Background.Protocols that incorporate donor-specific cell infusions using bone marrow, spleen, or blood transfusion continue to enhance allograft survival and often lead to tolerance in experimental models. Clinical benefits from these modalities have not been as striking, leading to ongoing study in this field. We have explored culture techniques for the in vitro selection and development of cellular effectors capable of enhancing allograft survival.Methods.Rat bone marrow or spleen cells cultured under a variety of conditions were screened for suppressor function. Bone marrow cells, nonadherent to plastic, cultured for 7 days with granulocyte-macrophage colony-stimulating factor, lipopolysaccharide, and with or without splenocytes were found to contain predominantly myeloid lineage cells and had the ability to suppress phytohemagglutinin or mixed lymphocyte reaction-induced splenocyte proliferation. Standard donor-specific peripheral blood transfusion was compared with cultured donor-specific bone marrow cells, splenocytes, or marrow cells cultured with splenocytes (cocultured) administered intravenously at 1×107cells/kg the day before an ACI to Lewis heterotopic 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 without and 16.6 days with a donor-specific blood transfusion. Cocultured cells extended allograft survival (39.5 days), whereas bone marrow or splenocytes cultured alone did not. With Percoll gradient separation, two predominant culture subfractions, one with potent suppressor function and another with stimulator function, were identified. Flow cytometric analysis showed mixed populations enriched for macrophages but also including dendritic cells in both subfractions. The suppressive fraction extended allograft survival to 20.8 days and the stimulatory fraction was less effective, yet remixing of both fractions regained the full allograft survival advantage.Conclusions.In this model, the coculture of bone marrow cells and splenocytes with granulocyte-macrophage colony-stimulating factor and lipopolysac-charide produced functionally divergent subpopulations that synergistically enhanced allograft survival. The development of cellular effectors with enhanced ability to prolong allograft survival using in vitro culture techniques is possible, and provides a new therapeutic option in the use of cell infusion-based therapies.