| Applications of allogeneic hematopoietic stem cell (HSC) transplantation have been limited due to the morbidity and mortality associated with acute graft versus host disease (aGVHD). Many researches have shown that donor or recipient's tolerogenic dendritic cells (tDCs) could improve allograft survival in mouse models. But, it is no idea whether third-party-derived tDCs also could induce tolerance in a model of allogeneic bone marrow transplantation (allo-BMT). In this research, different dose of D1-tDCs were adoptive transferred into the aGVHD model in allogeneic BMT, which chose B6mice as donors and D2mice as recipients, and explored the role of third-party-derived tDCs in the prevention aGVHD in mice after allo-BMT.First, tDCs were cultured with low doses of GM-CSF, IL-10and TGF-β1from bone marrow cells of D1mice. The phenotype, expression of cytokines and function associated molecules were identified with FACS and RT-PCR. The results assessed by FACS indicated that tDCs expressed lower levels of MHC II and co-stimulatory molecules, such as CD80, CD86and CD40, even when stimulated by LPS. The results assessed by RT-PCR indicated that tDCs expressed low levels of IL-12p40and high levels of'immunosuppressive'molecules, such as IL-10, TGF-β, Fas Ligand, and arginase. Therefore, this type of cells remained to tolerogenic phenotypes.Mixed lymphocyte reaction (MLR) analyzed the influence of third-party-derived tDC on allo-CD4+T cells proliferation in vitro. The suppressive activity was tested by CFSE staining. Freshly isolated CD4+T cells from B6mice were used as responder cells and activated by mature DCs from D2mice alone or in the presence of third-party tDCs with the different doses. After4days, CFSE progressive dilution and PI positive-expression were used as the readouts of responder cells proliferation and apoptosis. The results indicated that at all the responders versus tDCs ratios, tDCs were able to promote the apoptotic death of responder cells and suppress proliferation of allo-CD4+T cells. While102tDCs, which CD4+T cells versus tDCs ratio was1000:1, could inhibit expansion most effectively and lead to massive death of allo-CD4+T cells. In the allogeneic MLR, third-party tDCs could suppress allo-CD4+T cells proliferation, which was relative to the dose of tDCs.In order to determine whether the third-party tDCs were able to induce tolerance in aGVHD mouse models, an aGVHD model was established which chose B6mice as donors and D2mice as recipients. The MHC molecules of these two mice are different completely. The recipients were given a lethal dose of total body irradiation. Bone marrow cells and spleen cells from donors were injected after24h. Different doses of the third-party-derived tDCs were adoptive transferred into the aGVHD models, and termed "tDCs-treated group". The aGVHD group was the control group. Survival time and clinical GVHD score were observed after allo-BMT. In the B6â†'D2mouse models, all aGVHD mice died within18days. Remarkably, if10third-party tDCs were transferred,60%mice survived at least60days. But when the doses of tDCs were reduced to103cells, only20%of mice survived past day60and when increased to105, all of the mice died within day37after allo-BMT. These results indicated that adoptive therapy by transfusing third-party tDCs could significantly prolong the survival time of recipients after allo-BMT, and this role was associated with the dose of tDCs.To determine how tDCs reduce aGVHD, the cytokines IFN-y, IL-4, IL-10and IL-12p70in serum were further examined on7d and21d after allo-BMT. There was no significant difference between the tDCs-treated groups and aGVHD group in the early time post-BMT. But, on the21d, it was found that the levers of IL-10in serum were obviously higher in the mice treated with104tDCs than the mice transferred other doses of tDCs, and the IFN-y production is lower than the other two tDC-treated groups. From these results, we believed that the role of third-party tDCs in prevention aGVHD was associated with high IL-10secretion. |
PDF Full Text Request