IL-7联合IL-2对脐血CD4^+CD25^-T细胞体外诱导扩增影响的研究

Effects of IL-7 and IL-2 on CD4^+CD25^-T cells proliferation in vitro

目的:初步探讨IL-7联合IL-2对脐血CD4+CD25-T细胞体外扩增的促进作用,建立稳定的体外培养扩增脐血CD4+CD25-Tregs的培养体系,比较诱导扩增后的CD4+CD25+Tregs和自然分选的CD4+CD25+Tregs对PBMCs功能活性的影响。方法:采用免疫磁珠分选法分选出脐血CD4+CD25-T细胞和CD4+CD25+T细胞;加入不同浓度的细胞因子IL-7结合适当浓度的IL-2作为诱导剂,分析IL-7体外诱导CD4+CD25-T细胞增殖的有效性及最适浓度。我们利用流式细胞术检测体外扩增后的CD4+CD25-T细胞表型变化;MTS法检测体外诱导扩增的CD4+CD25+Tregs及自然分选的CD4+CD25+Tregs分别对成人外周血中单个核细胞增殖的抑制作用;RT-PCR方法分析体外诱导扩增的CD4+CD25+Tregs及自然分选的CD4+CD25+Tregs FOXP3基因、IL-10基因和TGF-β基因的cDNA表达的变化。结果:经3周体外培养、各组细胞均有明显的扩增。IL-7诱导组的扩增最强。体外抑制试验显示体外扩增的Tregs对成人外周血中单个核细胞有明显的抑制作用,IL-7联合IL-2诱导CD4+CD25-T细胞生成的CD4+CD25+Tregs具有较自然分选的CD4+CD25+Tregs稍弱的免疫抑制功能,其中IL-7浓度为4 ng/ml,IL-2浓度为2 000 U/ml时诱导CD4+CD25-T细胞生成的CD4+CD25+Tregs杀伤活性最强。结论:成功建立了CD4+CD25+Tregs的体外培养体系,联合应用IL-7、大剂量的IL-2和CD3/CD28单抗是体外诱导扩增CD4+CD25-T细胞成为CD4+CD25+Tregs的优选方法,并且扩增倍数高、可持续高表达CD4及CD25细胞表型。

Objective：To explore the promoting effects of IL-7 and IL-2 on CD4^＋CD25^-T cells proliferation in vitro and construct a stable culture system in vitro for CD4^＋CD25^＋regulatory T cells from human umbilical cord blood.To compare the inhibiting effects between induced proliferated CD4^＋CD25^＋Tregs and naturally isolated CD4^＋CD25^＋Tregs on PBMCs functional activity.Methods：CD4^＋CD25^-T cells and CD4^＋CD25^＋T cells were isolated from human umbilical cord blood mononuclear cells by magnetic activated cell sorting （ MACS） system and then expanded in vitro.Four different concentration levels of IL-7 combined with proper concentration of IL-2 were added as inducer and the efficiency and optimal concentration of IL-7 on inducing,CD4^＋CD25^-T cells were analyzed via 4 different methods.Flow cytometry method was used to detect the changes of CD4^＋CD25^-T cells.The inhibitory effect of expanded CD4^＋CD25^＋T cells on peripheral blood mononuclear cells （PBMCs） was tested by MTS.The expressions of Foxp3,IL-10 and TGF-βgenes in CD4^＋CD25^＋T cells were test by RT-PCR.Results：The CD4^＋CD25^＋T cells from each groups were expanded significantly after three weeks of culture.The results indicated that use of IL-7 combined with IL-2 resulted in the highest cell expansion comparing to the other groups.It was shown by the inhibitory test that the expanded CD4^＋CD25^＋regulatory T cells could inhibit the proliferation of PBMCs ,but IL-7 induced CD4^＋CD25^＋regulatory T cells exerted weaker suppressor activity than natural regulatory T cells .Only IL-7 （4 ng/ml） and IL-2 （2 000 U/ml） induced CD4^＋CD25^＋regulatory T cells showed the strongest killing activity.Conclusion：We successfully expand CD4^＋CD25^＋regulatory T cells in vitro.The protocol is established in which the use of mAbCD 3/CD28 combined with IL-7 and IL-2 resulted in the highest cell expansion ,and intensely expressed cell phenotype of CD 4 and CD25.