In vitro amplification of CD4~+CD25~+ regulatory T cells from the healthy volunteers

Objective:To establish a practical and convenient approach and experimental foundation for the in vitro expansion of CD4+CD25+cells and infusion of donor-derived CD4+CD25+cells for future treatment of acute graft versus host disease(aGVHD).Methods:CD4+T cells,CD4+CD25-T cells and CD4+CD25+T cells were isolated from healthy volunteers by MACS and then expanded in vitro.Phenotype of the T cell lines and expression of the FOXP3 was determined by FACS.The inhibitory effect of expanded CD4+CD25+T cells on auto-and allo-CD4+T cells was tested by MTT method.Results:The Treg cells from all the three groups were expanded significantly after three weeks of culture.The proliferation rates in the purity of CD4+CD25+Treg were 20±8 folds.However,the proportion of Treg cells was decreased from 89.28%±2.43% to 76.36%±2.38% during the culture.The CD4+CD25-T cells grew more rapidly and the proliferation rates were increased by 110±21 folds,with the percentage of raising from 0.55%±0.27% to 53.73%±3.81%.In the group of CD4+T cells group,the cells were 89±8 folds by with a percentage of Treg cells increasing cells from 10.63%±3.87% to 70.93%±1.71%.It was shown by the inhibitory test that the expanded CD4+CD25+T cells could inhibit the proliferation of auto-and allo-CD4+T cells in vitro in a cell dose-dependent manner.No significant difference of the inhibition effect was found in auto-and allo-groups of Treg cells.Conclusion:We successfully expand CD4+CD25+Tregs in vitro.The cells expanded have inhibitory effects in vitro.As the method is simple,we can conveniently obtain lots of high purity of CD4+CD25+ Tregs from CD4+T cell.Probably in future,the cells may be used in clinical trials.