《免疫学》（英文版） Chapter 10 T-Cell Maturation Activation, and Differentiation

8536dch102218/29/022:03 PM Page221mac83Mac83:379k T-Cell aturation,chapter 10 Activation and Differentiation HE ATTRIBUTE THAT DISTINGUISHES ANTIGEN recognition by most T cells from recognition by B ells is MHC restriction. In most cases, both the maturation of progenitor T cells in the thymus and the acti 901090 vation of mature T cells in the periphery are influenced by the involvement of MHC molecules. The potential antigeni 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 and CD8* subpopulations that exhibit class II and class I MHC restriction, respectively. Activation of mature peripheral T cells begins with the Engagement of TcR by Peptide: MHC Initiates interaction of the T-cell receptor( TCr)with an antigenic Signal Transduction peptide displayed in the groove of an MHC molecule. Al TCR, its low avidity necessitates the involvement of corecep-. T-Cell Maturation and the Thymus tors and other accessory membrane molecules that strengthen the TCR-antigen- MHC interaction and trans- Thymic Selection of the T-Cell Repertoire duce the activating signal Activation leads to the prolifera- TH-Cell Activation tion and differentiation of t cells into various types of T-Cell Differentiation ffector cells and memory T cells. Because the vast majority of thymocytes and peripheral T cells express the aB T-cell a Cell Death and T-Cell Populations receptor rather than the y8 T-cell receptor, all references to the T-cell receptor in this chapter denote the ap receptor un- Peripheral y8 T-Cells less otherwise indicated. Similarly, unless otherwise indi- cated, all references to T cells denote those aB receptor- bearing T cells that undergo MHC restriction As indicated in Chapter 2, the thymus occupies a central ole in T-cell biology. Aside from being the main source of all Tcells, it is where T cells diversify and then are shaped into effective primary T-cell repertoire by an extraordinary pair of T-Cell Maturation and the thymus selection processes. One of these, positive selection, permits the survival of only those T cells whose TCRs are capable of Progenitor T cells from the early sites of hematopoiesis begin recognizing self-MHC molecules. It is thus responsible for to migrate to the thymus at about day ll of gestation in mice the creation of a self-MHC-restricted repertoire of T cells. and in the eighth or ninth week of gestation in humans. In a The other, negative selection, eliminates T cells that react manner similar to B-cell maturation in the bone marrow, T- too strongly with self-MHC or with self-MHC Plus self- cell maturation involves rearrangements of the germ-line peptides. It is an extremely important factor in generating TCR genes and the expression of various membrane mark- a primary T-cell repertoire that is self-tolerant ers In the thymus, developing T cells, known as thymocytes, As shown in Figure 10-1, when T-cell precursors arrive at proliferate and differentiate along developmental pathways the thymus, they do not express such signature surface mark that generate functionally distinct subpopulations of mature ers of T cells as the T-cell receptor, the CD3 complex, or the T cells coreceptors CD4 and CD8. In fact, these progenitor cells have

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 ■ T-Cell Maturation and the Thymus ■ Thymic Selection of the T-Cell Repertoire ■ TH-Cell Activation ■ T-Cell Differentiation ■ Cell Death and T-Cell Populations ■ Peripheral 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 and CD8 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  T-cell receptor rather than the T-cell receptor, all references to the T-cell receptor in this chapter denote the  receptor un￾less otherwise indicated. Similarly, unless otherwise indi￾cated, all references to T cells denote those  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 ζ ζ γδε 8536d_ch10_221 8/29/02 2:03 PM Page 221 mac83 Mac 83:379_kyw:

8536d_ch10_221 8/27/02 1:37 PM Page 222 Mac 109 Mac 109: 1254_Bh Goldsby et al./Immunology 5e 222 PART II Generation of B-Cell and T-Cell Responses VISUALIZING CONCEPTS Surface markers Hematopoictic stem cell c-Kit CD25 (HSC) CD44 precursor Blood migration ◎ T-cell precursor TCR locus Pro-t cell 厂Dp ◎ (double TCR RAG Pre-T cell chain expression- Vp-Dp:)B (double CD3 Pre-1 IGURE 10-1 Development of aB Vp-Dp:) ProT cell TCR a CD4 T cells in the mouse. T-cell precursors (double arrive at the thymus from bone mar- chain row via the bloodstream, undergo de- velopment to mature T cells, and are exported to the periphery where they CD8+ CD4+ can undergo antigen-induced activa- tion and differentiation into effector T. cell ells and memory cells. Each stage blood of development is characterized by agespecific intracellular events and Peripheral CD8+ the display of distinctive cell-surfac tissues markers not yet rearranged their TCR genes and do not express pro- on early-stage DN cells. During this period, the cells are pro- arrangement. After arriving at the thymus, these T-cell cells stop expressing c-Kit, markedly reduce CD44 expres precursors enter the outer cortex and slowly proliferate Dur- sion, turn on expression of the recombinase genes RAG-I ing approximately three weeks of development in the thy- and RAG-2 and begin to rearrange their TCR genes. Al mus, the differentiating T cells progress through a series of though it is not shown in Figure 10-1, a small percentage stages that are marked by characteristic changes in their cell-(<5%)of thymocytes productively rearrange the y-and surface phenotype. For example, as mentioned previously, 8-chain genes and develop into double-negative CD3 y8 thymocytes early in development lack detectable CD4 and T cells In mice, this thymocyte subpopulation can be detected CD8. Because these cells are CD4 CD8, they are referred to by day 14 of gestation, reaches maximal numbers between as double-negative (DN)cell ays 17 and 18, and then declines until birth( Figure 10-2) Even though these coreceptors are not expressed during Most double-negative thymocytes progress down the ap the dN early stages, the differentiation program is progress- developmental pathway. They stop proliferating and begin to ing and is marked by changes in the expression of such cell rearrange the TCr B-chain genes, then express the p chain surface molecules as c-Kit, CD44, and CD25. The initial thy- Those cells of the aB lineage that fail to productively re- oocyte population displays c-Kit, the receptor for stem-cell range and express B chains die. Newly synthesized B chains growth factor, and CD44, an adhesion molecule involved in combine with a 33-kDa glycoprotein known as the pre-Ta homing; CD25, the B-chain of the IL-2 receptor, also appears chain and associate with the CD3 group to form a novel com-

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 CD4CD8, 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 -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 (5%) of thymocytes productively rearrange the - and -chain genes and develop into double-negative CD3 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  developmental pathway. They stop proliferating and begin to rearrange the TCR -chain genes, then express the  chain. Those cells of the  lineage that fail to productively re￾arrange and express  chains die. Newly synthesized  chains combine with a 33-kDa glycoprotein known as the pre-T chain and associate with the CD3 group to form a novel com￾VISUALIZING CONCEPTS FIGURE 10-1 Development of  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-Tα TCR β chain TCR α chain CD4 and CD8 CD4 or CD8 CD8+ CD4+ CD8+ CD4+ 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 Dβ-Jβ Vβ-Dβ-Jβ Vβ-Dβ-Jβ and Vα-Jβ TCR locus rearrangement Tc cell 8536d_ch10_221 8/27/02 1:37 PM Page 222 Mac 109 Mac 109:1254_BJN:Goldsby et al. / Immunology 5e:

8536ach1022-2478/28/023:58 PM Page223mac76mac76:3854 T-Cell Matur resses further rearrangement of TCR B-chain genes 16 Thymocytes Iting in allelic exclusio Renders the cell permissive for rearrangement of the oB Th the cD4 +o+ After advancing to the double-positive(DP)stage, where th cd4 and cD8 tes begin to proliferate. However, during this proliferative phase, TCR a-chain gene rearrangement does not occur; oth the RAg-1 and RAG-2 genes are transcriptionally ac 14 15 16 17 18 19 Birth Adult tive, but the RAG-2 protein is rapidly degraded in proliferat- ing cells, so rearrangement of the a-chain genes cannot take place. The rearrangement of a-chain genes does not begin FIGURE 10-2 Time course of appearance of yo thymocytes and until the double-positive thymocytes stop proliferating and tB thymocytes during mouse fetal development. The graph shows RAG-2 protein levels increase. The proliferative phase prior the percentage of CD3* cells in the thymus that are double-negative to the rearrangement of the a-chain increases the diversity of (CD48)and bear the y8 T-cell receptor(black) or are double. the T-cell repertoire by generating a clone of cells with a sin positive(CD48)and bear the ap T-cell receptor(blue) gle TCR B-chain rearrangement. Each of the cells within this clone can then rearrange a different a-chain gene, thereby generating a much more diverse population than if the orig plex called the pre-T-cell receptor or pre-TCR(Figure 10-3). inal cell had first undergone rearrangement at both the B- nd a-chain loci before it proliferated In mice, the tCR o nizes some intra-thymic ligand and transmits a signal chain genes are not expressed until day l6 or l7 of gestation: rough the CD3 complex that activates signal-transduction pathways that have several effects receptor begin to appear at day 17 and reach maximal levels about the time of birth(see Figure 10-2). The possession of a a Indicates that a cell has made a productive TCR B-chain complete TCR enables Dp thymocytes to undergo the rigors rearrangement and signals its further proliferation and of positive and negative selection maturation T-cell development is an expensive process for the host. in estimated 98% of all thymocytes do not mature-they die by apoptosis within the thymus either because they fail to Pre-TCR make a productive TCR-gene rearrangement or because they TCR fail to survive thymic selection. Double-positive thymocytes Pre-To that express the aB TCR-CD3 complex and survive thymic selection develop into immature single-positive CD4 thymocytes or single-positive CD8 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 Cell becomes Stops additional Thymic Selection of the TCR C-chain locus arrangements T-Cell Repertoire (allelic exclusion) Stimulates Random gene rearrangement within tCR germ-line DNA proliferation combined with junctional diversity can generate an enor- of cD and mous TCR repertoire, with an estimated potential diversity CD8 coreceptors ceeding 10 for the aB receptor and 10 for the y8 recep- tor Gene products encoded by the rearranged TCR genes hay FIGURE10-3Structure and activity of the pre-T-cell receptor(pre- no inherent affinity for foreign antigen plus a self-MHCmol TCR). Binding of ligands yet to be identified to the pre-TCR generates ecule; they theoretically should be capable of recognizing sol- intracellular signals that induce a variety of processes uble antigen(either foreign or self), self-MHC molecules,or

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: ■ Indicates that a cell has made a productive TCR -chain rearrangement and signals its further proliferation and maturation. T-Cell Maturation, Activation, and Differentiation CHAPTER 10 223 ■ Suppresses further rearrangement of TCR -chain genes, resulting in allelic exclusion. ■ Renders the cell permissive for rearrangement of the TCR  chain. ■ Induces developmental progression to the CD48 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 -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 -chain genes cannot take place. The rearrangement of -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 -chain increases the diversity of the T-cell repertoire by generating a clone of cells with a sin￾gle TCR -chain rearrangement. Each of the cells within this clone can then rearrange a different -chain gene, thereby generating a much more diverse population than if the orig￾inal cell had first undergone rearrangement at both the - and -chain loci before it proliferated. In mice, the TCR - chain genes are not expressed until day 16 or 17 of gestation; double-positive cells expressing both CD3 and the  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  TCR-CD3 complex and survive thymic selection develop into immature single-positive CD4 thymocytes or single-positive CD8 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 1015 for the  receptor and 1018 for the 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 thymocytes and  thymocytes during mouse fetal development. The graph shows the percentage of CD3 cells in the thymus that are double-negative (CD48) and bear the T-cell receptor (black) or are double￾positive (CD48) and bear the  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 14 15 16 17 18 CD3+ cells, % Days of gestation 19 Birth Adult αβ Thymocytes γδ Thymocytes Pre-TCR TCR β γ δ ς ς 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) S S S S S S S S 8536d_ch10_221-247 8/28/02 3:58 PM Page 223 mac76 mac76:385_reb:

8536dch10221-2478/29/0210:232 AM Page224mac114Mac114:24 d shift:1268tm:8536 224 PART II Generation of B-Cell and T-Cell Responses antigen plus a nonself-MHC molecule Nonetheless, the most EXPERIMENT 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 a Positive selection for thymocytes bearing receptors (A X B)FI(H-2 capable of binding self-MHC molecules, which results in MHC restriction. Cells that fail positive selection are Lethal x-irradiation eliminated within the thymus by apoptosis Strain-B thymus graft(H-2b) Negative selection that eliminates thymocytes bearing (A x B)F, hematopoictic sten high-affinity receptors for self-MHC molecules alone or cells(H-2a/b) self-antigen presented by self-MHC, which results in ← Infect with lcm virus self-tolerance Both processes are necessary to generate mature T cells that are self-MHC restricted and self-tolerant. As noted already me 98% or more of all thymocytes die by apoptosis within the thymus. The bulk of this high death rate appears to reflect LCM-infected LCM-infected a weeding out of thymocytes that fail positive selection be- rain-A cells strain-B cells cause their receptors do not specifically recognize foreign No killing 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 CONTROL ments by r. M. Zinkernagel and his colleagues(Figure 10-4). These researchers implanted thymectomized and ir Infect with LCM virus radiated(A X B)FI mice with a B-type thymus and then reconstituted the animals immune system with an intra venous infusion of F, bone-marrow cells. To be certain that (A X BF 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 X B)FI pleen cells bone-marrow transplant mature within a thymus that ex- sses only B-haplotype MHC molecules on its stromal cells. Would these(A X B)Fi T cells now be MHC- LCM-infected restricted for the haplotype of the thymus? To answer this strainA cells question, the chimeric mice were infected with LCM virus Killing and the immature T cells were then tested for their ability to kill LCM-infected target cells from the strain A or strain B FIGURE 10-4 Experimental demonstration that the thymus selects mice. As shown in Figure 10-4, when Tc cells from the or maturation only those T cells whose T-cell receptors recognize chimeric mice were tested on LCM virus infected target antigen presented on target cells with the haplotype of the thymus cells from strain A or strain B mice, they could only lyse Thymectomized and lethally irradiated (A X B)F, mice were grafted LCM-infected target cells from strain B mice. These mice with a strain-B thymus and reconstituted with (A X B)Fi bone- have the same MHC haplotype, B, as the implanted thymus. marrow cells. After infection with the LCM virus, the CTL cells were Thus, the MHC haplotype of the thymus in which T cells assayed for their ability to kill C-labeled strain-A or strain-B target develop determines their MHC restriction. cells infected with the LCM virus. Only strain-B target cells were Thymic stromal cells, including epithelial cells, macro- lysed, suggesting that the H-2b grafted thymus had selected for phages, and dendritic cells, play essential roles in positive and maturation only those T cells that could recognize antigen combined negative selection. These cells express class I MHC molecules with H-2bMHC molecules and can display high levels of class II mHC also. The interac ion of immature thymocytes that express the TCR-CD3 Positive Selection Ensures MHC Restriction complex with populations of thymic stromal cells results in positive and negative selection by mechanisms that are under Positive selection takes place in the cortical region of the thy intense investigation. First, we'll examine the details of each mus and involves the interaction of immature thymocytes selection process and then study some experiments that pro- with cortical epithelial cells(Figure 10-5). There is evidence vide insights into the operation of these processes. that the T-cell receptors on thymocytes tend to cluster with

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: ■ 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. ■ 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 B) F1 mice with a B-type thymus and then reconstituted the animal’s immune system with an intra￾venous infusion of F1 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 B) F1 bone-marrow transplant mature within a thymus that ex￾presses only B-haplotype MHC molecules on its stromal cells. Would these (A B) F1 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 TC 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 B) F1 mice were grafted with a strain-B thymus and reconstituted with (A B) F1 bone￾marrow cells. After infection with the LCM virus, the CTL cells were assayed for their ability to kill 51Cr-labeled strain-A or strain-B target cells infected with the LCM virus. Only strain-B target cells were lysed, suggesting that the H-2b grafted thymus had selected for maturation only those T cells that could recognize antigen combined with H-2b MHC molecules. Lethal x-irradiation Thymectomy EXPERIMENT (A × B)F1 (H–2a/b) Strain-B thymus graft (H–2b) (A × B)F1 hematopoietic stem cells (H–2a/b) Infect with LCM virus Spleen cells CONTROL Infect with LCM virus (A × B)F1 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:

8536ach1022-2478/28/023:58 PM Page225mac76mac76:3854 T-Cell Matur During positive selection, the RAG-1, RAG-2, and TdT T-cell precursor proteins required for gene rearrangement and modification continue to be expressed. Thus each of the immature thymo- tes in a clone expressing a given p chain have an opportu Rearrangement of TCR genes nity to rearrange different TCR a-chain genes, and the resulting TCRs are then selected for self-MHC recognition Only those cells whose ap TCR heterodimer recognizes T-cell receptor self-MHC molecule are selected for survival. Consequentl the presence of more than one combination of ap TCR thymocyte chains among members of the clone is important because it increases the possibility that some members will"pass"the Positive selection of Death by apoptosis test for positive selection. Any cell that manages to rearrange cells whose receptor/ of cells that do not interact an a chain that allows the resulting ap TCR to recognize self- binds mhc molecules with MHC molecules MHC will be spared; all members of the clone that fail to do so will die by apoptosis within 3 to 4 days Class i and /or class Il MHC molecules 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 mocytes undergo negative selection by an interaction with thymic stromal cells. During negative selection, dendritic ls and macrophages bearing class I and class II MHC mol selection a ecules interact with thymocytes bearing high-affinity recep- high-affinity recepto rs for self-antigen plus self-MHC molecules or for for self- MHC or self-MHC self-antigen self-MHC molecules alone(see Figure 10-5). However, the precise details of the process are not yet known. Cells that ex 原画喜 perience negative selection are observed to undergo death by apoptosis. Tolerance to self-antigens encountered in the thy nus is thereby achieved by eliminating T cells that are reac- TH cell Tccell Mature CD +or Experiments Revealed the Essential Elements CDS* Tlymphocytes 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 Dendritic cell thymus came from experimental studies with knockout mice In the Incapable of producing functional class I or class II MHC FIGURE 10-5 Positive and negative selection of thymocytes in the molecules (Table 10-1). Class l-deficient mice were found to thymus. Thymic selection involves thymic stromal cells (epithelial have a normal distribution of double-negative, double-posi- cells, dendritic cells, and macrophages), and results in mature T cells tive, and CD4 thymocytes, but failed to produce CD8* thy hat are both self-MHC restricted and self-tolerant mocytes. Class II-deficient mice had double-negative, double-positive, and CD8 thymocytes but lacked CD4 thymocytes. Not surprisingly, the lymph nodes of these class II-deficient mice lacked CD4tT cells. Thus, the absence of MHC molecules on the cortical cells at sites of cell-cell con- class I or II MHC molecules prevents positive selection of tact. Some researchers have suggested that these interactions CD8 or CD4 T cells, respectively allow the immature thymocytes to receive a protective signal Further experiments with transgenic mice provided addi- that prevents them from undergoing cell death; cells whose tional evidence that interaction with MHC molecules plays a receptors are not able to bind MHC molecules would not in- role in positive selection. In these experiments, rearranged teract with the thymic epithelial cells and consequently aB-TCR genes derived from a CD8 T-cell clone specific for would not receive the protective signal, leading to their death influenza antigen plus H-2 class I MHC molecules were in- by jected into fertilized eggs from two different mouse strains

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  chain have an opportu￾nity to rearrange different TCR -chain genes, and the resulting TCRs are then selected for self-MHC recognition. Only those cells whose  TCR heterodimer recognizes a self-MHC molecule are selected for survival. Consequently, the presence of more than one combination of  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  chain that allows the resulting  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 thymocytes, but failed to produce CD8 thy￾mocytes. Class II–deficient mice had double-negative, double-positive, and CD8 thymocytes but lacked CD4 thymocytes. Not surprisingly, the lymph nodes of these class II–deficient mice lacked CD4 T cells. Thus, the absence of class I or II MHC molecules prevents positive selection of CD8 or CD4 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 -TCR genes derived from a CD8 T-cell clone specific for influenza antigen plus H-2k 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+ TH cell TC 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: