体外诱导的Helios＋调节性T细胞的特征分析

Phenotypic and functional properties of in vitro-induced Helios＋ regulatory T cells

目的本研究拟对体外诱导的Helios＋调节性T细胞（induced Helios＋ regulatory T cell，Helios＋ iTreg）的特征进行分析，并比较其与正常人外周血固有Helios＋ Treg（natural Helios＋ Treg, Helios＋ nTreg）的差异。方法采集并分选正常人外周CD4＋CD25－效应性T细胞（effector T cell，Teff），体外以抗CD3/抗CD28合并IL-2及TGF-β刺激诱导Helios＋ iTreg生成，以外周血Helios＋ nTreg作为对照，通过流式细胞术和real-time PCR分别分析其表型特征Foxp3＋ Treg特异性去甲基化区（Treg-specific demethylated region，TSDR）的甲基化水平。结果体外诱导的Helios＋ iTreg高表达CD45RA，而Helios＋ nTreg几乎不表达CD45RA；体外诱导的Helios＋ iTreg较Helios＋ nTreg CD127、ICOS和PD-1的水平明显升高，IL-2和IFN-γ的表达水平明显升高，但CXCR3、CCR4等趋化因子受体的水平则明显下降；另外CD25的表达水平也明显下降；诱导型Helios＋ iTreg TSDR的甲基化水平明显低于Helios＋ nTreg。结论诱导型Helios＋ iTreg具有与Helios＋ nTreg不同的特征，提示其在生理及病理条件下可能发挥不同的免疫调节作用。

Objective To investigate the phenotypic and functional properties of in vitro-induced Helios＋ regulatory T cells （Helios＋ iTreg） and to analyze the differences between Helios＋ iTreg and Helios＋ thymic-derived natural Treg （Helios＋ nTreg）.MethodsCD4＋ CD25- effector T cells （CD4＋ CD25- Teff） that were separated from healthy subjects were cultured with anti-CD3 and anti-CD28 antibodies and stimulated with IL-2 and TGF-β to induce the generation of Helios＋ iTreg. Flow cytometry and real-time PCR were respectively used to analyze the differences in phenotype and CpG methylation in Treg-specific demethylated region （TSDR） between Helios＋ iTreg and Helios＋ nTreg derived from the same donor.ResultsCD45RA was highly expressed on Helios＋ iTreg, but not on Helios＋ nTreg. Additionally, Helios＋ iTreg expressed significantly higher levels of CD127, ICOS and PD-1, but lower levels of CXCR3, CCR4 and CD25 than Helios＋ nTreg did. IFN-γ and IL-2 that expressed by Helios＋ iTreg were more than those by Helios＋ nTreg. It was also found that the level of CpG methylation in TSDR was much higher in Helios＋ iTreg than in Helios＋ nTreg.ConclusionBoth nTreg and iTreg expressed Helios, but the phenotypic and functional properties of the two Treg subsets were different. It was suggested that Helios＋ iTreg and Helios＋ nTreg might play different roles in the immune system.