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 transgeneone 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: