In vitro cell expansion and function detection of human peripheral blood mononuclear cell derived CD4~+CD25~+ regulatory T cells

In this study, we aim to establish an in vitro expansion method for CD4+CD25+regulatory T cells(Treg) and detect its function. CD4+CD25+Treg was separated and purified from peripheral blood mononuclear cell by flow cytometry with 98% purity and 95% of purified cells were Foxp3 positive. The purified CD4+CD25+Treg was further stimulated in vitro with IL-2 and anti-CD3/CD28 monoclonal antibody for 1 to 6 weeks. Cell proliferation of stimulated Treg was detected by radioactive H3incorporation assay and a thousandfold increase was found after 6-week culture. Furthermore, the expression of Foxp3 and the secretion of IL-10/TGF-β were found significantly lower in stimulated Tregs than those in non-stimulated. In conclusion, this study successfully established an expansion method for CD4+CD25+Tregs, and Treg lose their Foxp3 marker after in vitro expansion.