THE PURPOSE OF THIS STUDY IS TO ESTABLISH A RATIONALE FOR THE SELECTION AND USE OF CURRENTLY AVAILABLE IMMUNOSUPPRESSIVE AGENTS BASED ON THE MODIFYING EFFECTS THEY EXERT ON THE PURINE METABOLISM OF BIOCHEMICALLY DEFINED IMMUNOCOMPETENT CELLS. We propose to define differences in purine metabolism between lymphocyte subsets differentiated by Wrights stain morphology, cell surface markers, and in vitro immune function. These techniques will then be used to characterize cells infiltrating Nucleopore chambers as an allograft rejection model and to correlate the immunologic and biochemical effects of purine analogs, steroids, and Cyclosporine on these cells. Initial studies will use spleen cells from genetically defined mice. Ficoll-Hypaque separation of mononuclear cells followed by plastic adherence and nylon wool separation will be used to obtain adherent, non-adherent, and B-cells. Non-adherent cells will be further separated by antibody-complement subset depletion and phenotypically characterized by cell sorter analysis. Antigen specific cytotoxic cells will be recovered on monolayers of fibroblasts syngeneic with the immunizing allogeneic strain. Cytotoxicity, suppressor, and helper function will be assessed (using standard techniques of mixed leukocyte culture, cell mediated lympholysis, and cell addition studies) to confirm the phenotype of separated cells and evaluate specificity for the immunizing antigen. These highly immunologically defined cells will then be studied biochemically with regard to their purine salvage pathways to determine inherent differences by which they handle purine salvage in the resting and functionally active state. Compounds will be selected based on their ability to inhibit purine metabolism in specific places and expected to be more toxic to certain cells than others based on their baseline differences in purine metabolism. These selected drugs will be given alone or in combination or sequentially to allograft Nucleopore chamber bearing mice. This will test the effect in vivo of these drugs in abrogating cellular rejection as defined by decreased infiltration of the chambers.
