Tk Induces CD4+CD25-T Cells Differentiated Into CD4+CD25+Foxp3+T Cells And Promotes CD4+CD25+Treg Proliferation And Differentiation In Vitro

To elucidate the mechanisms underlying the Tk-induced immunosuppression and to explore the feasibility to the treatment and prevention of rejection in organ transplantation specifically by using Tk as a immune regulator, this study is designed to cover following two aspects.Part 1 Tk induces CD4+CD25-T cells differentiated into CD4+CD25+Foxp3+TcellsAs reported that Tk at low concentration (100ng/ml) could induce immunosuppression by down- regulated the CD80 and LFA-1 molecules expression on dendretic cells (DC) and prevent DC from maturation. We presumed that Tk could drive CD4+CD25-Tcells differentiated into CD4+CD25+Foxp3+T cells and induced immunosuppression.Methods1. CD4+CD25-T cells was isolated by MACS (magnetic activated cell sorting )and the purity of these isolated cells was detected by FACS (fluorescence activated cell sorting).2. C57BL/6 CD4+CD25-T cells were sitimulated by irradiated Balb/c splenic mononuclear cells (SMC) in the presence of or absence of Tk at lOOng/ml for 4 weeks. The rate of CD4+CD25+T cells was also detected by FACS.3. CD4+CD25+T cells isolated from culture system by MACS were detected for their Foxp3 expression by RT-PCR.4. The secretion of IL-10 and TGF-β by CD4+CD25+T cells stimulated by anti-CD3 was assayed by ELISA.5. The immunosuppressive effect of CD4+CD25+T cells induced by Tk was analyzed by the test of T cells proliferation assays.6. The antigen specificity of immunosuppressive in CD4+CD25+T cells induced by Tk was analyzed by mixed lymphocyte reaction (MLR).