T lymphocytes develop in the thymus through a combination of positive and negative selection; this process allows selective maturation of T cells deemed useful for self without causing autoimmunity. Thymic selection events are extremely stringent and result in differentiation of only a tiny fraction of immature T cells; the rest of the cells are presumed to die in situ by apoptosis, a process of programmed cell death. Since the thymus continuously generates large numbers of immature T cells, apoptosis is presumed to be widespread in the normal thymus during ongoing positive and negative selection. Conventional techniques, however, fail to detect prominent cell death in the normal thymus, which makes it difficult to studies the events that control apoptosis of thymocytes. This problem can now be overcome by using a recently established TUNEL staining method which specifically detects apoptotic cells, both in vivo and in vitro. In order to obtain information on the events that control thymocyte. apoptosis. two areas of investigation are proposed. First, using the TUNEL staining method precise information will be sought on the events that cause death of immature T cells in the thymus. This will be done by searching for apoptotic cells in the thymuses of various gene knockout and transgenic mice in situations where either positive or negative selection predominates or no selection occurs. In addition, the stage of differentiation at which immature thymocytes undergo apoptosis in the normal thymus will be defined; this will involve determining the relationship between apoptosis and cell division in the thymus using a combination of TUNEL staining and BrdU labeling procedures. Second, information will be sought on the capacity of monolayers of thymic epithelial cells to inhibit spontaneous apoptosis of immature thymoyctes cultured in vitro. Particular emphasis will be placed on determining whether the inhibition of apoptosis by thymic epithelium involves cell to cell contact or the release of soluble factors. Obtaining basic information on the processes that control T cell generation are crucial for reaching a clear understanding of autoimmune disease and the rejection of neoplastic cells.
