Training Plan: The clonal deletion of autoreactive thymocytes (negative selection) results from the induction of programmed cell death or apoptosis. This process is important for achieving tolerance within an individual. While loss of self-tolerance may result in autoimmune disease, the induction of tolerance to alloantigens may result in permanent organ graft acceptance. Recent studies suggest that thromboxane A2 (TxA2) produced in the thymic microenvironment may regulate thymic maturation. Thromboxane synthase, the final catalytic enzyme involved in TxA2 synthesis, is expressed within the thymus. Moreover, the thymus expresses high levels of the thromboxane receptor (TxR), far above that of other tissues tested. Finally, TxR expression has been localized to immature thymocytes and exposure of these cells to TxA2 induces apoptosis. Therefore, the hypothesis is that TxA2 binds to its receptor and, through specific signaling pathways, regulates cellular processes important to T cell maturation and selection. The goals of this project are to identify the biochemical characteristics and ontogeny of the TxR in thymocytes and identify the function of TxA2 in thymocyte development. To investigate this hypothesis, three specific aims are proposed: (1) To characterize the signal transduction pathways coupled to the TxR in thymocytes. In these studies, the signal transduction pathways coupled to the TxR in freshly isolated thymocytes will be identified by microphysiometry and active second messenger coupling confirmed by standard biochemical assays. (2) To identify which signals activated by TxR cause apoptosis in thymocytes. These studies will directly determine which of the second messengers (identified in specific aim I) mediate apoptosis of thymocytes in vitro. (3) To define the effects of TxA2 on thymocyte maturation in vitro. Using fetal thymic organ culture as a model of thymic development, the applicant will directly test the role of TxA2 in influencing the development of T cells. T cell receptor transgenic mice will be used to specifically examine the role of TxA2 in controlling negative selection of autoreactive T cell precursors. This project is expected to provide novel information regarding the expression and function of the TxR in thymocytes and the effective role of TxA2 in regulating T cell maturation. It is hoped that understanding of the role of lipid mediators in the thymic development may suggest new strategies in preventing autoimmune disease and in the induction of transplantation tolerance to organ allografts.
