Generation of Suppressive Blood Cells for Induction of Donor- Specific Tolerance in a Rat Heart Transplant Model: A Clinically Relevant Model

Abstract

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.