The goal of this application is to elucidate mechanisms by which bone marrow grafts are rejected in mice. Achieving this goal will help understand the rejection of allogeneic marrow grafts in man and the design of approaches to overcome rejection for therapeutic benefit. This application is based on results obtained during the previous project period in which a mechanism responsible for acute rejection of marrow grafts was shown to be due to cells that co-expresses NK1 and CD3. The objective of this application is to test several hypotheses, the first of which is that NK1 CD3 cells express specific cytotoxicity. Others are that NK1 CD3 cells utilize T-cell receptors or NK cell receptors in rejection and cytolytic function and that MHC class I antigens are recognized. NK1+ CD3+ will be purified from spleen of normal or nude mice by fluorometric cell sorting. Cultures supplemented with cytokines to stimulate cell proliferation and cytotoxicity will be established. Cytolytic activity on tumor and lymphoblast targets will be assayed to investigate specificities. Specificity of in vitro target cell lysis will be compared to specificity of marrow rejection. To probe the participation of TCR, its function will be blocked by anti-TCR F(ab')2 fragments. If inhibition is seen, NK1+ CD3+ cells from TCR transgenic mice will be isolated and cytotoxic specificities examined and compared to those of NK1+ CD3- cells. The function of NK receptors on NK1+ CD3+ cells will be probed by using F)ab')2 fragments specific for NK receptors, i.e., NK1.1, Ly49, 5E6 to block target recognition and lysis. Both NK1+ CD3+ and NK1+ CD3- cells will be assayed for cytotoxicity on targets from MHC recombinant or transgenic mice to map specificity or transfectants expressing various MHC class I specificities. MHC class I specific F(ab')2 fragments will be used for in vitro blocking to map epitope specificities. NK1+ CD3+ cells from TCR transgenic mice lacking distinct TCR specificities will be used to examine whether MHC class I antigens are recognized and what their specificity is. Attempts will be made to interfere with recognition of MHC class I in vivo during development of NK1+ CD3+ cells and it will be examined how this affects specificity of marrow graft rejection and cytotoxic specificity of effectors. The role of NK1+ CD3- cells in marrow rejection will be examined by exploring the specificity of marrow rejection in SCID mice and in case it is MHC specific, it will be mapped to a specific MHC region. NK1+ CD3- cells from SCID mice will be isolated and specificity of target cell lysis compared with that of bone marrow rejection. Precise MHC epitope specificity of effectors will also be determined. Finally, the specificity of NK1+ CD3- and NK1+ CD3+ cells from normal mice will be compared with that of cells from TCR transgenic mice to examine whether in normal mice NK1 receptors may play a dominant role in acute marrow graft rejection.
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