This proposal focuses on various aspects of T cell differentiation in vivo, with particular emphasis on how H-2 molecules select T cells in the thymus and then control the function of these cells in the peripheral lymphoid tissues. Three main areas of investigation are proposed. 1) T cell development in the thymus. Previous work from this lab has led to a model in which H-2-restricted T cells are selected in the thymus in two steps: i) positive selection by cortical epithelial cells followed by ii) negative selection (deletion of high affinity auto H-2-reactive cells) by bone marrow (BM)-derived cells in the medulla. To assess this model of thymic selection, various experiments are proposed, including 1) using monoclonal antibodies (mAb) specific for different T cell markers to determine the precise anatomical distribution of mature vs. immature thymocytes in frozen sections of thymus, b) establishing whether stem cells are situated only in the medulla or in both the cortex and medulla, c) studying the mechanism of positive selection, e.g. by culturing thymic stem cells with cloned lines of epithelial cells, d) investigating why """"""""unselected"""""""" T cells die in situ, and e) determining whether epithelial cells contribute to negative selection. 2) H-2 tolerance in the extrathymic environment. Parent to Fl BM chimeras prepared with supralethal irradiation are essentially devoid of host BM-derived cells. However, the T cells from these chimeras show substantial, though incomplete tolerance to host-type H-2 determinants, which suggests that post-thymic contact with H- 2 determinants on host stromal (non-BM-derived) cells is tolerogenic. Further information on this question will be sought by i) purifying CD4+ and CD8+ cells from parent to Fl chimeras and assessing tolerance to host-type H-2 determinants with several different assay systems, including induction of graft-versus-host disease, ii) assessing the affinity of chimera T cells retaining anti-host reactivity, and iii) searching for tolerance induction when normal parental strain T cells are transferred to parent to Fl chimeras. 3) B cells as antigen-presenting cells in vivo. Data from several models has shown that B cells play a decisive role in presenting antigen to unprimed T cells in the environment of lymph nodes. To pursue this finding we hope to establish whether the in vivo antigen-presenting cells (APC) function of B cells is (a) controlled by antigen-specific cells and (b) requires prior T cell contact with non-B APC.
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