Graft-versus-host disease (GVHD) in allogeneic bone marrow transplantation (BMT) recipients can be prevented by T-cell depletion (TcD) of the allograft, but this results in increased frequencies of graft rejection and leukemia relapse. The applicant has investigated the use of irradiated allogeneic leukocytes as a novel method to support engraftment and preserve the GVL effect of donor lymphocytes without causing GVHD. Preliminary work established at 7.5 Gy radiation inhibits the proliferative potential of allogeneic T-cells while preserving their in vitro cytotoxicity and their capacity to facilitate engraftment by TcD allogeneic bone marrow in vivo. TcD of irradiated allogeneic splenocytes eliminated the graft facilitating activity. Multiple injection of irradiated allogeneic splenocytes increased the rate of leukemia-free survival in mice transplanted with allogeneic TcD bone marrow and congenic leukemia cells compared to recipients of allogeneic TcD BMT and leukemia alone. The overall hypothesis of these studies is that irradiated donor leukocytes provide differential short-term allo-reactivity against host leukemia cells and promote donor hematopoietic engraftment without producing GVHD in the BMT setting. Possible mechanisms of action of irradiated allogeneic leukocytes include enhanced cytolytic activity against normal and leukemic host hematopoietic cells; production of cytokines that mediate an anti-proliferative or cytopathic effect against host-type cells; or enhancement of the allo-reactivity of co-administered non-irradiated or radio-resistant donor cells. To investigate these mechanisms are proposed three specific aims. In the first specific aim, the applicant will identify the cells responsible for the graft-facilitating and GVL activity of irradiated allogeneic lymphocytes. He will use splenocytes enriched or depleted for T or NK-cell populations and mutant SCID and beige mice lacking T and NK cells respectively to determine whether these cells mediate the graft-facilitating and GVL effect. The anti-leukemia activity of irradiated allogeneic splenocytes will be tested using recipients co-transplanted with a congenic myeloid leukemia line or mrp-8/bcl-2/lpr transgenic mice that spontaneously develop myeloid leukemia. In the second specific aim, the applicant will determine the molecular mechanisms of the anti-leukemia activity of irradiated leukocytes testing the contribution of cell contact-dependent cytolysis vs. the release of cytopathic or cytostatic soluble factors by irradiated cells. In the third specific aim, the applicant will test whether priming donors of allogeneic splenocytes by allo-immunization or with cytokines enhances the graft facilitating and GVL activity of irradiated lymphocytes. Multiple injections of irradiated leukocytes will be used to establish initial engraftment; subsequent infusion of non-irradiated donor leukocytes will be used to eradicate residual host-type cells when the risk for developing acute GVHD is reduced.
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