BONE-MARROW AND SPLENOCYTE COCULTURE-GENERATED CELLS ENHANCE ALLOGRAFT SURVIVAL

Background. Protocols that incorporate donor-specific cell infusions u sing bone marrow, spleen, or blood transfusion continue to enhance all ograft survival and often lead to tolerance in experimental models. Cl inical benefits from these modalities have not been as striking, leadi ng to ongoing study in this field. We have explored culture techniques for the in vitro selection and development of cellular effecters capa ble of enhancing allograft survival. Methods. Rat bone marrow or splee n cells cultured under a variety of conditions were screened for suppr essor function, Bone marrow cells, nonadherent to plastic, cultured fo r 7 days with granulocyte-macrophage colony-stimulating factor, lipopo lysaccharide, and with or without splenocytes were found to contain pr edominantly myeloid lineage cells and had the ability to suppress phyt ohemagglutinin or mixed lymphocyte reaction-induced splenocyte prolife ration. Standard donor-specific peripheral blood transfusion was compa red with cultured donor-specific bone marrow cells, splenocytes, or ma rrow cells cultured with splenocytes (cocultured) administered intrave nously at 1 x 10(7) cells/kg the day before an ACI to Lewis heterotopi c heart transplant, Cyclosporine was administered at 10 mg/kg on day - 1 and 2.5 mg/kg on days 0-6 relative to transplantation. Results. Mean allograft survival in cyclosporine-treated animals was 8.5 days witho ut and 16.6 days with a donor-specific blood transfusion. Cocultured c ells extended allograft survival (39.5 days), whereas bone marrow or s plenocytes cultured alone did not, With Percoll gradient separation, t wo predominant culture subfractions, one with potent suppressor functi on and another with stimulator function, were identified. Plow cytomet ric analysis showed mixed populations enriched for macrophages but als o including dendritic cells in both subfractions. The suppressive frac tion extended allograft survival to 20.8 days and the stimulatory frac tion was less effective, yet remixing of both fractions regained the f ull allograft survival advantage. Conclusions. Tn this model, the cocu lture of bone marrow cells and splenocytes with granulocyte-macrophage colony-stimulating factor and lipopolysaccharide produced functionall y divergent subpopulations that synergistically enhanced allograft sur vival, The development of cellular effecters with enhanced ability to prolong allograft survival using in vitro culture techniques is possib le, and provides a new therapeutic option in the use of cell infusion- based therapies.