibits T-cell activation. a73 TCR engagement with antigenic peptide-MHC may in- duce activation or clonal anergy. The presence or absence of the co-stimulatory signal (signal 2) determines whether activation results in clonal expansion or clonal anergy. a73 Naive T cells are resting cells (G 0 ) that have not encountered antigen. Activation of naive cells leads to the generation of ef- fector and memory T cells. Memory T cells, which are more easily activated than naive cells, are responsible for secondary responses. Effector cells are short lived and perform helper, cytotoxic, or delayed-type hypersensitivity functions. a73 The T-cell repertoire is shaped by apoptosis in the thymus and periphery. a73 H9253H9254 T cells are not MHC restricted. Most in humans bind free antigen, and most have the same specificity. They may function as part of the innate immune system. References Ashton-Rickardt, P. G., A. Bandeira, J. R. Delaney, L. Van Kaer, H. P. Pircher, R. M. Zinkernagel, and S. Tonegawa. 1994. Evidence for a differential avidity model of T-cell selection in the thymus. Cell 74:577. Drappa, M. D., A. K. Vaishnaw, K. E. Sullivan, B. S. Chu, and K. B. Elkon. 1996. Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associ- ated with autoimmunity. New England Journal of Medicine 335:1643. Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T-cell mem- ory. Annu.Rev.Immunol.16:201. Ellmeier, W., S. Sawada, and D. R. Littman. 1999. The regulation of CD4 and CD8 coreceptor gene expression during T-cell de- velopment. Annu.Rev.Immunol.17:523. Hayday, A. 2000.H9253H9254 Cells: A right time and right place for a con- served third way of protection. Annu. Rev. Immunol. 18:1975. Herman, A., J. W. Kappler, P. Marrack, and A. M. Pullen. 1991. Superantigens: mechanism of T-cell stimulation and role in immune responses. Annu.Rev.Immunol.9:745. Lanzavecchia, A., G. Lezzi, and A. Viola. 1999. From TCR en- gagement to T-cell activation: a kinetic view of T-cell behavior. Cell 96:1. Myung, P. S., N. J. Boerthe, and G. A. Koretzky. 2000. Adapter proteins in lymphocyte antigen-receptor signaling. Curr. Opin. Immunol. 12:256. Osborne, B. A. 1996. Apoptosis and maintenance of homeostasis in the immune system. Curr. Opin. Immunol. 8:245. Osborne, B., A. 2000. Transcriptional control of T-cell develop- ment. Curr. Opin. Immunol. 12:301. Owen, J. J. T., and N. C. Moore. 1995. Thymocyte–stromal-cell interactions and T-cell selection. Immunol. Today 16:336. Salomon, B., and J. A. Bluestone. 2001. Complexities of CD28/B7: CTLA-4 costimulatory pathways in autoimmunity and transplantation. Annu.Rev.Immunol.19:225. Schreiber, S. L., and G. R. Crabtree. 1992. The mechanism of ac- tion of cyclosporin A and FK506. Immunol. Today 13:136. Thompson, C. B. and J. C. Rathmell. 1999. The central effectors of cell death in the immune system. Annu.Rev.Immunol. 17:781. Vaishnaw, A. K., J. R. Orlinick, J. L. Chu, P. H. Krammer, M. V. Chao, and K. B. Elkon. 1999. The molecular basis for apoptotic defects in patients with CD95 (Fas/Apo-1) mutations. Journal of Clinical Investigation 103:355. USEFUL WEB SITES http://www.ncbi.nlm.nih.gov/Omim/ http://www.ncbi.nlm.nih.gov/htbinpost/Omim/getmim The Online Mendelian Inheritance in Man Web site contains a subsite that features ten different inherited diseases associ- ated with defects in the TCR complex or associated proteins. 244 PART II Generation of B-Cell and T-Cell Responses 8536d_ch10_221-247 8/29/02 10:23 AM Page 244 mac114 Mac 114:2nd shift:1268_tm:8536d: http://www.ultranet.com/~jkimball/BiologyPages/A/ Apoptosis.html http://www.ultranet.com/~jkimball/BiologyPages/B/ B_and_Tcells.html These subsites of John Kimball’s Biology Pages Web site pro- vide a clear introduction to T-cell biology and a good basic discussion of apoptosis. http://www.bioscience.org/knockout/knochome.htm Within the Frontiers in Bioscience Database of Gene Knock- outs, one can find information on the effects of knockouts of many genes involved in the development and function of cells of the T cells. Study Questions CLINICAL FOCUS QUESTION Over a period