The development of immature T cells into mature, antigen-specific, functional T lymphocytes is central to immune defenses against pathogens. It is essential that this process be efficient, lest autoreactive T lymphocytes develop and migrate to the peripheral lymphoid system where they can mediate autoimmune disease. Class I MHC molecules present on epithelial cell surfaces play a pivotal role in the development of thymocytes. The long-term goals of the proposed studies are to elucidate the subcellular organization of thymic epithelium and to determine if a specialized distribution of MHC molecules on specific membrane domains of thymic epithelial cells plays a role in the development of thymocytes. Glycosylphosphatidylinositol (GPI)-tethered proteins are targeted to specialized membrane domains in certain cell types, including neurons and epithelial cells. A GPI-tethered class I MHC molecule has been reported to be defective in mediating the development of thymocytes whose antigen receptors are specific for that class I MHC molecule. To determine if thymic epithelium has specialized membrane domains, mutant forms of the mouse H-2Db class I MHC molecule, including a GPI-tethered form, will be generated and introduced into the germline of mice. These transgenic mice will then be mated to other transgenic mice which express a T-cell receptor specific for H-Y antigen + H-2Db to generate double transgenic mice. The development of T lymphocytes in single and double transgenic female and male mice will be analyzed. The submembranic localization of wild type and mutant H-2Db molecules will be examined to determine if a GPI tether causes a redistribution of H-2Db on the surfaces of thymic epithelial cells. Because GPI-anchored proteins also differ from wild type class I MHC molecules in terms of their trafficking to and from the cell surface, the endocytosis and recycling of wild type and mutant H-2Db molecules will be studied. Finally, since many GPI-tethered proteins have been shown to form protein complexes with tyrosine protein kinases known to be involved in signal transduction pathways, biochemical studies will be performed to determine if H-2Db-GPI molecules associate with protein kinases. These studies provide a model system for analyzing the role that the thymic architecture plays in T lymphocyte development. These studies have implications for malfunctions of the immune system such as autoimmunity and immunodeficiency diseases.
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