抗原特异性调节性T细胞体外诱导扩增及其功能测定

Induction and expansion of antigen specific regulatory T cells in vitro

目的体外诱导扩增抗原特异性CD4+CD25+T细胞,测定其免疫抑制功能,验证其抗原特异性。方法应用免疫磁珠法(MACS)分选C57BL/6鼠CD4+CD25+T细胞。CD4+CD25+T细胞在Balb/c鼠抗原刺激条件下,体外扩增获得Balb/c鼠抗原特异性的CD4+CD25+T细胞(iCD4+CD25+T),流式细胞术测定其纯度,RT-PCR方法测定其FoxP3mRNA。测定在Balb/c小鼠(A组)或昆明鼠(B组)抗原刺激条件下,iCD4+CD25+T抑制T细胞增殖的效率,验证iCD4+CD25+T的抗原特异性。ELISA法测定A、B组上清液中IL-10和TGF-β的含量。结果MACS分离的C57BL/6小鼠CD4+CD25+T细胞纯度为99.6%。扩增3周,iCD4+CD25+T纯度达85%,扩增数目达900倍,高表达Foxp3基因。当iCD4+CD25+T∶CD4+CD25-T为1∶1、1∶2时,A组抑制率明显高于B组(82.2%±3.29%vs11.6%±1.2%,54.4%±3.5%vs5.4%±1.7%,P<0.01)。A组IL-10和TGF-β的分泌量均高于B组(P<0.01)。结论在体外成功诱导扩增了具有免疫抑制功能的抗原特异性的CD4+CD25+调节性T细胞。

Objective To set up a system of inducing and expanding antigen specific CD4^＋CD25^＋ regulatory T cells （iCD4^＋CD25^＋ T）, and to determine their properties of suppressing T cell proliferation. Methods CD4^＋CD25^＋ regulatory T cells were purified from C57BL/6 mouse and using MACS. Balb/c antigen specific CD^4＋CD25^＋T （iCD4^＋CD25^＋ T） were induced and expanded in the presence of Balb/c APC,and their antigen specific suppressive function was determined by suppressing proliferation of T cells in the presence of Balb/c APC （ Group A） or kunming APC （ Group B） . The contents of IL-10 and TGF-β in supernatant in Group A or Group B were determined using ELISA. Results CD4^＋CD25^＋ T sorted by MACS yield a purity of 99.6%. After 3 weeks of culture in our system, the number of CD4^＋CD25^＋ T which expressed high number of Foxp3 mRNA increased by 900- folds, when the ratio of iCD4^＋CD25^＋ T and B6 CD^4＋CD25^- T cells was 1 ： 1 and 1 ： 2, suppress rate was higher in group A than in group B（82. 2% ±3.29% vs 11.6% ± 1.2% ,54.4%±3.5% vs 5.4% ± 1.7% ,P 〈0. 01）. The contents of IL-10 and TGF-β in supernatant in Group A was higher than in Group B （P =0. 00）. Conclusion The system of inducing and expanding antigen specific CD4^＋CD25^＋ T cells is set up successfully.