Bmscs Joint Bone Marrow Transplantation Chimera And Forming Mechanism Of Induction Of Immune Tolerance

BackgroundAllogeneic organ transplantation has became a common and final clinicaltreatment for organ failure. Chemotherapy, radiotherapy and immunesuppressivetherapy improve allogeneic transplant survival rate. However, these protocolsoften lead to serious complications including chronic organ failure.Therefore,many studies have aimed to find better methods to induce immune tolerance.Mixed hematopoietic chimerism via bone marrow transplantation has beendemonstrated as an excellent method to induce specific tolerance to donorallografts. However, routine clinical application of this approach is impeded bythe toxicity of the myeloablative or cytoreductive recipient conditioning whichis required to allow transient engraftment of MHC-mismatched bone marrow.As irradiation and cytotoxic drugs are associated with considerable medical risks such as profound leukopenia, their elimination from immune toleranceprotocols is preferred for clinical translation. Whether the goal of anon-cytoreductive mixed chimerism with conventional BM doses can beachieved is still remained elusive.Bone marrow-derived mesenchymal stem cells （BMSCs） can be obtained andexpanded easily in vitro. The inherent immunosuppressive properties and lowimmunogenicity of BMSCs suggest their therapeutic potential inallotransplantation rejection. The therapeutic exploitation of BMSCs haspronounced effects in autoimmune, systemic inflammatory response syndromeand GVHD models. At present, the lack of non-toxic recipient conditioningprotocols for establishing mixed chimerism via bone marrow transplantation isarguably hindering the widespread clinical application of this approach toachieving tolerance. Therefore, we investigated whether mixed chimerism couldbe established by IBM-BMT combined with BMSCs treatment without anyadditional cytoreductive conditioning. The balance of Th17/Tregs plays a keyrole in immune rejection and tolarence formation after allotransplantation.Therefore, we investigated the effect and mechanism of BMSCs on T celldifferentiation induced by alloantigen stimulationin vitro.Materials and Methods1^st. The recipient mice （C57BL/6） accepted BMSCs from donor mice （Balb/c）through daily tail vein injection for four days followed by IBM-BMTimmediately. Full-thickness skin grafts from donor mice as well as from thethird party mice （ICR） were transplanted to the dorsum of the recipient miceafter the combined IBM-BMT with BMSCs treatment. The immune tolerancewas assessed by the survival time of skin allografts. The establishment of mixed chimerism and cytokine expression profile in recipient peripheral blood weredetermined by flow cytometry and enzyme-linked immunosorbent assay,respectively.2^nd. CD4⁺T cells derived recipient mice were isolated and cultured in vitro. Inorder to investigate the effect of BMSCs on Th17/Tregs differentiation fromnaive T cells induced by alloantigen stimulation,one-way MLR was performedbetween recipient CD4⁺T cells and unseparated splenocytes, derived from donormice, treated with mitomycin. And levels of IL-1β, IL-2, IL-6, IL-10, IL-17,TGFβ1and IDO in the MLR system were detected to explore the mechanism ofBMSCs on Th17/Tregs differentiation.3^rd.1-D-MT was used to inhibit the function of IDO in BMSCs. Then the effectof IDO in BMSCs on Th17/Tregs differentiation from naive T cells induced byalloantigen stimulation was investigated.Results1^st. IBM-BMT combined with BMSCs treatment led to stable mixed chimerismand donor-specific skin graft tolerance. The flow cytometry analysis revealedthat recipient mice developed20-25%chimerism levels among the myeloidlineage. The skin allografts survived more than1year and the hair re-grewnormally on the grafts. Overwhelming infiltration of lymphocytes andgranulocytes were observed and located at the basal layer of the skin grafts fromthird party mice, while much less immune cell infiltration was observed in donorskin grafts. Cytokine profile showed that IBM-BMT combined with BMSCstreatment prolonged humoral tolerance in recipient chimeras.Hypo-responsiveness to the donor cells was observed in the combinedIBM-BMT and BMSC treated group, which showed a significant difference to hyper-responsive reactivities in other groups. Hyper-responsiveness to the thirdparty antigens was observed in all groups at both time points.2^nd. MLR results showed that alloantigenic stimulator could promote thedifferentiation of na ve T cell to Th17/Tregs. The ratio of Th17/Tregs decreasedremarkably when BMSCs existed. And BMSCs could up-regulate the cytokinelevels of IL-1β, IL-2, IL-6, IL-17and down-regulate IL-10, TGFβ1and IDO.3^rd. When the function of IDO in BMSCs was inhibited by1-D-MT, the effect ofBMSCs on differentiation of na ve T cell to Th17/Tregs induced by alloantigenicstimulator was blocked remarkably. However, the cytokine levels of IL-6andTGFβ1were not affected by IDO.ConclusionsOur results demonstrate that donor specific immune tolerance can be effectivelyinduced by IBM-BMT combined with BMSCs treatment without any additionalcytoreductive recipient treatment. This approach provides a promising allografttransplantation strategy when the donor bone marrow is available. Thedecreased Th17cells and increased Tregs may due to the effect of BMSCs onna ve T cell differentiation. And IDO played a key role in this process.