of several years, a group of children and adolescents are regularly dosed with com- pound X, a life-saving drug. However, in addition to its beneficial effects, this drug interferes with Fas-mediated signaling. a. What clinical manifestations of this side effect of com- pound X might be seen in these patients? b. If white blood cells from an affected patient are stained with a fluorescein-labeled anti-CD4 and a phycoerythrin- labeled anti-CD8 antibody, what might be seen in the flow-cytometric analysis of these cells? What pattern would be expected if the same procedure were performed on white blood cells from a healthy control? 1. You have a CD8 H11001 CTL clone (from an H-2 k mouse) that has a T-cell receptor specific for the H-Y antigen. You clone the H9251H9252TCR genes from this cloned cell line and use them to pre- pare transgenic mice with the H-2 k or H-2 d haplotype. a. How can you distinguish the immature thymocytes from the mature CD8 H11001 thymocytes in the transgenic mice? b. For each transgenic mouse listed in the table below, indi- cate with (H11001) or (H11002) whether the mouse would have im- mature double-positive and mature CD8 H11001 thymocytes bearing the transgenic T-cell receptor. 3. Antigenic activation of T H cells leads to the release or induc- tion of various nuclear factors that activate gene transcrip- tion. a. What transcription factors that support proliferation of activated T H cells are present in the cytoplasm of resting T H cells in inactive forms? b. Once in the nucleus, what might these transcription fac- tors do? 4. You have fluorescein-labeled anti-CD4 and rhodamine- labeled anti-CD8. You use these antibodies to stain thymo- cytes and lymph-node cells from normal mice and from RAG-1 knockout mice. In the diagrams below, draw the FACS plots that you would expect. T-Cell Maturation, Activation, and Differentiation CHAPTER 10 245 Transgenic Immature Mature mouse thymocytes thymocytes female male female maleH-2 d H-2 d H-2 k H-2 k CD8 H11001 c. Explain your answers for the H-2 k transgenics. d. Explain your answers for the H-2 d transgenics. 2. Cyclosporin and FK506 are powerful immunosuppressive drugs given to transplant recipients. Both drugs prevent the formation of a complex between calcineurin and Ca 2+ /calmodulin. Explain why these compounds suppress T- cell–mediated aspects of transplant rejection. Hint: see Figure 10-11. CD8 Normal mice RAG-1 knockout mice Thymus Normal mice RAG-1 knockout mice Lymph node CD4 CD4 CD4 CD4 CD8 CD8 CD8 5. In order to demonstrate positive thymic selection experi- mentally, researchers analyzed the thymocytes from normal H-2 b mice, which have a deletion of the class II IE gene, and from H-2 b mice in which the class II IA gene had been knocked out. a. What MHC molecules would you find on antigen-pre- senting cells from the normal H-2 b mice? b. What MHC molecules would you find on antigen-pre- senting cells from the IA knockout H-2 b mice? c. Would you expect to find CD4 H11001 T cells, CD8 H11001 T cells, or both in each type of mouse? Why? 6. In his classic chimeric-mouse experiments, Zinkernagel took bone marrow from mouse 1 and a thymus from mouse 2 and transplanted them into mouse 3, which was thymec- tomized and lethally irradiated. He then challenged the re- constituted mouse with LCM virus and removed its spleen cells. These spleen cells were then incubated with LCM-in- fected target cells with different MHC haplotypes, and the lysis of the target cells was monitored. The results of two 8536d_ch10_221-247 8/29/02 10:23 AM Page 245 mac114 Mac 114:2nd shift:1268_tm:8536d: such experiments using H-2 b strain C57BL/6 mice and H-2 d strain BALB/c mice are shown in the table on the above. a. What was the haplotype of the thymus-donor strain in experiment A and experiment B? b. Why were the H-2 b target cells not lysed in experiment A but were lysed in experiment B? c. Why were the H-2 k target cells not lysed in either experi- ment? 7. Fill in the blank(s) in each statement below (a–k) with the most appropriate term(s) from the following list. Terms may be used once, more than once, or not at all. 8. You wish to determine the percentage of various types of thymocytes in a sample of cells from mouse thymus using the indirect immunofluorescence method. a. You first stain the sample with goat anti-CD3 (primary antibody) and then with rabbit FITC-labeled anti-goat Ig (secondary antibody), which emits a green color. Analysis of the stained sample by flow cytometry indicates that 70% of the cells are stained. Based on this result, how many of the thymus cells in your sample are expressing antigen-binding receptors on their surface? Would all be expressing the same type of receptor? Explain your an- swer. What are the remaining unstained cells likely to be? b. You then separate the CD3 H11001 cells with the fluorescence- activated cell sorter (FACS) and restain them. In this case, the primary antibody is hamster anti-CD4 and the sec- ondary antibody is rabbit PE-labeled anti-hamster-Ig, which emits a red color. Analysis of the stained CD3 H11001 cells shows that 80% of them are stained. From this re- sult, can you determine how many T C cells are present in this sample? If yes, then how many T C cells are there? If no, what additional experiment would you perform in order to determine the number of T C cells that are pre- sent? 9. Many of the effects of engaging the TCR with MHC-peptide can be duplicated by the administration of ionomycin plus a phorbol ester. Ionomycin is a Ca 2+ ionophore, a compound that allows calcium ions in the medium to cross the plasma membrane and enter the cell. Phorbol esters are analogues of diacylglycerol (DAG). Why does the administration of phor- bol and calcium ionophores mimic many effects of TCR en- gagement? 10. What effects on cell death would you expect to observe in mice carrying the following genetic modifications? Justify your answers. a. Mice that are transgenic for BCL-2 and over-express this protein. b. Mice in which caspase 8 has been knocked out. c. Mice in which caspase 3 has been knocked out. 11. Several basic themes of signal transduction were identified and discussed in this chapter. What are these themes? Con- sider the signal-transduction processes of T-cell activation and provide an example for each of six of the seven themes discussed. 246 PART II Generation of B-Cell and T-Cell Responses a. Lck and ZAP-70 are ______ . b. ______ is a T-cell membrane protein that has cytosolic domains with phosphatase activity. c. Dendritic cells express ______ constitutively, whereas B cells must be activated before they express this mem- brane molecule. d. Calcineurin, a ______ , is involved in generating the ac- tive form of the transcription factor NFAT. e. Activation of T H cells results in secretion of ______ and expression of its receptor, leading to proliferation and differentiation. f. The co-stimulatory signal needed for complete T H -cell activation is triggered by interaction of ______ on the T cell and ______ on the APC. g. Knockout mice lacking class I MHC molecules fail to produce thymocytes bearing ______ . h. Macrophages must be activated before they express ______ molecules and ______ molecules. i. T cells bearing ______ are absent from the lymph nodes of knockout mice lacking class II MHC molecules. j. PIP 2 is split by a ______ to yield DAG and IP 3 . k. In activated T H cells, DAG activates a ______ , which acts to generate the transcription factor NF-H9260B. l. ______ stimulates and ______ inhibits T-cell activation when engaged by ______ or on antigen-presenting cells. Thymectomized, x-irradiated recipient Lysis of LCM-infected target cells Experiment Bone-marrow donor A B C57BL/6 H11003 BALB/cC57BL/6 H11003 BALB/c C57BL/6 H11003 BALB/cC57BL/6 H11003 BALB/c H11001H11002H11002 H11002H11002H11001 H-2 b H-2 k H-2 d protein phosphatase(s) CD8 Class I MHC CD45 protein kinase(s) CD4 Class II MHC B7 CD28 IL-2 IL-6 CTLA-4 8536d_ch10_221-247 8/29/02 2:31 PM Page 246 mac114 Mac 114:2nd shift:1268_tm:8536d: T-Cell Maturation, Activation, and Differentiation CHAPTER 10 247 8536d_ch10_221-247 8/28/02 3:58 PM Page 247 mac76 mac76:385_reb: