《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:

9536d_ch102212478/28/023:58 PM Page226mac76ma76:385e 226 RT II Generation of B-Cell and T-Cell Respons TABLE10·1 Effect of class I or lI MHC deficiency tured in vitro with antigen-presenting cells expressing the on thymocyte populations H-Y antigen, the thymocytes were observed to undergo apoptosis, providing a striking example of negative selection KNOCKOUT MICE Some Central Issues in Thymic Sel Class I Class ll Cell type deficient deficient Remain Unresolved Although a great deal has been learned about the develop CD4 CD mental processes that generate mature CD4 and CD8 T CD4+CD8+ cells, some mysteries persist. Prominent among them is a seeming paradox: If positive selection allows only thymo CD8 cytes reactive with self-MHC molecules to survive, and nega- Plus sign indicates normal distribution of indicated cell types in thymus cytes, then no T cells would be allowed to mature. Since this Minus sign indicates absence of cell type. 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 one with the H-2 haplotype and one with the H Experimental evidence from fetal thymic organ culture type(Figure 10-6). Since the receptor transgenes (FTOC)has been helpful in resolving this puzzle. In this sy ready rearranged, other TCR-gene rearrangement tem, mouse thymic lobes are excised at a gestational age of day suppressed in the transgenic mice; therefore, a high percent- 16 and placed in culture. At this time, the lobes consist pre- age of the thymocytes in the transgenic mice expressed the dominantly of CD48 thymocytes. Because these immature, T-cell receptor encoded by the transgene. Thymocytes double-negative thymocytes continue to develop in the organ expressing the TCR transgene were found to mature into culture, thymic selection can be studied under conditions that CD8* T cells only in the transgenic mice with the H-2 class permit a range of informative experiments. Particular use has I MHC haplotype (i.e, the haplotype for which the transgene receptor was restricted). In transgenic mice with a different CD8 IHC haplotype(H-2), immature, double-positive thyme tes expressing the transgene were present, but these thy Influenza. oocytes failed to mature into CD8 T cells. These findings infected ≈○ clone confirmed that interaction between T-cell receptors on im mature thymocytes and self-MHC molecules is required for Class I mhc ositive selection In the absence of self-mHc molecules,as (H2) in the H-2 transgenic mice, positive selection and subse aB-TCR genes 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 aB TCR trans- gene specific for the class I D MHC molecule plus H-Y anti- gen, a small protein that is encoded on the Y chromosome Thymocytes transgenic transgenic and is therefore a self-molecule only in male mice. In this periment, the MHC haplotype of the transgenic mice was In transgenics H-2, the same as the MHC restriction of the transgene- TCR*/CD4+8* encoded receptor. Therefore any differences in the selection TCR+/CD8+ of thymocytes in male and female transgenics would be re- lated to the presence or absence of H-Y antigen. FIGURE 10-6Effect of host haplotype on T-cell maturation in mice Analysis of thymocytes in the transgenic mice revealed carrying transgenes encoding an H-2 class I-restricted T-cell recep that female mice contained thymocytes expressing the H-Y- tor specific for influenza virus. The presence of the rearranged TCR ecific TCR transgene, but male mice did not(Figure 10-7). transgenes suppressed other gene rearrangements in the transgen- In other words, H-Y-reactive thymocytes were self-reactive ics; therefore, most of the thymocytes in the transgenics expressed in the male mice and were eliminated. However, in the female the aB T-cell receptor encoded by the transgene Immature double- ansgenics, which did not express the H-Y antigen, these positive thymocytes matured into CD8* T cells only in transgenics cells were not self-reactive and thus were not eliminated. with the haplotype(H-2)corresponding to the MHC restriction of When thymocytes from these male transgenic mice were cul- the TCR transgene

one with the H-2k haplotype and one with the H-2d 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 T cells only in the transgenic mice with the H-2k class I MHC haplotype (i.e., the haplotype for which the transgene receptor was restricted). In transgenic mice with a different MHC haplotype (H-2d ), immature, double-positive thymo￾cytes expressing the transgene were present, but these thy￾mocytes failed to mature into CD8 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-2d 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  TCR trans￾gene specific for the class I Db 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-2b , 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 and CD8 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 CD48 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 CD4CD8   CD4CD8   CD4   CD8   * 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-2b 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  T-cell receptor encoded by the transgene. Immature double￾positive thymocytes matured into CD8 T cells only in transgenics with the haplotype (H-2k ) corresponding to the MHC restriction of the TCR transgene. Thymocytes in transgenics TCR+/CD4+8+ TCR+/CD8+ H–2k transgenic + + + − Influenza￾infected target cell TC-cell clone (H-2k) CD8 Class I MHC (H-2k) αβ-TCR genes H–2d transgenic 8536d_ch10_221-247 8/28/02 3:58 PM Page 226 mac76 mac76:385_reb:

9536d_ch102212478/28/023:58 PM Page227mac76ma76:385e T-Cell Matur CTL H-Y specific H-2Db restricted peptic Clone TCR Male cell(H-2D) a and B genes Female cell(H-2Db) Use to make a H-Y TCR transgenic mice FIGURE 10-7Experimental demonstration that gative selection of thymocyte gen plus self-MHC. In this experiment, H-2male and female prepared carrying transgenes specific for H-Y antigen plus the D" mol Male h-2Db Female h-2Db ecule. This antigen is expressed only in males. FACS analysis of thymocytes from the transgenics showed that mature CD8" T cells expressing the transgene Thymocytes were absent in the male mice but present in the fe- CD4-8 male mice, suggesting that thymocytes reactive with CD4+8+ a self-antigen(in this case, H-Y antigen in the male mice)are deleted during thymic selection [ Adapted from H won Boehmer and P. Kisielow, 1990, Science CDSt 248:1370 been made of mice in which the peptide transporter, TAP-1, concentrations of peptide. At low peptide concentrations, has been knocked out. In the absence of TAP-1, only low levels few MHC molecules bound peptide and the avidity of the of MHC class I are expressed on thymic cells, and the develop- TCR-MHC interaction was low. As peptide concentrations ment of CD8* thymocytes is blocked. However, when exoge- were raised, the number of peptide-MHC complexes dis- nous peptides are added to these organ cultures, then played increased and so did the avidity of the interaction. In peptide-bearing class I MHC molecules appear on the surface this experiment, very few CD8 cells appeared when peptide of the thymic cells, and development of CD8* T cells is re- was not added, but even low concentrations of the relevant stored. Significantly, when a diverse peptide mixture is added, peptide resulted in the appearance of significant numbers of the extent of CD8 T-cell restoration is greater than when a CD8 T cells bearing the transgenic TCR receptor. Increas- ingle peptide is added. This indicates that the role of peptide ing the peptide concentrations to an optimum range yielded is not simply to support stable MHC expression but also to be the highest number of CD8 T cells. However, at higher con recognized itself in the selection process centrations of peptide, the numbers of CD8 T cells pro- q Two competing hypotheses attempt to explain the Para- duced declined steeply. The results of these experiments dox of MHC-dependent positive and negative selection. The show that positive and negative selection can be achieved avidity hypothesis asserts that differences in the strength of with signals generated by the same peptide-MHC combina- the signals received by thymocytes undergoing positive and tion. No signal (no peptide) fails to support positive selec negative selection determine the outcome, with signal tion. A weak signal (low peptide level)induces positive strength dictated by the avidity of the TCR-MHC-peptide in- selection. However, too strong a signal(high peptide level) teraction. The differential-signaling hypothesis holds that the results in negative selection outcomes of selection are dictated by different signals, rather The differential-signaling model provides an alternative than different strengths of the same signal explanation for determining whether a T cell undergoes posi- The avidity hypothesis was tested with TAP-1 knockout tive or negative selection. This model is a qualitative rather mice transgenic for an aB TCR that recognized an LCM virus than a quantitative one, and it emphasizes the nature of the peptide-MHC complex. These mice were used to prepare fe- signal delivered by the TCR rather than its strength. At tal thymic organ cultures(Figure 10-8). The avidity of the core of this model is the observation that some MHC-peptide TCR-MHC interaction was varied by the use of different complexes can deliver only a weak or partly activating signal

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 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 T cells is re￾stored. Significantly, when a diverse peptide mixture is added, the extent of CD8 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  TCR 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 cells appeared when peptide was not added, but even low concentrations of the relevant peptide resulted in the appearance of significant numbers of CD8 T cells bearing the transgenic TCR receptor. Increas￾ing the peptide concentrations to an optimum range yielded the highest number of CD8 T cells. However, at higher con￾centrations of peptide, the numbers of CD8 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-2b male and female transgenics were prepared carrying TCR transgenes specific for H-Y antigen plus the Db mol￾ecule. This antigen is expressed only in males. FACS analysis of thymocytes from the transgenics showed that mature CD8 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-2Db Female H-2Db H-Y expression Thymocytes CD4−8− CD4+8+ CD4+ CD8+ + + + + + − − + + + + + + Clone TCR α and β genes Female cell (H-2Db Male cell (H-2D ) b) CTL H-Y specific H-2Db restricted H-Y peptide α β × 8536d_ch10_221-247 8/28/02 3:58 PM Page 227 mac76 mac76:385_reb:

9536d_ch102212478/28/023:58 PM Page228mac76ma76:385e 228 PART 11 Generation of B-Cell and T-Cell Responses while others can deliver a complete signal. In this model, pos- pression was artificially raised to twice its normal level, the itive selection takes place when the TCRs of developing thy- concentration of mature CD8 cells in the thymus was one mocytes encounter MHC-peptide complexes that deliver thirteenth of the concentration in control mice bearing nor- weak or partial signals to their receptors, and negative selec- mal levels of CD8 on their surface. Since the interaction of T tion results when the signal is complete. At this point it is not cells with class I MHC molecules is strengthened by partici- ossible to decide between the avidity model and the differen- pation of CD8, perhaps the increased expression of CD tial-signaling model; both have experimental support. It may would increase the avidity of thymocytes for class I mole be that in some cases, one of these mechanisms operates to the cules, possibly making their negative selection more likely. complete exclusion of the other. It is also possible that no sin- Another important open question in thymic selection is gle mechanism accounts for all the outcomes in the cellular how double-positive thymocytes are directed to become ei- teractions that take place in the thymus and more than one ther CD4 8 or CD4 8 T cells. Selection of CD4 8 thy- mechanism may play a significant role. Further work is re- mocytes gives rise to class I MHC-restricted CD8 T cells quired to complete our understanding of this matter. and class II-restricted CD4+T cells. Two models have been The differential expression of the coreceptor CD8 also can proposed to explain the transformation of a double-positive ffect thymic selection. In an experiment in which CD8 ex- precursor into one of two different single-positive lineag (a) Experimental procedure-fetal thymic organ culture(FTOC) Place in Porous membrane Growth medium (b) Development of CDS* CD4- MHC I-restricted cell Thymocyte Degree of Thymus Thymic CD8+ T-cell FIGURE 10-8 Role of peptides in selection. donor peptide added development Thymuses harvested before their thymocyteNormal populations have undergone positive and negative selection allow study of the develop- ment and selection of single positive Norma CD4"CD8 and CD4" CD8T)T cells(a) Peptide Outline of the experimental procedure for in vitro fetal thymic organ culture(FTOC).(b) The development and selection ofTCR-transgenic CD8"CD4 class I-restricted T cells depends TAP.1 deficient on TCR peptide-MHC I interactions. TAP. None Minimal knockout mice are unable to form peptide- MHC complexes unless peptide is added The mice used in this study were transgenic for the a and B chains of a TCR that recog- Weak signal nizes the added peptide bound to MHC molecules of the TAP. knockout/TCR trans Approaches genic mice. Varying the amount of added pep- normal tide revealed that low concentrations of peptide, producing low avidity of binding, re- sulted in positive selection and nearly normal Strong signal levels of CD4"CD8" cells. High concentra- tions of peptide, producing high avidity of binding to the TCR, caused negative selection, Minimal and few CD4-CD8+ t cells (Adapted from Ashton Rickardt et al.(1994) e25:651

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 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 CD48 or CD48 T cells. Selection of CD48 thy￾mocytes gives rise to class I MHC–restricted CD8 T cells and class II–restricted CD4 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 (CD4CD8 and CD4CD8) T cells. (a) Outline of the experimental procedure for in vitro fetal thymic organ culture (FTOC). (b) The development and selection of CD8CD4 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  and  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 CD4CD8 cells. High concentra￾tions of peptide, producing high avidity of binding to the TCR, caused negative selection, and few CD4CD8 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:

8536ach1022-2478/28/023:58 PM Page229mac76mac76:3854 T-Cell Matur INSTRUCTIVE MODEL CD4+8 D8 E-◎ CDiloghi CD4-8+T cell STOCHASTIC MODEL g+ class I MHC CD4 CD4+8+ CLogh Ag+ class I MHC Apoptosis of the CD4 and CD8 coreceptors in thymic se- lection of double positive thymocytes leading single positive T cells. According to the Able to bind structive model, interaction of one coreceptor Ag+ class II MHC with MHC molecules on stromal cells results Random CD4+8-Tcell in down-regulation of the other coreceptor. Not able to bind According to the stochastic model, down Ag class II MHC → Apoptosis regulation of CD4 or CD8 is a random process (Figure 10-9). The instructional model postulates that the and differentiating into memory cells or effector cells. Many multiple interactions between the TCR, CD8 or CD4 of the gene products that appear upon interaction with anti coreceptors, and class I or class II MHC molecules instruct gen can be grouped into one of three categories depending the cells to differentiate into either CD8 or CD4 single- on how early they can be detected after antigen recognition positive cells, respectively. This model would predict that a ( Table 10-2) class I MHC-specific TCR together with the CD8 would generate a signal that is different from the signal in-. Immediate genes, expressed within half an hour of duced by a class II MHC-specific TCR together with the tigen recognition, encode a number of transcription CD4 coreceptor. The stochastic model suggests that CD4 or factors, including c-Fos, c-Myc, c-Jun, NFAT, and NF-KB CD8 expression is switched off randomly with no relation to Early genes, expressed within 1-2 h of antigen the specificity of the TCR. Only those thymocytes whose recognition, encode IL-2, IL-2R(IL-2 receptor), IL-3, TCR and remaining coreceptor recognize the same class of IL-6, IFN-Y, and numerous other proteins MHC molecule will mature At present, it is not possible to. Late genes, expressed more than 2 days after antigen choose one model over the other recognition, encode various adhesion molecules These profound changes are the result of signal-transduction TH-Cell Activation pathways that are activated by the encounter between the TCR and MHC-peptide complexes. An overview of some of The central event in the generation of both humoral and cell- the basic strategies of cellular signaling will be useful back pansion of TH cells. Activation of Tc cells, which is generally by T cells. preciating the specific signaling pathways used mediated immune responses is the activation and clonal ex- ground for app similar to TH-cell activation, is described in Chapter 14.TH- cell activation is initiated by interaction of the TCR-CD3 Signal-Transduction Pathways Have Several mplex with a processed antigenic peptide bound to a class Features in Common II MHC molecule on the surface of an antigen-presenting cell. This interaction and the resulting activating signals also The detection and interpretation of signals from the environ involve various accessory membrane molecules on the TH ment is an indispensable feature of all cells, including those of cell and the an tigen -presenting c ell. Interaction of a TH cell the immune system. Although there are an enormous number with antigen initiates a cascade of biochemical events that in- of different signal-transduction pathways, some common duces the resting TH cell to enter the cell cycle, proliferating themes are typical of these crucial integrative processes

