The Preliminary Study On The Expansion Of Natural CD4+CD25+Foxp3+ Regulatory T Cells And Induction Of Induced CD4+CD25+ Regulatory T Cells Ex Vivo

As a necessary subset of natural occurring regulatory T cells,CD4+CD25+Foxp3+ regulatory T cells(Treg) play an important role in maintaining the homeostasis of the immune system.More and more evidences confirm that CD4+CD25+regulatory T cells is of great importance to maintaining immunologic homeostasis,monitoring of tumor immunity and autoimmune disease.However,the number of natural Treg cells in body is very small.They consist 5%~ 10%of human CD4+T cells,while the mouse is only 1%~ 2%.Therefore,it is necessary to establish an effective method of expanding Treg cells ex vivo in order to meet the needs of clinical infusion.At present,most experiments expend Treg cells in vitro by anti-CD3,CD28 monoclonal antibody and exogenous IL-2,but the efficiency is very low and can not meet the clinical demand.In addition,co-culture with dendritic cells can also amplify Treg cells.CD4+CD25+Foxp3+regulatory T cells were purified from splenocyte(Balb/c or C57/B6) with immunomagnetic beads(MACS),and proliferated using homologous or heterologous mature DC,immature DC and regulatory DC.The phenotype and purity of Treg cells were determined by flow cytometry.Immunosuppressive function of Treg cells were veriflcated using CD4+CD25-effective T cells.In addition, induced CD4+CD25+regulatory T cells were induced from CD4+CD25-T cells by TGF-β,IL-2 and anti-CD3/CD28 monoclonal antibody.As a result,the purity of Treg cells separated by MACS reached 93.57%,and they could be expended effectively by homologous and heterologous DC.The effect of homologous mDC's stimulation is the most obvious.What's more,the purity of Treg cells expended by homologous mDC reached 94.16%,and expressed Foxp3 highly.When the ratio of CD4+CD25+regulatory T cells and effective T cell were 1:1,Treg cells could suppress the proliferation of effective T cells effectively.And the inhibitory effect of expended Treg cells is stronger than fresh purified Treg cells.Meanwhile,Foxp3 expression of CD4+CD25-T cells separated by MACS is 17.62%.The expression of CTLA-4 and CD62L associated with Treg cells' function is 1.37%and 73.61% respectively.After stimulation of TGF-β,IL-2 and anti-CD3/CD28 monoclonal antibody,the expression of Foxp3,CTLA-4 and CD62L up regulate,is 98.70%, 72.1%and 87.51%respectively.When the ratio of iTreg cells and effective T cell were 0.5:1 and 1:1,iTreg cells could suppress the proliferation of effective T cells effectively.In addition,iTreg can be expended effectively by homologous and heterologous DC.The effect of heterologous rDC's stimulation is the most obvious. This study provides a theoretical basis for the amplication of Treg cells to treatment of allogeneic transplantation tolerance and autoimmune diseases.