母胎界面MDSCs通过Gal-9-Tim-3调控uNK功能介导免疫耐受的机制研究

Immune tolerance at maternal-fetal interface during early pregnancy is crucial for successful pregnancy and represents an essential issue in reproductive immunology. Uterine natural killer cells (uNK), the major population of decidual lymphocytes during the first trimester, play an important role in regulating pregnancy immune tolerance by interacting with dendritic cell, monocytes and T cells. The model of pregnancy tumor-bearing mice shows that myeloid-derived suppressor cells (MDSCs) expand significantly during pregnancy and promote immune tolerance and tumor metastasis via inhibiting the cytotoxic activity of NK cells. In view of the similarity between pregnancy immune tolerance and tumor immune tolerance in immunology, we speculate that MDSCs expanded at maternal-fetal interface may promote the formation of immune tolerance via regulating the function of uNK. Recent studies suggest that MDSCs induce T cell exhaustion and anergy via Gal-9-Tim-3 pathway. Our results showed that Tim-3 mainly expressed in uNK compared with other cell types in human decidual tissue, and the expression level of Tim-3 is significantly higher than that of periphery NK cells. Based on these results, we will detect the level of MDSCs at maternal-fetal interface for the first time as far as we know, and investigate the roles of Gal-9-Tim-3 pathway on the regulatory mechanism of MDSCs to NK cytotoxicity, cytokines expression and apoptosis. Through the research of this subject, we will further reveal the additional regulatory mechanism of the immune tolerance at maternal-fetal interface and provide new strategies and targets for prevention and treatment of pathological pregnancy.

母-胎界面免疫耐受的建立是成功妊娠的关键，uNK是妊娠早期蜕膜淋巴细胞的主要群体，通过与DCs、单核细胞及T细胞等相互作用在调节妊娠免疫耐受中发挥重要作用。妊娠荷瘤小鼠模型的研究显示，髓系来源抑制细胞（MDSCs）在妊娠发生时大量扩充，并通过抑制NK杀伤活性促进免疫耐受及肿瘤转移。我们前期结果发现人蜕膜组织中MDSCs大量聚集，然而其在调节母-胎界面免疫耐受中的作用及机制仍不清楚。最近的研究表明MDSCs可通过Gal-9-Tim-3通路调节T细胞耗竭和失能，我们课题组对人蜕膜组织Tim-3的表达进行检测，发现Tim-3主要在uNK中表达。基于以上分析，本课题拟在前期研究的基础上，首次分析母-胎界面MDSCs的水平，并围绕Gal-9-Tim-3通路探讨MDSCs对NK细胞杀伤、细胞因子表达及凋亡的调控作用，旨在进一步揭示母-胎界面免疫耐受的调节机制，为病理性妊娠的防治提供新的策略及靶点。

母体对半同种异体胎儿免疫耐受的建立是成功妊娠的关键，一旦打破即可导致习惯性流产等病理性妊娠发生。揭示母-胎界面免疫耐受的建立机制将为病理性妊娠的防治提供新的策略及靶点。研究表明母-胎界面免疫细胞功能具有区域特性，蜕膜NK细胞（dNK）在妊娠早期母胎界面大量聚集，其功能、转录特征与外周NK（pNK）显著不同。另外母胎界面T细胞则发生Th2极化，同时Treg细胞大量募集发挥免疫抑制作用。近年来研究发现髓系来源抑制细胞（MDSCs）在妊娠早期外周及母胎界面大量聚集，然而其在母胎免疫中的确切功能及与病理性妊娠的关系仍不清楚。本项课题中，我们首次系统分析了妊娠过程中外周及母胎界面MDSCs的水平，发现妊娠发生时外周血MDSCs较未孕对照显著增加，而习惯性流产患者外周MDSCs水平较正常妊娠组则显著降低。在蜕膜组织局部MDSCs大量聚集，并可通过Tim-3-Gal-9通路诱导NK细胞凋亡，抑制NK细胞IFN-γ和TNF-α水平，并抑制NK细胞对滋养层细胞系HTR-8的杀伤。此外我们比较分析Tim-3在正常妊娠、习惯性流产患者及健康对照pNK细胞中的表达，结果表明妊娠发生时pNK细胞Tim-3的表达显著上调，习惯性流产患者pNK表达Tim-3的水平则较正常妊娠降低；蜕膜组织当中Tim-3主要在dNK中表达，且dNK表达Tim-3的水平显著高于pNK细胞。同时我们发现母-胎界面微环境通过TGF-β1上调NK细胞Tim-3的表达，并且Tim-3+ dNK细胞表达更高水平的活化标志分子：CD94、CD69和CD11b。利用Tim-3-Fc抗体阻断Tim-3可显著抑制dNK细胞IFN-γ和TNF-α的水平。此外我们的研究发现，Tim-3通过破坏脱颗粒进而抑制NK细胞对滋养层细胞系HTR-8/SVneo的杀伤。本课题首次分析妊娠过程中MDSCs的水平，并围绕 Gal-9-Tim-3通路探讨MDSCs对NK细胞杀伤、细胞因子表达及凋亡的调控作用，进一步补充妊娠免疫耐受的调节机制，为病理性妊娠的防治提供新的见解及策略，同时也为器官移植、肿瘤、自身免疫性疾病等免疫相关疾病的治疗提供实验依据及新的治疗靶点。