(Figure 10-9). The instructional model postulates that the multiple interactions between the TCR, CD8 or CD4 coreceptors, and class I or class II MHC molecules instruct the cells to differentiate into either CD8 or CD4 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. TH-Cell Activation The central event in the generation of both humoral and cell￾mediated immune responses is the activation and clonal ex￾pansion of TH cells. Activation of TC cells, which is generally similar to TH-cell activation, is described in Chapter 14. TH￾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 TH cell and the antigen-presenting cell. Interaction of a TH cell with antigen initiates a cascade of biochemical events that in￾duces the resting TH 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): ■ 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-B ■ Early genes, expressed within 1–2 h of antigen recognition, encode IL-2, IL-2R (IL-2 receptor), IL-3, IL-6, IFN-, and numerous other proteins ■ 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 CD4lo8hi CD4hi8lo CD4+8+ CD4+8+ CD4lo8hi CD4hi8lo 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:

8536d_ch10_221 8/27/02 1: 37 PM Page 230 Mac 109 Mac 109: 1254_BpLGoldsby et al./Immunology 5e 230 PART I1 Generation of B-Cell and T-Cell Responses LE 10-2 Time course of gene expression by TH cells following interaction with antigen Time mRNA Ratio of activated to Gene product Function expression begins Location nonactivated cells MMEDIATE Protooncoger 5 min Nucleus >100 c-Jun Cellular oncogene; 15-20mn Nucleus transcription factor NFAT Transcription factor 20 min Nucleus Cellular oncogene 30 min Nucleus NF-KB Transcription factor 30 min Nucleus >10 EARLY tokine 30 min 100 45 min 1000 Insulin receptor Hormone receptor 1-2h TGF-B 100 4-6h >10 IL-4 ccc 100 IL-5 100,?,?, SOURCE: Adapted from G. Crabtree, Science 243: 357 a Signal transduction begins with the interaction between a signal and its receptor. Signals that cannot penetrate the hydrolysis of GTP or exchange for GDP turns off the cell membrane bind to receptors on the surface of the signal by returning the G protein to an inactive form. cell membrane. This group includes water-soluble There are two major categories of G proteins. Small G signaling molecules and membrane-bound ligands proteins consist of a single polypeptide chain of about (MHC-peptide complexes, for example). Hydrophobi 21 kDa. An important small G protein, known as Ras, signals, such as steroids, that can diffuse through the cell is a key participant in the activation of an importan embrane are bound by intracellular receptors. proliferation-inducing signal-transduction cascade triggered by binding of ligands to their receptor tyrosine Signals are often transduced through G proteins, kinases. Large G proteins are composed of a,B, and Y membrane-linked macromolecules whose activities subunits and are critically involved in many processes, controlled by binding of the guanosine nucleotides GTP including vision, olfaction, glucose metabolism, and and gDP which act as molecular switches. Bound GTP phenomena of immunological interest such as leukocyte turns on the signaling capacities of the G protein

■ 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. ■ 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 , , and 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 TH 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  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-B Transcription factor 30 min Nucleus  10 EARLY IFN- Cytokine 30 min Secreted  100 IL-2 Cytokine 45 min Secreted  1000 Insulin receptor Hormone receptor 1 h Cell membrane 3 IL-3 Cytokine 1–2 h Secreted  100 TGF- Cytokine  2 h Secreted  10 IL-2 receptor (p55) Cytokine receptor 2 h Cell membrane  50 TNF- Cytokine 1–3 h Secreted  100 Cyclin Cell-cycle protein 4–6 h Cytoplasmic  10 IL-4 Cytokine  6 h Secreted  100 IL-5 Cytokine  6 h Secreted  100 IL-6 Cytokine  6 h Secreted  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  100 VLA-1, VLA-2, VLA-3, VLA-5 Adhesion molecules 7–14 days Cell membrane  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: