Tim-3+Treg细胞在母胎界面聚集并调控母胎免疫耐受的机制研究

Successful pregnancy relies on the accurate regulation of the maternal-fetal immune system. There is considerable evidence suggesting that Treg cells play a key role in the induction of immune tolerance to fetal antigens. Compared to Tim-3-Treg cells, Tim-3+Treg cells exhibit the enhanced suppressing function and proliferative activity. Tim-3+Treg cells might be highly efficient suppressors of effector T (Teff) cells. Our recent study shows that a large number of Tim-3+Treg cells were infiltrating in the decidua of mice at the early stage of pregnancy, and highly expressed the suppressing molecules. However, it remains unclear about the factors and mechanism on the accumulation of Tim-3+Treg cells. We hypothesize that IL-27 and Gal-9 secreted by trophoblast cells may promote the expression of Tim-3 and activate the Tim-3 signaling pathway on CD4+T cells and Treg cells, which contribute to the accumulation of Tim-3+Treg cells and the regulation of Treg/Teff balance at the maternal-fetal interface. In this study, by clinical observation, in-vitro study and animal model experiments, we continue to explore the factors that promote the accumulation of Tim-3+Treg cells and the underlying mechanism. Moreover, by setting up abortion prone mice model, we will further investigate the therapeutic effects by targeting Tim-3 signaling. We aim to elucidate the important molecular immunological mechanism of maternal-fetal immune tolerance, to discover the potential therapeutic target, and to provide supporting evidence for the early clinical prevention and treatment of pregnancy-related complications.

母胎界面免疫耐受的建立是成功妊娠的必要条件。调节性T（Treg）细胞能促进母胎耐受的建立，其数量和功能的正常是维持成功妊娠的关键。最新研究表明Tim-3+Treg细胞是一类特殊的有更强抑制能力的细胞亚群。近期本课题组研究发现Tim-3+Treg细胞在早期妊娠小鼠的子宫蜕膜大量聚集，且高表达抑制性功能分子。然而，其在母胎界面聚集的机制及发挥何种作用尚不清楚。我们推测：滋养层细胞通过分泌IL-27和Gal-9训导CD4+T细胞和Treg细胞上Tim-3的表达并激活其介导的信号通路，使Tim-3+Treg细胞在母胎界面聚集并调节Treg/Teff平衡，从而促进母胎耐受的建立与维持。本项目拟通过临床现象观察、体外实验和动物模型多层面来揭示Tim-3+Treg细胞在早期妊娠母胎界面聚集并参与母胎免疫耐受的机制，以期发掘潜在的治疗靶标，为制定妊娠相关性疾病的早期防治策略提供新的理论依据。

本项目探讨了Tim-3+Treg细胞在母胎界面聚集、诱导母胎免疫耐受的机制和干预不明原因复发性流产（RPL）的策略。我们采用流式细胞术检测Tim-3+Treg细胞在小鼠不同妊娠阶段的动态变化，发现其在E6.5天子宫局部大量聚集，远高于脾脏组织；相对于Tim-3-Treg细胞，Tim-3+Treg细胞表达更高水平的抑制分子（CTLA-4和PD-1），具有更强的免疫抑制功能。且在子宫引流淋巴结中Tim-3+ CD4+ T细胞和Tim-3+ Treg细胞表达更多的CCR4和CCR5，提示子宫局部Tim-3+Treg可能来源于子宫引流淋巴结。在异基因妊娠小鼠模型中阻断Tim-3信号通路使胚泡吸收率明显增加，表明Tim-3+Treg细胞在小鼠早期妊娠建立和维持中发挥着重要作用。临床样本中探究不明原因RPL与正常妊娠人外周和蜕膜组织中CD4+T细胞和Treg细胞上Tim-3的表达差异；发现Tim-3在早期妊娠蜕膜CD4+ T细胞表达，几乎不在外周CD4+ T细胞表达，表明Tim-3的表达具有区域特性。此外，蜕膜Tim-3+Treg细胞比例在RPL中明显降低，与其训导分子IL-27和Gal-9在绒毛组织中的表达异常有关。机制层面，通过体外共培养外周血CD4+T细胞和原代滋养层细胞，发现原代滋养层细胞通过分泌IL-27和Gal-9促进Treg 细胞上Tim-3的表达。进一步通过可溶性IL-27受体或抗Tim-3单克隆抗体（阻断IL-27或Tim-3信号通路）处理共培养细胞，发现IL-27在Tim-3表达和Tim-3通路的激活中起重要作用。通过流式细胞术和转录组测序分析，发现蜕膜Treg细胞中转录因子Blimp-1高表达，其表达量与Tim-3呈正相关。体外研究发现，IL-27能明显上调Treg细胞上Blimp-1和Tim-3的表达，且主要通过激活STAT1信号通路实现。进一步采取Luciferase方法验证了Blimp-1能结合在Tim-3分子启动子区域，上调Tim-3的表达。此外，利用LPS诱导的流产小鼠模型过继转输Blimp-1+Treg细胞，发现对小鼠妊娠结局有一定的挽救效应。本项目率先揭示了蜕膜Tim-3+Treg细胞的区域免疫特性、在母胎界面聚集并诱导免疫耐受的机制，同时探索了干预RPL的策略，这对完善母胎免疫耐受理论和制定RPL干预新措施有重要的理论参考价值。