MITOMYCIN C-TREATED DENDRITIC CELLS SUPPRESS ALLOREACTIVE T CELLS AND INDUCE SPECIFIC UNRESPONSIVENESS TO HEART ALLOGRAFT IN RATS: MEDIATION OF SUPPRESSION BY DOWNREGULATION OF ICAM-1, CD80 AND CD86 (2004)

P912 Aims: Although dendritic cells (=DCs) strongly stimulate the immune response, it has been shown that, under certain circumstances, these cells can also induce strong immunosuppression. Generation of tolerogenic donor DCs provides an efficient tool for suppression of graft rejection. Mitomycin C (=MMC) is an alkylating agent which partially inhibits protein synthesis and influences the immunogenicity of transplanted cells. Here we use MMC for generation of tolerogenic rat DCs and analyze their mechanism of action on T cells. Methods: Monocyte–derived DCs were generated in the presence of GM–CSF, IL–4, maturated with TNF–α/PGE2, incubated with MMC (20μg/ml) and extensively washed. DC phenotype as well as cell death were analyzed by flow cytometry. The stimulatory capacity of MMC–DCs was tested in an allogeneic T–cell proliferation (BNRT1n [wng]224[/wng] LEWRT1l) assay in-vitro. In a lymph node assay, LEW rats were injected into the footpad with 106 BN- derived DCs and one week later T-cell proliferation was assessed by weighing the popliteal lymph nodes. In an other experiment, 106 MMC–treated BN-DCs were injected intraportaly into LEW rats. One week later the recipients received a heterothopic BN or DART1avl (third party) heart transplant and graft survival was monitored by daily palpation and EKG. Differences with P < 10-2 (rank sum test) were considered statistically significant. Results: MMC-treated allogeneic DCs lose their T-cell stimulatory capacity “in vitro” and this effect can neither be attributed to MMC–induced death of DCs, nor to the T-cell inhibitory action of possible MMC traces in cell culture supernatants. Most importantly, suppressed T cells cannot be restimulated with fresh donor DCs, whereas third party restimulation is possible. FACS analysis showed that MMC-treatment selectively downregulates adhesion (ICAM-1) and costimulatory (CD80, CD86) molecules on the DC cell surface whereas other cell membrane proteins (MHC–II, OX62, ED-2) remain unaffected. Functional blocking of these molecules with specific monoclonal antibodies (= mAbs) confers to DCs the same T-cell suppressive properties in-vitro as treatment with MMC. In-vivo, rats injected with allogeneic DCs pre-treated with MMC or mAbs to ICAM-1, CD80, CD86 showed a suppressed local lymph node reaction. When prospective graft recipients received MMC–treated donor DCs they presented a significantly prolonged heart allograft survival (MST= 30 days) as compared to animals receiving placebo (MST = 9 days) or untreated donor DCs (MST = 6 days) (both P≤10-5). Most notably, a similar prolongation of allograft survival could be induced using DCs pre–incubated with mAbs specific for ICAM–1, CD80, and CD86 (MST = 52 days, P=10-4). Along with our findings in the lymph node assay and the in-vitro data, this result indicates that downregulation of ICAM–1, CD80, and CD86 was responsible for MMC-mediated suppression of allograft rejection. Prolongation of allograft survival by MMC-DCs was donor–specific, since no significant effect on third-party heart allograft survival was noted (untreated: MST = 8 days; MMC: 11 days, P = N.S). Conclusions: Our results show that MMC treatment converts DCs into T-cell suppressive cells able to induce donor–specific prolongation of rat heart allograft survival. This mechanism of suppression is mediated by downregulation of ICAM–1, CD80 and CD86 on DCs. If a similar effect could be defined in man, MMC-DCs might prove to be an efficient tool for immunosuppressive therapy in clinical transplantation.