The immediate objective of this project is to elucidate the resistance of cytotoxic T lymphocytes (CTLs) to lysis by their own pore-forming protein, perforin. Perforin is the principal lytic effector molecule of highly activated CTLs and natural killer cells. CTLs are highly resistant to lysis by their own cytolytic granules or by isolated perforin. The nature of factors mediating this resistance has not been defined. The primary objective is to characterize genes and proteins expressed by CTLs that protect them from perforin-mediated lysis. Murine tumor cells have been transfected with a CTL-derived cDNA and have been selected for resistance to perforin. Genes from the CTL-derived cDNA library that protect recipient cells from perforin will be identified, sequenced and analyzed. In order to generate monoclonal antibodies against protective proteins, hamster cell lines will be similarly transfected with murine CTL cDNA and selected for resistance to perforin. The cell lines will be used to immunize congenic hamsters. As another approach, protein from CTL membrane will be fractionated and reconstituted into liposomes. Protein fractions that protect liposomes from perforin will be subjected to further rounds of fractionation. Thus, protective protein(s) will be purified. CTL membrane proteins that associate with perforin will be characterized by their cross-linking with perforin. Protein that bind perforin may be protective of CTLs, especially those polypeptides that are specifically expressed in CTL membranes. Binding of perforin to sensitive cell lines and to resistant CTLs will be measured. Perforin isomerizes and inserts into target membrane. The rate of insertion and channel formation will be compared between sensitive and resistant cells. Such measurements are useful for understanding mechanisms of resistance. Cytotoxic T lymphocytes are essential for the eradicating of virally infected cells and possibly of many neoplasia. The mechanisms of CTL mediated cytolysis and the means by which cell can resist CTL attack are the broad objectives of this effort.
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