Immune tolerance is maintained, in part, by a process of clonal elimination in the population of immature lymphocytes. In particular, there is a well defined population of thymocytes that expresses both CD4 and CD8 (termed double positives or DPs), and this immature subset of thymocytes specifically undergoes elimination when challenged with antigen. We have defined two steps in this killing process: one is mediated by an interaction between the T cell antigen receptor and antigen peptide bound to a major histocompatibility complex (MHC) molecule, and a second is antigen-independent and mediated by an unknown interaction involving an antigen presenting cell (APC). A second phase of clonal elimination involves engulfment of dying or targeted thymocytes, and finally, a large number of cells fragment their DNA in response to elicitors of negative selection. This proposal is aimed at understanding these processes of induced or programmed cell death (PCD) that are involved in the elimination of self-antigen reactive thymocytes. Experiments are proposed that will dissect the two steps in killing and the processes of engulfment and DNA fragmentation. TCR-mediated signals can be delivered in the absence of a second step using either anti-TCR antibodies or chemically-fixed, antigen-pulsed APCs. Thymocytes thus challenged will then undergo a killing step by incubation with various types of APCs including small B cells, macrophages, and MHC class II- expressing fibroblasts. For these two steps several parameters will be analyzed. Cellular ultrastructure and morphology will be examined by light and transmission electron microscopy. DNA fragmentation will be analyzed by the TdT-mediated incorporation of dUTP at newly generated 3' chromosomal ends. We will determine the cell types that can mediate engulfment of dying thymocytes, and whether engulfment is mediated by previously described mechanisms. We will determine whether the second killing step is carried out by known receptor/co-receptor interactions. If not, we will produce new monoclonal Abs to the receptors involved by screening for activity in the thymocyte deletion assay. Finally, we will carry out a comprehensive analysis of gene expression, first looking at genes known to play some role in PCD, and then screening for new genes by randomly primed PCR and differential display. These experiments will yield important new results concerning negative selection, thymocyte death, and cellular elimination. We feel that we have a unique perspective on the process of thymocyte death, and our ability to carry out experiments that take into account cellular subsets and antigen- mediated negative selection will allow us to clarify significant aspects of the process of negative selection leading to immunological self- tolerance.
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