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.

Cells have been previously used in experimental models for tolerance induction in organ transplantation and autoimmune diseases. One problem with the therapeutic use of cells is standardization of their preparation. We discuss an immunosuppressive strategy relying on cells irreversibly transformed by a chemotherapeutic drug. Dendritic cells (DCs) of transplant donors pretreated with mitomycin C (MMC) strongly prolonged rat heart allograft survival when injected into recipients before transplantation. Likewise, MMC-DCs loaded with myelin basic protein suppressed autoreactive T cells of MS patients in vitro and prevented experimental autoimmune encephalitis in mice. Comprehensive gene microarray analysis identified genes that possibly make up the suppressive phenotype, comprising glucocorticoid leucine zipper, immunoglobulin-like transcript 3, CD80, CD83, CD86, and apoptotic genes. Based on these findings, a hypothetical model of tolerance induction by MMC-treated DCs is delineated. Finally, we describe the first clinical application of MMC-treated monocyte-enriched donor cells in an attempt to control the rejection of a haploidentical stem cell transplant in a sensitized recipient and discuss the pros and cons of using MMC-treated antigen-presenting cells for tolerance induction. Although many questions remain, MMC-treated cells are a promising clinical tool for controlling allograft rejection and deleterious immune responses in autoimmune diseases.

Indoleamine 2,3-dioxygenase (IDO), an enzyme involved in the catabolism of tryptophan, is expressed in certain cells and tissues, particularly in antigen-presenting cells of lymphoid organs and in the placenta. It was shown that IDO prevents rejection of the fetus during pregnancy, probably by inhibiting alloreactive T cells, and it was suggested that IDO-expression in antigen-presenting cells may control autoreactive immune responses. Degradation of tryptophan, an essential amino acid required for cell proliferation, was reported to be the mechanism of IDO-induced T cell suppression. Because we wanted to study the action of IDO-expressing dendritic cells (DCs) on allogeneic T cells, the human IDO gene was inserted into an adenoviral vector and expressed in DCs. Transgenic DCs decreased the concentration of tryptophan, increased the concentration of kynurenine, the main tryptophan metabolite, and suppressed allogeneic T cell proliferation in vitro. Kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid, but no other IDO-induced tryptophan metabolites, suppressed the T cell response, the suppressive effects being additive. T cells, once stopped in their proliferation, could not be restimulated. Inhibition of proliferation was likely due to T cell death because suppressive tryptophan catabolites exerted a cytotoxic action on CD3+ cells. This action preferentially affected activated T cells and increased gradually with exposure time. In addition to T cells, B and natural killer (NK) cells were also killed, whereas DCs were not affected. Our findings shed light on suppressive mechanisms mediated by DCs and provide an explanation for important biological processes in which IDO activity apparently is increased, such as protection of the fetus from rejection during pregnancy and possibly T cell death in HIV-infected patients.