The long term goal of this project is to fully characterize the effector mechanisms used by cytotoxic lymphocytes to kill target cells, and to develop strategies to inhibit the activity of the cytotoxic cells in disease states like graft versus host disease, while preserving ability to kill virus infected cells and tumor cells. In this study they will define the roles of granzymes (gzms) A-G for the induction of target cell apoptosis, and define the cytotoxic potential of cells that express granzyme B. To accomplish these goals, they propose the following specific aims: 1) To create new loss-of-function murine models for gzms B-G and to define contributions to cell-mediated cytotoxicity in vitro and in vivo. They will use homologous recombination in embryonic stem cells to create mutant mice that lack gzm B only, that lack the entire gzm B-G cluster, or that lack only gzms C-G. These mice will be intercrossed with granzyme A-/- mice to create mice doubly deficient for the gzm A and B. They will characterize and compare cytotoxic lymphocyte functions in these mice, using in vitro and in vivo models of acute graft versus host disease (GvHD), viral clearance and tumor clearance. 2) They will define the biology and cytotoxic repertoire of lymphocytes that express gzm B. They will utilize homologous recombination in ES cells to make mutant mice that contain an enhanced green fluorescent protein (GFP) cassette in the 5' untranslated (UT) region on the murine gzm B gene. They will analyze and purify GFP+ lymphocytes from these mice using flow cytometric methods, and study the biologic properties of GFP+ cells (i.e., those expressing gzm B) that have been activated in vitro and in vivo. 3) They will create mice that are deficient for gzm B-expressing cells to determine the role that these cells play in GvHD, viral clearance, and tumor clearance. They will utilize homologous recombination in ES cells to make mice that contain an attenuated diphtheria toxin cDNA (or herpes simplex virus thymidine kinase gene) in the 5' UT of the murine gzm B gene. If these mice delete all gzm B-expressing cells, they will be able to fully evaluate the role that this cellular compartment plays in acute GvHD, viral clearance and tumor clearance.
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