Our previous studies showed that incubation of dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders them immunosuppressive. Donor-derived MMC-DCs injected into the recipient before transplantation prolonged heart allograft survival in rats. Whereas generation of DCs is labour-intensive, peripheral blood mononuclear cells (PBMCs) can be easily harvested. Here we analyse under which conditions DCs can be replaced by PBMCs and study their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs abrogated their suppressive effect, showing that monocytes are the active cell population. Suppression of rejection was donor-specific. Recipients treated with MICs showed an increased number of CD4+CD25+FoxP3+ Tregs in their peripheral blood and FoxP3-cell infiltration of heart allograft. Most important, tolerance could be transferred to syngeneic recipients with blood cells, whereas depletion of Tregs annihilated the effect. This argues for mediation of suppression by CD4+CD25+FoxP3+ Tregs. Cell culture studies showed that MMC-treated monocytes give rise to myeloid cells, which do neither resemble monocytes, nor DCs but show morphological and phenotypic similarities with monocytic myeloid-derived suppressor cells (MDSCs) occurring in some forms of cancer. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs were applied for the first time in a patient with therapy-resistant rejection of a haploidentical stem cell transplant, showing that they can be easily generated and used in humans. In conclusion, we describe here a simple method for in vitro generation of monocytic suppressor cells for potential use in clinical organ transplantation.

Purpose: Our previous studies showed that the chemotherapeutic drug mitomycin c (MMC) renders strongly stimulatory dendritic cells (DCs) suppressive. Donor-derived MMC DCs induced specific tolerance in rat heart allograft recipients. Since in clinical transplantation (Tx) peripheral blood mononuclear cells (PBMCs) are easier available than DCs we tried to replace DCs by PBMCs. Materials and methods: Donor blood (1ml) or PBMCs (108) were incubated for 30 min. with MMC, washed and injected i.v. into recipients 1 week before allogeneic heart Tx (DA to PVG). Blood and spleen cells (SPCs) of tolerant recipients were analyzed by FACS for regulatory cells (Tregs) and adoptively transferred into syngeneic animals transplanted with DA grafts. Grafts were immunohistochemically analyzed for cellular infiltration, vascular lumen narrowing and complement (C4d) deposition. The cytokine profile of recipient blood was determined by multiplex immunoassay. Results: MMC-treated donor blood injected into recipients prolonged allograft survival (34.43 ± 3.95 vs. 8.56 ± 0.27 in untreated and 21 ± 4.16 days in donor-blood transfused recipients). A stronger effect up to tolerance (50% of the animals) was obtained when donor blood was replaced with MMC- PBMCs (64.8 ± 16.8 vs. 8.56 ± 0.27 in untreated and 34.43 ± 3.95 days in PBMC treated recipients). The effect was abrogated by elimination of monocytes from PBMCs. Third-party heart allograft survival showed no prolongation indicating donor-specific tolerance. Tolerated grafts had cellular infiltrates with a significantly increased number of Foxp3+ cells and decreased deposition of CD4d in blood vessels in comparison to rejected grafts. FACS analysis of PBMCs and SPCs of tolerant animals revealed an increased percentage of CD4+CD25+Foxp3+ Tregs when compared to rejecting animals (PBMCs: 6.12 + 0.99 vs. 5.52 + 0.28%, p > 0.05; SPCs: 8.31 + 1.11 vs. 6.74 + 0.13%, p = 0.02). Both, PBMCs and SPCs of tolerant animals prolonged allograft survival up to tolerance by adoptive transfer into syngeneic recipients. Cytokine profile analysis suggests a Th2 deviation early after transplantation. Non-significant reduction of vascular lumen in allografts (as measure for chronic rejection) of tolerant animals in comparison to syngeneic ones was observed. Conclusions: A single pretransplant infusion of MMC-PBMCs is able to induce donor-specific suppression up to tolerance in a heart allotransplant model without concomitant use of immunosuppressants. The cell subpopulation which induces suppression is mainly monocytes. Suppression might be mediated by CD4+CD25+Foxp3+ Tregs, since these cells were found in increased number in blood, spleen and grafts of tolerant animals, and tolerance can be adoptively transferred by PBMCs and SPCs. MMC-PBMCs reduce but do not fully prevent chronic rejection. The described model has clinical relevance.

Background. Several approaches have been proposed to pharmacologically ameliorate hepatic ischemia/reperfusion injury (IRI). This study was designed to evaluate the effects of a preconditioning oral nutritional supplement (pONS) containing glutamine, antioxidants, and green tea extract on hepatic warm IRI in pigs. Methods. pONS (70 g per serving, Fresenius Kabi, Germany) was dissolved in 250 mL tap water and given to pigs 24, 12, and 2 hrs before warm ischemia of the liver. A fourth dose was given 3 hrs after reperfusion. Controls were given the same amount of cellulose with the same volume of water. Two hours after the third dose of pONS, both the portal vein and the hepatic artery were clamped for 40 min. 0.5, 3, 6, and 8 hrs after reperfusion, heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), portal venous flow (PVF), hepatic arterial flow (HAF), bile flow, and transaminases were measured. Liver tissue was taken 8 hrs after reperfusion for histology and immunohistochemistry. Results. HR, MAP, CVP, HAF, and PVF were comparable between the two groups. pONS significantly increased bile flow 8 hrs after reperfusion. ALT and AST were significantly lower after pONS. Histology showed significantly more severe necrosis and neutrophil infiltration in controls. pONS significantly decreased the index of immunohistochemical expression for TNF-α, MPO, and cleaved caspase-3 (P < 0.001). Conclusion. Administration of pONS before and after tissue damage protects the liver from warm IRI via mechanisms including decreasing oxidative stress, lipid peroxidation, apoptosis, and necrosis.