Studies On Effects Of IL-10 Gene Modified-dendritic Cells Induced Type 1 T Regulatory Cells On Prevention Of Graft Versus Host Disease And Preserving Graft Versus Leukemia

Background and Aim:Allogeneic hematopoietic stem cell transplantation is the most efficient strategy to cure leukemia and other hematological malignancies,but GVHD is one of the mostcommon causes of morbi-mortality after allo-HSCT.However,in addition to GVHD,the donor-derived T cells also could induce the beneficial GVL response,where donor T cells recognized and eradicated residual leukemic cells.Therefore,prevention of GVHD while maintaining GVL is the research hotspot in allo-HSCT setting.Tregs play a crucial role in the induction of peripheral tolerance to self and foreign antigens.In generally,Tregs are divided into two categories:one is nTregs and another one is i Tregs.The main subset of the latter is Tr1 cells.It is very difficult to obtain a large scale of nTregs in vitro to meet clinical requirements,In this study,we designed the experiment,in which Tr1 cells can be induced through the stimulation of donor naive CD4⁺T cells by IL-10 gene-modified DCs transfected with lentiviral vector carrying IL-10 gene and evaluated the effect of Tr1 cells on prevention of graft versus host disease and preserving graft versus leukemia.Methods:Donor naive CD4+T cells were co-cultured with recipient IL-10 gene modified DCs for 3 days,Tr1 cells were harvested and purified using a CD4+T cell positive enrichment kit.Immunophenotype of Tr1 cells were analysed by FACS and the secretion of cytokines were detected by ELISA.The suppressive effect of Tr1 cells was analyzed using a CD8+T cell and CD4+T proliferation assay and mixed lymphocyte response(MLR).In addition,the effects of Tr1 cells on occurence of GVHD and GVL were investigated in an murine BMT model.Results:1.Lentiviral vectors carrying IL-10 gene were successfully constructed and transfected into DCs,which maintained morphology and phenotype of mature DCs,and IL-10 gene modified-DC(DC_LV-IL-10)could secrete high levels IL-10.2.Large numbers of Tr1 cells can be induced through the stimulation of donor naive CD4⁺T cells by recipient DC_LV-IL-10.These Tr1 cells not only expressed CD4and CD25,but also co-expressed CD49b,LAG-3 without expression of Foxp3,which are the marker of Tr1 cells.Meanwhile,our data shown that these Tr1 cells also expresse IL-10 and IFN-?.3.Recipient DC_LV-IL-10 co-cultured with donor naive CD4+T cells inducing Tr1cells secreted high levels IL-10 and IFN-?,and low levels IL-4?IL-2,which were the major factors for induced Tr1 cells to exert an immunoregulation function.4.They markedly inhibited CD4+and CD8+T cells proliferation and MLR in vitro,which were in a dose-dependent manner.The immunosuppressive effects could be neutralized by anti-IL-10 antibody,indicating that immunosuppressive effects of Tr1 cells are IL-10 dependly.5.Tr1 cells could significantly reduce the severity of clinic and pathological GVHD and extend survival of mice in a murine BMT model.6.Tr1 cells infusion significantly reducthe severity of GVHD,and did not affect the cytotoxicity of donor T cells to leukemia cells.Meanwhile,Th1 to Th2 shifted in recipient mice receiving Tr1 cells,indicating that Tr1 cells not only could extenuate graft versus host disease but preserve graft versus leukemia effect also.Conclusion:1.Donor naive CD4⁺T cells co-cultruing with recipient IL-10 gene-modified DCs could induce large numbers of Tr1 cells,which played a key role of specific immunosuppressive functions.Induced Tr1 cells could markedly inhibit CD8+and CD4+T cells proliferation and alleviate MLR in vitro.2.Tr1 cells infusions result in milder GVHD while maintaining GVL in an MHC mismatched leukemia murine allogeneic BMT model.Meantime we found a Th1 to Th2 shift in recipient mice maybe played a key role to exert function on immune system.