The Preliminary Investigation Of The Mechanism Of MDSC Induced Immune Tolerance After Allo-HSCT

[Objective] In order to evaluate possible effects of MDSCs on aGVHD development and clinical outcomes, this study systematically detected the dynamic changes of MDSCs accumulation in patients during the first 100 days after allo-HSCT, and investigated the levels of other cell types and relative cytokines during MDSCs accumulation.[Methods] Peripheral blood was obtained from all patients at time points of engraftment,28d,35d,49d, 63d,77d and 91 d after allo-HSCT. For patients developing aGVHD, peripheral blood was collected when aGVHD occurred and after aGVHD treated, respectively. For healthy controls, peripheral blood was collected from volunteers. All the analyses were performed on freshly harvested cells. Cell were stained with the relevant mAbs and analyzed on a Becton Dickinson FACS Calibur. Data were analyzed with Cell Quest software. MDSCs were isolated by magnetic beads separation. For measuring the serum concentrations of IL-6, TNF-?, Arg-1, HO1, iNOS, IL-10, IL-10 samples were analyzed using ELISA kits.[Results] Accumulation of MDSCs in the graft and in peripheral blood when engraftment might contribute to patients'overall immune suppression and result in the successful control of severe aGVHD and long-term survival without influence on risk of recurrence after allo-HSCT. But MDSCs levels in the graft had more favorable predictive abilities. Furthermore, MDSCs frequencies significantly increased in patients developing aGVHD after allo-HSCT. It might be caused by secondary inflammatory response, especially related to high concentrations of IL-6 and TNF-?. But this accumulation would not be able to counterbalance the aggravation of aGVHD and would not have influence on clinical outcomes and risk of relapse.[Conclusion] MDSCs might be considered as a potential new approaches to regulate transplant rejection and achieve long-term survival.[Objective] Establishment of the in vitro culture system of MDSC induced by G-CSF and investigation the function of myeloid-derived suppressor cells in graft-versus-host disease after mouse allogeneic bone marrow transplantation.[Methods] In order to establish the in vitro culture system of MDSC, we cultured mice bone marrow cells for different incubation times with different concentrations of G-CSF. After we purified the G-CSF induced MDSC, we infused them into transplant recipients with bone marrow cells and T cells of donors when the day of bone marrow transplantation. The clinical outcomes of transplantaion were observed.[Results] The most appropriate concentration of G-CSF for in vitro MDSC cultrue is 100 ng/mL and the most appropriate incubation time is 4 days. The most appropriate concentration of G-CSF for in vivo inducing MDSC is 5?g per mice once a day, and the most appropriate inducing time is 3 days. Accumulation of MDSC in the graft could contribute to overall immune suppression and result in the successful control of severe aGVHD and long-term survival in allo-BMT mice model.[Conclusion] MDSCs might be considered as a potential new approaches to regulate transplant rejection and achieve long-term survival.[Objective] Establishment of the in vitro culture system of MDSC induced by recombinant G9 protein and investigation the function and mechanism of G9-induced MDSC in graft-versus-host disease after mouse allogeneic bone marrow transplantation.[Methods] In order to establish the in vitro culture system of MDSC, we cultured mice bone marrow cells for different incubation times with different concentrations of recombinant G9 proten. The morphology and the phenotype of purified G9 induced MDSC were identify by May-Grunwald-Giemsa staining and Flow Cytometry analysis, respectively. Purified G9 induced MDSC were co-cultured with T cells with or without tranwell system for 72 hours, then observe the inhibition effect on proliferation of T cells. The expression of Arg-1, iNOS, Tim3 genes were detected by real-time PCR. The apoptosis and differentiation of G9 induced MDSC were dectected by Flow Cytometry analysis. After we purified G9 induced MDSC, we infused them into transplant recipients with bone marrow cells and T cells of donors when the day of bone marrow transplantation. The clinical outcomes of transplantaion were observed.[Results]1. The most appropriate concentration of G9 for in vitro MDSC cultrue is 300 ng/mL and the most appropriate incubation time is 4 days.2. Markers for hematopoietic stem cell (CD34) and mature myeloid cells (CD14, CD11c)were negative on G9 induced MDSC. Co-stimulatory molecules CD86 and CD86 were found negative. The G9 induced MDSC expressed MHC-?, F4/80 and Tim3. It has a "ring-shaped" morphology.3. The proliferation of G9 induced MDSC is depended on a pathway related to Tim3 expression.But G9 induced MDSC are differentiated from GMP and the expression of Tim3 on G9 induced MDSC is not involved in the proliferation of MDSC.4. G9 induced MDSC have abilities to suppress the proliferation of T cells through cell-to-cell contact.5. The expression of Arg-1, iNOS and Tim3 genes are upregulated in G9 induced MDSC.6. G9 induced MDSC have abilities to maintain the immunosuppressive function for about 7 days which is longer than that of normal MDSC.7. Accumulation of G9 induced MDSC in the graft could contribute to overall immune suppression and result in the successful control of severe aGVHD and long-term survival in allo-BMT mice model.[Conclusion] The role of G9 on MDSC proliferation is connfirmed. And G9 induced MDSC suppress the proliferation and activation of T cell is an entirely new finding in mechanisms of G9 in GVHD after allo-BMT except G9/Tim3 pathway.