Programmed cell death (apoptosis) is a basic biological process used to modify cell repertoires during development, and also involved in cancer, anti-neoplastic therapy, and AIDS. The antigen-specific repertoire of lymphocytes is established through processes of apoptosis, rescue from apoptosis, and proliferation. Signals triggered by the engagement of antigen-specific receptors on lymphocytes are crucial for these processes. These signals guide the development of immature lymphocytes and also control mature T cells. Much less is known about the mechanisms which connect T cell receptor (TCR)-derived signals to apoptosis than about the cellular interactions which guide repertoire formation in the thymus and T cell activation in the periphery. Our long-term goal is to understand the pathways which link cell surface-derived signals with apoptosis of thymocytes. The theme of this proposal is that biochemical and molecular biologic investigations into the genes which connect cell surface-derived signals to apoptosis will be facilitated by the development of a genetic approach to studying these pathways. We have identified a thymic lymphoma cell line which resembles immature thymocytes and which undergoes apoptosis as a result of TCR cross-linking or lectin treatment, and an immature thymocyte lymphoma of known TCR specificity which undergoes negative selection in response to physiologic TCR engagement by antigen with major histocompatibility complex (MHC) protein. Following insertional mutagenesis with a gene-trap retrovirus and selection by lectin treatment, we have generated a panel of apoptosis-resistant mutants. Since mutations created by gene-trap retroviruses are tagged by the virus the process of identifying mutant genes is accelerated compared to other methods of mutagenesis.
Our specific aims are to characterize mutations which lead to resistance to apoptosis in thymocyte mutants already selected after gene- trap mutagenesis. We also propose to improve this technology, using the thymocyte cell line of known T cell receptor specificity. Apoptosis- resistant thymocyte mutants represent unique reagents which ultimately will be used to investigate the order of gene action along pathways between the TCR and cell death.
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