外泌体AOC3招募CD4+T细胞诱导PD-L1表达进而介导异位子宫内膜免疫逃逸的机制

The pathogenesis of endometriosis has not yet been elucidated. The survival of ectopic endometrial tissue probably related to the abnormal immune system in EMs patients. Based on previous work, by analyzing the exosomal protein profiling from ectopic cells, this study found that AOC3 was aberrantly high expressed in the exosoms. By further exploring previous reports, it was found that AOC3 could recruit CD4+T cells to infiltrate into surrounding lesions Moreover, we found that CD4+T cells isolated from EMs patients and their secreted exosomes could induce the expression of PD-L1 in ectopic endometrial cells. Therefore, we hypothesized that exosomal AOC3 secreted by ectopic endometrium collected CD4+T cells to infiltrate around ectopic tissues, and CD4+T cells could secrete specific exosome miRNA to induce the expression of PD-L1 in ectopic endometrium tissues caused immune escape. We will overload AOC3 amino acid sequence into exosome to detect the tendency to CD4+T cells. MiR sequencing was used to search for specific exo-miR from CD4+T cell induced PD-L1 expression. The molecular pathway mediating immune escape were determined by IP and RNA PULL DOWN techniques, and the indicators were verified and analyzed by various experiments. In addition, we will confirm that exosomal AOC3 in vaginal secretion can be used as a non-invasive diagnostic marker for EMs using large sample size verification.

子宫内膜异位症（EMs）的病因至今未被阐明。异位定植的内膜组织得已存活，可能是EMs患者体内的免疫系统处于异常。本研究以前期工作为基础，通过分析异位细胞外泌体蛋白质谱，发现在外泌体中异常高表达的蛋白AOC3，已有文献支持AOC3可招募CD4+T细胞外渗至病灶。同时我们发现EMs患者的CD4+T细胞外泌体可诱导异位内膜表达PD-L1。因此，我们假设：异位内膜组织分泌外泌体AOC3募集CD4+T细胞至异位组织，而CD4+T细胞可分泌特异性外泌体miRNA诱导异位内膜表达PD-L1造成免疫逃逸。我们将过载AOC3氨基酸序列至外泌体，检测其对CD4+T细胞的趋向性。MiR测序寻找特异性诱导PD-L1表达的CD4+T细胞外泌体miRNA。通过IP及RPD技术确定介导免疫逃逸的分子通路，并在体内和体外实验中对各项指标进行分析。另外，将通过大样本量确定阴道分泌物中外泌体AOC3可作为EMs诊断标志物。

子宫内膜异位症，因为其复杂的免疫环境，临床上仍然缺乏有效的药物治疗与无创诊断标志物。本项目中，我们首先利用了体外三维原代细胞模型、FISH结合免疫荧光、原代免疫细胞共培养和构建双重药物输送系统等手段，发现了子宫内膜异位症的病灶中确实存在免疫逃逸的现象，肥大细胞衍生的外泌体LINC00632协助雌激素受体调控免疫检查点蛋白VISTA/PSGL-1在子宫内膜异位症细胞中的异常高表达，同时，我们也揭示了在子宫内膜异位病灶中免疫检查点VISTA/PSGL-1能够引起效应T细胞的自噬性的耗竭。同时在本项目期间中，我们也利用了测序手段对子宫内膜异位组织来源的外泌体进行了微小RNA和蛋白组学的测序，发现了异位病灶中的肥大细胞来源的外泌体也会高表达tRF-Leu-AAG-001，这种特殊的tRNA微小片段，可以促进周围血管的成管能力和炎症因子的表达增加。我们从子宫内膜异位症患者和对照组患者的阴道分泌物中成功提取了外泌体，并验证了白带外泌体MSN蛋白，tRF-Leu-AAG-001，以及has-exo-miRNA202-3p的表达，结果显示异位症患者的白带外泌体中这三种不同的生物标志物均在异位症患者的阴道分泌物白带中异常升高，并且阴道分泌物外泌体tRF-Leu-AAG-001的表达量与子宫内膜异位症的临床评分呈现正相关。本项目的研究结果可能将为子宫内膜异位症的免疫治疗提供新的临床思路，为异位症无创诊断的提供新的候选标志物。