In vitro conversion of CD4+ CD25-T cells to CD4-CD8-regulatory T cells / 中华普通外科杂志

ABSTRACT

Objective To optimize the condition of converting murine naive CD4+ CD25-T cells ( effector T cells,Teffs) to CD4-CD8-double negative regulatory T cells ( DN Tregs) in vitro.Methods Naive Teffs from C57BL/6 mouse were isolated with magnetic activated cell sorting( MACS)and co-cultured with DBA/2 mature dendritic cells (mDCs) with different doses of recombinant murine interleukin-2 (IL-2).The percentage of converted DN Tregs was examined by flow cytometry after 6 days.Purified DN Tregs were co-cultured with CFSE labeled Teffs.The proliferation rate of Teffs were evaluated by flow cytometry.Results Without IL-2,the percentage of CD4-CD8-T cells was 6.21％ ± 2.03％.With IL-2,the percentage was 14.77％ ± 2.15％ ( 25 ng/ml),21.29％ ± 2.68％ (50 ng/ml),43.45％ ±4.45％ (75 ng/ml),and 28.59％ ±3.05％ ( 100 ng/ml) respectively.The IL-2 concentration of 75 ng/ml signilicantly enhanced the conversion of Teffs to DN Tregs ( separately t =10.700,8.288,6.158,3.932,all P ＜ 0.05).Highly purified DN Trega significantly suppress the proliferation of Teffs in vitro.Conclusions Teffs are converted to DN Trega in vitro with the LPS-activated allogeneic mDCs and that 75 ng/ml of IL-2 is the optimal concentration for the conversion of Teffs to DN Trogs in vitro.