Effect Of Rapamycin On The Expansion Of Human Cd4~+Cd25~+Regulatory T Cells In Vitro

As a necessary subset of natural occurring regulatory T cells, CD4+CD25+regulatory T cells (Tregs) play an important role in modulating the immune system and maintaining peripheral tolerance. It has been shown that CD4+CD25+regulatory T cell is the master negative regulators of immune response, which can effectively inhibit autoimmune diseases, allergic reaction and allogeneic transplantation rejection. Immunotherapy based on Tregs infusion is therefore a potential treatment for many immune disorders. This approach has been proven successful in animal models of hematopoietic stem cell transplantation, solid organ transplantation and autoimmunity. However, the number of natural Treg cells in body is very small. They consist5%～10%of human CD4+T cells, while the mouse is only1%～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.CD4+CD25+regulatory T cells were purified from adult peripheral blood with immunomagnetic beads (MACS), and proliferated using IL-2and Anti-CD3/CD28monoclonal antibody. The phenotype and purity of Treg cells were determined by flow cytometry. Immunosuppressive function of Treg cells were determined by using proliferation inhibition assay of CD4+CD25-effective T cells. In addition, the method to amplify CD4+CD25+Tregs in vitro based on IL-2and Anti-CD3/CD28monoclonal antibody. We set up three groups:in group one, CD4+CD25+Tregs were cultured with IL-2and CD3/CD28antibody-coated beads; in group two, all culture condition was the same as group one except the addition of100nmol/L rapamycin; in group three, CD4+CD25+Tregs were further sorted into CD4+CD25+CD127-Tregs on Day12, and then were cultured for the second cycle, and the culture condition was the same with group two. As a result, the purity of CD4+CD25+Treg cells and CD4+CD25-T cells separated by MACS reached respectively91%and98%. All Treg cells in the three groups amplified significantly. Compare with the first group, the expansion ratio of the latter two groups is lower (1.0×103vs. 1.8×103), but the cell purity increased significantly (92%vs.80%). The expanded CD4+CD25+Tregs of the latter two groups still retain the cell anergy. Although the latter two groups showed a stronger inhibiting activity than the first group, there were no significant differences between the latter two groups. Moreover, compare with the first group, the latter two groups expressed lower levels of IL-2and IFN-y. The results of this study show that rapamycin inhibited the expansion of CD4+effective T cells which were contaminated in CD4+CD25+Tregs, therefore the use of rapamycin effectively improved the cell purity of expanded CD4+CD25+Tregs in vitro and the immunosuppressive function.Finally, we also preliminary investigate the methods used for cryopreservation and thawing of expanded CD4+CD25+Treg cells in vitro, which will provide experimental basis for clinical application.