Combined Effects Of IL-7and IL-2on CD4~+CD25~-T Cells Proliferation In Vitro

Objectives:Currently, the worldwide common autoimmune diseases include rheumatoid arthritis,thyroiditis, vitiligo, pernicious anemia, type Ⅰ diabetes mellitus, systemic lupuserythematosus and so on. These diseases are all closely related to the amount ofCD4+CD25+regulatory T cells in the body. Studies confirmed that the patient with higherdisease activity had the lower amount of CD4+CD25+regulatory T cells in his body. Therefore,CD4+CD25+regulatory T cell has great significance for the application in the adoptiveimmunotherapy of autoimmune diseases. However, due to the low proliferative activity ofCD4+CD25+regulatory T cell as well as its extremely low proportion and absolute number inthe body and the instability of Foxp3expression in the induced CD4+CD25+regulatory T cells,the source of CD4+CD25+regulatory T cells becomes the bottleneck for the treatment ofautoimmune diseases, and limits the clinical application of CD4+CD25+regulatory T cells.Thus, in this study, we devoted to establish an in vitro culture system inducing thetransformation of CD4+CD25-T cells to the CD4+CD25+regulatory T cells in order toconstruct solid foundation for the clinical application of CD4+CD25+regulatory T cells.Methods:CD4+CD25-T cells and CD4+CD25+T cells were isolated from human umbilical cord bloodmononuclear cells by magnetic activated cell sorting (MACS) system and then expanded invitro. Four different concentration levels of IL-7combined with proper concentration of IL-2were added as inducer. The efficiency and optimal concentration of IL-7on inducing,CD4+CD25-T cells were analyzed via4different methods. Flow cytometry method was usedto detect the changes of CD4+CD25-T cells. The inhibitory effect of expanded CD4+CD25+Tcells on peripheral blood mononuclear cells (PBMCs) and CIK cells from human umbilicalblood was tested by MTS. The expressions of Foxp3、IL-10and TGF-β genes in CD4+CD25+T cells were test by RT-PCR.Results:The CD4+CD25+T cells from each groups were expanded significantly after three weeks of culture. The results indicated that use of IL-7combined with IL-2resulted in the highest cellexpansion comparing to the other groups. The inhibitory test showed that the expandedCD4+CD25+regulatory T cells could inhibit the proliferation of PBMCs and CIK cells fromhuman umbilical blood, but IL-7induced CD4+CD25+regulatory T cells exerted weakersuppressor activity than natural regulatory T cells. Only IL-7(4ng/ml) and IL-2(2000U/ml)induced CD4+CD25+regulatory T cells showed the strongest killing activity.Conclusion:The CD4+CD25+regulatory T cells were successfully expanded in vitro. It is concluded that aprotocol is established in which the use of mAbCD3/CD28combined with IL-7and IL-2resulted in the highest cell expansion, and intensely expressed cell phenotype of CD4andCD25.