Amplification of mice CD4+ CD25+ regulatory T cells in vitro and it's function detection

Objective To detect the immune function of CD4 + CD25 +T cells (Tregs),amplified in vitro.Methods We used the micro-magnetic beads to isolate Tregs.Two means were adopted to amplify Tregs (anti-CD3mAb group and allo-APC group).The function of Tregs was detected with the Transwell system,cytokine detection and CFSE marker.Results The purity of Tregs is 89.5％.The amplification times were not significantly deferent between the two groups.The counts per minute(CPM) of CD4 + CD25 +T cell,CD4 + T cell and CD4 + CD25T cell is 1 470,12 700 and 14 300 respectived.In mixed lymphocyte reaction (MLR),when the ratio of CD4/CD25+ T cell is 1∶ 1 and 8∶1,suppression ratio is 79％ and 33％.We labeled CD4+T cells with CFSE,the percent is 83.7 ％ without Tregs,and 31.7 ％ with Tregs (P =0.0006).The proliferation inhibition ratio inducedby is 5％ in Transwell system and 95％ in Non-Transwell system.The concentration of IL-2 in Transwell system is significantly higher than that in Non-Transwell(158.33 ± 2.08) pg/mL vs (23.00 ± 2.00) pg/mL,P ＜ 0.0001).Conclusion The method of micro-magnetic beads can be used to isolate Tregs.Tregs can be amplified effectively and inhibit CD4 +T cells proliferation.Tregs amplified in vitro still have immune with function with enhanced immune-suppressive effect. Key words: CD4+ CD25+ regulatory T cells; In vitro amplification