This competitive continuation grant request is based largely on three major areas of advances derived from our research in the previous grant period: transgene technology and perforin knock out, perforin promotor analysis, and perforin induction studies. Using homologous recombination in embryonal stem cells of mice the perforin gene was deleted. Perforin deficiency creates a dramatic deficiency in the ability of mice to clear viral infections through the generation of cytotoxic T-cells and NK cells. The perforin promotor analysis using conventional Cat assays demonstrated its strict cell specificity to activated CD8+ T-cells. Specificity is achieved by active repression of perforin transcription in perforin negative cells. Using the perforin promotor to drive the expression of human perforin cDNA we plan to reconstitute, by transgene technology, the perforin knock out mice with human perforin. By using various promotor deletion and recombination constructs the expression of human perforin in knock out mice will be modulated so as to eliminate or enhance perforin expression in constitutively or inducibly perforin expressing cells. The biological effects of altered perforin expression will be determined in vitro and in vivo in the hope that less restricted perforin expression may allow the rejection of immune challenges that are normally lethal. The perforin promotor will also be used to drive diphtheria toxin transgenes, resulting in the deletion of perforin expressing cells. This will allow us to address the question of the role of constitutively perforin expressing cells such as NK-cells endometrial and decidua granulated cells and double negative alpha-beta- and gamma-delta-TCR T-cells. To delineate perforin independent killing mechanisms both the perforin knock out mice and mice in whom perforin expressing cells have been deleted by diphtheria toxin will be used. In perforin deficient T-cells, perforin independent pathways will be studied without perforin interference in cells normally expressing perforin. In diphtheria toxin transgenic mice, T-cell activation will lead to the elimination of perforin expressing cells and killer function will be determined in the surviving population of cells that do not express perforin. Perforin independent killing mechanisms will be investigated in the presence of EGTA, focussing on Fas and its putative ligand on CTL. The Fas ligand will be defined using functional cytotoxicity inhibition assays and monoclonal antibodies. Finally the role of costimulatory molecules in perforin induction in CD4+ and CD8+ T-cells will be studied.
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