TGF-β1诱导PD-1调控泡型包虫病肝脏区域和系统CD8+T细胞耗竭的机制研究

Alveolar echinococcosis (AE), similar to the invasive growth of hepatocellular carcinoma, has not been effectively treated yet. The clinical manifestations of AE are closely related to immune function of CD8+ T cells. In our previous study, we found that expression of co-inhibitory molecule PD-1 was significantly increased in the liver and peripheral CD8+ T cells of AE patients, and was positively correlated to the expression of TGF-β1, whereas CD8+ T cells function decreased. In vitro TGF-β1 promotes the up-regulated expression of PD-1 in CD8+ T cells. Furthermore, MAPK signaling pathway was activated in liver lesions, suggesting that TGF-β1 may promote PD-1 expression through MAPK signaling pathway and induce CD8+ T cells exhaustion, resulting in immune escape and high pathogenicity of E.multilocularis. However, the detailed mechanism is to be elucidated. In this work, we plan to clarify the roles of TGF-β1 and PD-1 in CD8+ T cells exhaustion in AE patients, and the correlations with clinical typing. Through the establishment of E. multilocularis infection models in mice (wild type and deficient type), we try to understand the molecular mechanism of up-regulating PD-1 expression by TGF-β1 through MAPK signaling pathway, which mediates CD8+ T cell immune exhaustion. Finally, we will explore the feasibility and effectiveness for salvaging CD8+ T cells function by blocking TGF-β1/PD-1 or their signaling related molecules. These studies are of great significance for exploring new strategies for immunotherapy of hydatid disease.

泡型包虫病（AE），类似侵袭性生长的“肝癌”，至今无有效治疗手段。AE临床表现与CD8T细胞免疫功能密切相关。我们前期发现负性调节因子PD-1在AE病人肝脏局部和外周CD8T细胞表面表达显著增加，与TGF-β1的表达呈正相关，且CD8T细胞功能降低。体外实验证实TGF-β1诱导CD8T细胞PD-1表达上调。肝脏病灶周围MAPK信号通路被激活，这提示TGF-β1通过MAPK信号通路上调PD-1表达，进而诱导CD8T细胞耗竭，造成Em免疫逃逸和高致病性，但具体机制仍然不明。本项目拟利用临床标本，明确TGF-β1和PD-1在AE病人CD8T细胞耗竭中作用，与临床分型关系。利用泡球蚴小鼠（野生和缺陷）模型阐明TGF-β1通过MAPK信号通路上调PD-1表达，介导CD8T细胞耗竭分子机制。靶向阻断TGF-β1和PD-1或细胞转输，以证实恢复CD8T细胞功能，对探讨包虫病新的免疫治疗策略有重要意义。

泡型包虫病（Alveolar Echinococcoisis，AE）是由多房棘球绦虫（Echinococcus multilocularis，E. m，泡球蚴）的幼虫（六钩蚴）寄生于人体所致的一种“类肝癌样”致死性寄生虫病。宿主的免疫状态是影响泡球蚴感染寄生、病灶活性及疾病转归的重要因素。TGF-β1调控PD-1介导AE患者T细胞免疫耗竭的分子机制不明。本研究首先利用AE患者肝脏手术标本和外周血，鉴定肝脏形成“炎症微环境”，以T细胞亚群为主，高通量筛选分析发现病灶周围免疫抑制性分子呈上调表达（如TIGIT、PD-L2、CTLA4和IL-10等）；流式细胞术和ELISA检测发现AE患者肝脏局部和外周TGF-β1和T细胞表面PD-1上调表达，导致T细胞免疫耗竭；免疫组化染色显示MAPK（ERK1/2）通路激活，以单核细胞和成纤维细胞定位为主。通过体外培养AE患者分离的PBMC细胞，添加PD-1中和抗体阻断，发现T细胞分泌效应分子能力与IgG对照组无差异。其次，建立经肝门静脉小鼠模型，确定小鼠体内泡球蚴感染不同阶段时间-剂量效应，发现高剂量感染晚期肝脏病灶以T细胞浸润为主；利用免疫缺陷小鼠或抗体清除实验，证实T细胞缺陷促进肝脏泡球蚴病灶快速生长。建立泡型包虫病野生型和PD-1缺陷型小鼠模型，发现感染晚期肝脏和脾脏T细胞表面PD-1表达上调，导致T细胞功能下降；并且PD-1缺陷能够部分恢复T细胞功能，但未能抑制肝脏病灶生长；进一步分析PD-缺陷小鼠模型，发现肝脏和脾脏募集大量G-MDSC细胞，利用Gr-1中和抗体清除，促进T细胞分泌细胞因子，抑制肝脏病灶生长。最后建立泡型包虫病小鼠模型，利用TGF-β1、PD-1和PD-L1中和抗体分别进行晚期治疗，发现肝脏和脾脏T细胞部分恢复效应因子的分泌，但不能抑制肝脏病灶生长；通过PD-1联合MDSC清除（Gr-1中和抗体），可以恢复T细胞功能，提高T细胞的免疫监视和杀伤能力，证实用于治疗泡型包虫病小鼠模型的可行性和有效性。本研究揭示PD-1调控泡型包虫病肝脏和系统T细胞免疫耗竭分子机制，明确PD-1中和抗体联合MDSC清除（Gr-1）对泡型包虫病小鼠模型治疗的可行性，对研制新型药物，探索泡型包虫病新的临床治疗方法具有重要的科学意义。