原发免疫性血小板减少症中Foxp3+RORγT+调节性T细胞的鉴定和免疫调控功能研究

It has been proved that regulatory T cells (Tregs) harbor numerical and functional abnormalities in primary immune thrombocytopenia (ITP). Recent studies revealed that Tregs could express T effector cell markers under inflammation and become biphenotypic Treg with unique immunological features. We demonstrated elevated IL-17+ cell population among Tregs from peripheral blood of primary ITP patients, along with down-regulation of Treg specific transcriptional factor Foxp3 and up-regulation of Th17 effector molecule IL-17A, which indicated the existence of Foxp3+RORγT+ biphenotypic Treg in primary ITP. In an attempt to characterize the biological features of Foxp3+RORγT+ biphenotypic Treg, the present study was to investigate the immunephenotype, gene expression profile, genomic DNA methylation, and in-vitro immune modulation function of this cell population utilizing magnetic activated cell sorting, flow cytometry, gene expression microarray, methylated DNA immunoprecipitation sequencing, and Treg-Teff coculture. Upon establishing the methods for lineage identification and in-vitro purification of Foxp3+RORγT+ biphenotypic Treg, it could provide us novel vantage point for understanding Treg dysfunction in autoimmunity and new insights into the diagnosis and management of primary ITP.

原发免疫性血小板减少症（ITP）中存在调节性T细胞（Treg）数量和功能异常，近年来研究发现Treg在炎症微环境中可表达效应T细胞相应分子标志，形成双表型Treg，具有独特的免疫学特征。我们在前期研究中发现原发ITP患者外周血Treg中IL-17+细胞比例升高，Treg特异性转录因子Foxp3表达下调而Th17效应分子IL-17A上调，提示原发ITP中存在Foxp3+RORγT+双表型Treg，但其生物学特征尚不明确。本研究拟采用免疫磁珠分选、多重荧光流式细胞分析、基因表达芯片、甲基化DNA免疫共沉淀测序等技术，对原发ITP中Foxp3+RORγT+双表型Treg的免疫表型、基因表达特征、DNA甲基化特征以及体外免疫调控功能进行系统研究，确定其表面标志并建立体外分离纯化方法，从新的角度理解自身免疫性疾病中的Treg功能异常，为原发ITP的诊断和治疗提供新的思路。

原发免疫性血小板减少症（ITP）患者外周血存在多种免疫调控细胞异常，其中调节性T细胞（Treg）可出现功能性亚群分布异常、分泌功能异常、异常表达效应T细胞标志等。本研究发现ITP患者外周Treg细胞中Th17细胞效应分子IL-17表达上调但Th1细胞效应分子IFN-γ表达无显著变化，且可使用替代性表面标志CCR6对磁珠分选的CD4+CD25+CD127-细胞进行二次富集从而纯化Foxp3+RORγT+双表型Treg，但由于ITP患者外周血获取的双表型Treg细胞总数不足且活性明显降低，难以用于基因表达/DNA甲基化分析或体外免疫调控功能评价。ITP患者外周血存在CD68+CD163+ M2单核细胞，其绝对数量约为Foxp3+RORγT+双表型调节性T细胞的3-5倍，且在大剂量地塞米松方案治疗有效的ITP患者中可出现显著上调，可使用替代性表面标志CX3CR1富集CD68+CD163+ M2单核细胞，免疫磁珠纯化后的CX3CR1+细胞共表达CD68与CD163的细胞比例明显升高 (46.79 ± 4.71 % vs. 5.31 ± 0.51 %)。ITP患者的CX3CR1+ M2单核细胞中效应分子Arg-1转录水平上调，Arg-1与转录因子PPARγ的蛋白浓度均升高，DNA甲基转移酶DNMT3b转录水平下调，DNMT3b与DNMT1的蛋白浓度均降低。在被动免疫小鼠ITP模型中使用小剂量DNA甲基转移酶抑制剂地西他滨可通过抑制DNMT1与DNMT3b，降低PPARγ启动子的甲基化水平而促进PPARγ转录，促进前体巨噬细胞极化为M2巨噬细胞，从而改善ITP小鼠体内免疫微环境，提高其血小板水平。本研究后续拟在单细胞平台进一步探索CX3CR1+ M2单核细胞与CD4+CD25+CD127-CCR6+双表型Treg的特征和免疫调控功能。