CD8 is a heterodimeric cell surface glycoprotein expressed on the mature T lymphocyte subset that recognizes foreign antigen in association with class I major histocompatibility complex (MHC) proteins and has cytotoxic function. The main goal of this proposal is to further elucidate the role of CD in T cell activation and development by analysis of the function of the CD8beta chain in these processes. First, the mechanisms by which CD8beta stimulates T cell responses will be explored in several ways: Binding studies to a recombinant, soluble for of class I linked to beta/2- microglobulin will be used to determine whether CD8beta it can bind directly to class I MHC proteins. Transfectants expressing various forms of CD8beta and/or CD8alpha will be used to assess the role of CD8beta in the activation-dependent increase in the avidity of binding of CD8alphabeta to class I MHC. The direct or indirect interaction between CD8beta and other surface proteins that might enhance signaling will be analyzed in coimmunoprecipitation and blotting studies. The interaction of the CD8beta cytoplasmic tail with proteins that may be involved in signaling through CD8beta will be analyzed using the yeast two-hybrid system. A second major aim is to determine the physiological significance of CD8alpha-independent expression of CD8beta that we have previously identified in fetal and adult thymocytes. The presence of such forms of CD8beta will be analyzed by cell staining and flow cytometric analysis of thymocytes and peripheral lymphoid cells in normal and mutant mice, both with and without activation stimuli. The biochemical nature of such complexes will be explored to determine whether they indeed represent the CD8beta/TCRbeta disulfide-linked heterodimers we have observed in transfected cells. The function of such a complex and the cells bearing it will be examined by mAb treatment of fetal thymic organ cultures, by thymic reconstitution experiments using this population isolated from day 14 fetal thymus and by evaluation of the signaling capacity of CD8beta/TCRbeta heterodimers. An attempt will be made to generate a mutant form of CD8beta that no longer dimerizes with TCRbeta but retains dimerization and functional capacity with CD8alpha. If obtained, such a mutant would be used to distinguish effects of these two forms of dimers during thymocyte that has been identified on transfectants and that appears to be present on a subset of thymocytes. These studies will aid in our understanding of how cytotoxic T cells are activated to kill virally infected or tumor cells and may additionally provide insight into the regulation of their developmental pathway.
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