The long term goal of this project is to define the role(s) of granule associated serine proteases (granzymes) for the functions of cytotoxic lymphocytes. Cytotoxic T-lymphocytes (CTL) and natural killer cells (NK) contain granules that are directly cytotoxic for target cells. These granules contain the pore-forming molecule perforin and several serine proteases; granzymes A and B are the most abundant and most conserved proteases in these granules, ad are thought to play a major role in granule-mediated cytotoxicity since they can induce DNA fragmentation and apoptosis in target cells. To further define the role of granzymes for CTL and NK cell effector functions, the Applicant proposes the following Specific Aims: 1. Analyze the functions of CTL and NK cells derived from mice with null mutations in granzyme A, granzyme B, and both granzymes A and B. Targeted null mutations will be created using homologous recombination in embryonic stem cells. Mutant mice will be extensively characterized, and their CTL and NK comtpartments will be examined in vitro for their ability to induce apoptosis and /or cytolysis in allogeneic target cells, virus-infected cells and tumor cell lines. The Applicant already determined that CTL derived from granzyme B -/- mice are severely deficient in their ability to cause rapid DNA fragmentation and apoptosis in allogeneic target cells. Since CTL can also induce IL-1beta processing and secretion in association with apoptosis in allo-macrophages, the Applicant will also determine whether this pathway is altered in granzyme-deficient CTL. 2. Examine the role that granzymes A and B play in CTL- and NK cell- mediated viral clearance. Since CTL and NK cells are important for viral clearance, and since perforin-deficient mice have a profound defect in their ability to clear lymphocytic choriomeningitis virus (LCMV), cytotoxic granules must play a role in the clearance of LCMV infected cells. To define the role that granzymes A and B play in this process, granzyme A and B or A/B deficient mice will be subjected to infection with LCMV, murine cytomegalovirus (MCMV) and reovirus, and patterns of viral infection and clearance, IL-1 beta signaling, and histopathology will be carefully examined in vitro and in vivo. 3. Examine the roles of granzymes A and B in murine models of graft vs. host disease and hematopoietic graft rejection. To define the roles that granzymes A and B play in htese processes, the applicant will use established bone marrow transplant protocols using purified populations of CD4+ and CD8+ cells derived from granzyme A, B and A/B deficient mice to induce acute or chronic GVHD. The severityof GVHD wll be analysed using histopathologic and immunohistochemical methods. The Applicant also plans to determine whether granzyme- deficient NK cells are capable of normal rejection of hematopoietic stem cells.
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