Allogeneic BMT has been increasingly applied for the treatment of immune deficiencies, hematopoietic disorders, cancer therapy, and recently in attempts to induce immunological tolerance for solid organ transplantation. Unfortunately, without prophylactic measures, most allogeneic marrow transplant recipients will develop GVHD. This is a major complication that continues to prevent more successful clinical application. The overall focus of the proposed studies is to determine the role of cytotoxicity in GVHD. The applicants intend to address this issue by utilizing donor cell populations derived from mutant strains of mice with cytotoxic deficiencies to conclusively evaluate the importance of perforin- and Fas-dependent cytotoxicity in GVHD. Donor transplant populations will be derived from perforin-deficient mice, mice that contain a functional defect in the Fas-ligand, or mice with a double-defect lacking the ability to mediate both perforin and Fas-dependent cytotoxicity. The studies are designed to assess the importance of donor anti-host cytotoxicity during two phases of GVHD; the developmental phase, and during the pathogenesis (effector) phase. Concerning the developmental phase, studies will test the hypothesis that donor anti-host cytotoxicity is a determining factor that may be required for induction of and mortality in GVHD following allogeneic BMT. With respect to the pathogenesis phase, the study will define the involvement of perforin and/or Fas-mediated cytotoxic effector activity in principal target tissues of GVHD including skin, liver, gastrointestinal tract, and the lymphoid compartment. Studies will examine the ability of donor marrow inoculum containing cytotoxically defective cells to overcome host resistance and engraft, with the long term goal of successful immune reconstitution. The experiments examining engraftment will employ transplants between multiple histocompatibility complex (MHC) matched Ly5.2 congenic allogeneic donors and recipients, and use phenotypic and functional (CFU) assays to evaluate donor/host hematopoietic lineages and progenitor populations. Following engraftment, studies will examine immune reconstitution by investigating responsiveness in vitro, immune competence in vivo, the clearance of a viral pathogen, and the ability to reject allogeneic skin grafts.
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