This proposal represents a continuation of our investigations into the structure, function and physiological importance of perforin. Perforin is a component of the cytolytic mechanism of cytotoxic T cells (CTL) and natural killer (NK) cells. It is contained in cytoplasmic granules that are secreted during the cytolytic mechanism. Although it has been established that perforin and granules can play an important in cytolysis, it is still controversial whether perforin is essential for killing or even represents a major cytolytic component. Research in this area has been impeded previously by the unavailability of sensitive assays for perforin. Our success in the isolation and analysis of cDNA clones for both murine and human perforin opens new avenues into the exploration of the crucially important effector mechanisms of CTL and NK. Accordingly, this continuation application will focus on several new areas of research intimately related to perforin and cytotoxicity. The genomic structure of perforin will be investigated and compared to C9. The regulation of the induced expression of perforin will be studied and transcriptional or translational control mechanisms uncovered. The cell biology of the expression of perforin by transfection will be analyzed. In particular, the influence of granule factors on stability and cytotoxicity of perforin will be investigated. The requirement for a secretory apparatus for the mediation of cytotoxicity in transfected cells will be determined, In anti-sense transfection experiments, it will be determined whether perforin is essential for the cytolysis of target cells. In addition to its cytotoxicity, factors that protect cells from cytotoxicity by perforin (perforin inhibitor) will be isolated and analyzed. The role of these factors in self protection of CTL and NK will be investigated. The perofrin inhibitor will be purified and characterized. Its structure will be determined by obtaining and isolating cDNA clones from a human LAK cDNA library. Cumulatively, these studies will allow an unambiguous assessment of perforin's role in cell mediated cytotoxicity and the role of perforin inhibitors in killer cell self protection.