An Observation On Expanding CD4~+CD25~+ Regulatory T Cells In Vitro

Objective To evaluate the effect of cytokines on the proliferation and function of CD4+CD25+ regulatory T cell (Treg) in vitro.Methods Tregs were isolated from na?ve C57BL/6 mice spleen and lymphnodus,and labeled with CFSE. Mature dendritic cells (mDC) were isolated from DBA/2 mice and co-cultured with Tregs(Treg 1×105/hole,mDC 2.5×104/hole), with or without interleukin-2 (IL-2) (20ng/ml), interleukin-4 (IL-4) (20ng/ml), and interleukin-15 (IL-15) (150ng/ml). 6 days later,Treg proliferation and apoptosis were detected by flow cytometry (FACS). 6 days later,The co-culture increased Tregs'suppressive function was evaluated by MLR(mixed lymphocyte culture) using co-culture of CFSE labled na?ve CD4+CD25- T cells and proliferated Tregs. Foxp3 was detected by FACS to prove expanded Tregs still maintain their phenotype.Results Tregs stimulated by IL-2, IL-4, and IL-15 maintain suppressive function and have a higher precursor frequency (PF) (respectively, 31.3%, 28.9%, and 34.5%) than the control group (14.5%) and a higher proliferation index (PI) (respectively, 1.9, 1.7, and 1.8) than the control group (1.5) ,P<0.05. Tregs stimulated by IL-2, IL-4, and IL-15 have a lower apoptosis percentage (respectively, 12.8%, 11%, 12.7%) than the control group (28.9%),P<0.05.Expanded Tregs highly express Foxp3 (91.75%).Conclusion IL-2, IL-4, and IL-15 stimulate Treg proliferation, reduce apoptosis, and maintain suppressive function in vitro. Expanded Tregs still maintain their phenotype, highly express Foxp3.