In preliminary work, we conducted an extensive transmission electron microscopic survey of normal thymus as well as light microscopic studies on the architecture of the thymus by the use of monoclonal antibodies directed against the major histocompatibility complex, major surface determinants found on thymic lymphocytes, and cell surface gangliosides (with secondary reagents coupled to peroxidase). These studies were correlated with studies employing conventional enzyme histochemistry to create an overall description of the thymic microenvironment with emphasis on nonlymphoid components. Using this model, we hypothesized that microenvironments exist in the thymus that promote extensive proliferation of immature thymic lymphocytes perhaps leading to mature thymic lymphocytes of the medullary phenotype. We propose the creation of such thymic microenvironments in vitro by culture of thymic macrophages with the aid of CSF-1 and creation of tumors of other nonlymphoid components by induced carcinogenesis. As an alternative approach toward the creation of permanent cell lines of nonlymphoid thymic components, we have created a HAT-sensitive variant of the epithelial line CSG.211, and with this we propose the creation of hybrid cell lines that retain functional characteristics of nonlymphoid cell components. We propose the mixing of these cell populations in vitro in the presence of immature thymic lymphocytes in order to verify the hypothesis that environments exist that promote thymocyte proliferation and maturation. We also propose establishing a clonal history for immature cortical thymocytes under these conditions by """"""""ultraculture"""""""" of single cells. (LB)
