In Vitro Amplification Of Cd4+cd25+regulatory T Cell 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 in future treatment of acute graft versus host disease (aGVHD).[Method] CD4+T cells,CD4+CD25-T cells and CD4+CD25+T cells were isolated from healthy volunteers by MACS and expanded in vitro.Phenotype of the T cell lines and expression of the FOXP3 was determined by FACS.The inhibitory effect of amplified 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 amplified significantly after three weeks culture.The proliferation rates in the purity of CD4+ CD25+Treg was 20±8 folds,however,the proportion of Treg cells was decresesed from 89.28%±2.43% to 76.36%±2.38% during the culture.The CD4+CD25-T cells group were growth more rapidly and the proliferation rates was 110±21 folds increased,with a Treg cells percentage raised from 0.55%±0.27% to 53.73%±3.81%.In the CD4+T cells group,The cells were 89±8 folds increaded with a Treg cells percentage increased were from 10.63%±3.87% to 70.93%±1.71%.Inhibitory test showed that the amplified CD4+CD25+T cells could inhibit the proliferation of auto and allo CD4+T cells in vitro with a cell dose dependent manner.No significant difference was found of the treg cells inhibition effect in auto and allo groups. [Conclusion] We successfully amplified CD4+CD25+Tregs in vitro.the cells amplified had inhibitory effects in vitro,as a result of the purity of cells method is simple and we can obtain lots of high purity of CD4+CD25+Tregs from CD4+T cells group,in future the cells amplified may be used in clinical trials.