While ex vivo donor T cell depletion (TCD) is highly effective for lethal GVHD prevention, T cell removal may compromise beneficial donor T-cell responses. Rather than broadly eliminate T-cells or T-cell subsets, the applicant proposes to functionally alter in vitro the small proportion of murine donor CD4+ T cells with anti-host alloreactive potential. A common feature of his strategy will be in the in vitro blockade of the CD28/CTLA4 T-cell costimulatory pathway since there is evidence that T cells can be rendered specifically tolerant of alloantigen present during the blockade (termed anergy). In vitro assays will be used to screen for conditions that favor the anergy development. The applicant has established criteria for the acceptable level of reduction of donor anti- host and anti-third party alloreactivity which will be required before proceeding with the in vivo testing of the GVHD properties of anergic donor cells. His initial studies will be focused on CD4+ T cells since these appear to be most susceptible to anergy induction. Anergic CD4+ T cells will be infused in vivo without any supplemental in vivo GVHD preventive reagents. Because IL-2 responses can override anergy induction in some situations, the applicant has designed experiments to address whether IL-2 production will prevent anergy induction in vitro or GVHD prevention in vivo. If necessary, he will target, in the context of optimal CD28/CTLA4 blockade, a limited number of other pathways which can cause T cells to proliferate in the presence of CD28/CTLA4 blockade. By using appropriate specificity controls and examining responses to relevant peptides given in vivo post- transplantation, he will determine if in vitro anergy induction is translatable into beneficial in vivo effects. These principles and strategies will be applied with suitable modifications to a CD8+ T cell mediated GVHD system. To determine how these approaches have altered the in vivo GVHD potential of the donor cells, the applicant will need to examine specifically those donor T cells that are exclusively alloreactive with the host. Because equal to or less than 0.1% of donor T cells have anti- host alloreactivity, the applicant will use T cell receptor transgenic mice as donors since more than 90% of all T cells will have an obligate alloreactivity to the recipients. To determine if anergy is preserved in vivo, phenotypic and functional assays of early post-bone marrow transplantation (BMT) alloreactive donor T cells, isolated by thoracic duct cannulation (TDC), will be performed. A highly enriched source of donor T cells can be obtained by TDC which can be studied for alloantigen and anti-peptide responses.
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