NKG2A+NK细胞诱导抗病毒CD8+T细胞免疫耗竭及其机制研究

Reversal of CD8 + T cell immune exhaustion is the key to prevent chronic viral infection but remains to be explored. In our previous studies, an increased proportion of natural killer (NK) cells expressing inhibitory receptor NKG2A with exhausted CD8+T cells were found in peripheral blood in patients suffering from chronic hepatits B (CHB). Moreover, blocking the NKG2A increased activity of NK cells and restored anti-HBV CD8+ T cell proliferation and function in HBV-carrier mice, suggesting that NK cell exhaustion may be a prelude to CD8+T cell exhaustion. Therefore, the aim of the present project is to define the role of NKG2A+NK cell exhaustion involved in leading to CD8+ T cell immune exhaustion. Firstly, we will further explain that NKG2A+ NK cells are involved in inducing CD8+ T cell immune exhaustion in patients infected with HBV and mouse models of HBV and LCMV. Secondly, the underlying mechanism of higher expression of NKG2A resulting in NK cell exhaustion and the character of exhausted NK cells will be revealed preliminarily. Thirdly, and then we will observe the time series of NK cell exhaustion and CD8+T cell exhaustion, and the regulation of CD8+T cell by exhausted NK cells compared with normal NK cells. At last, we will investigate whether blocking NKG2A can inhibit the secretion of IL-10 or enhance the secretion of IFN-gamma from NK cells by anti-NKG2A blocking antibody, and observe whether CD8+T cell exhaustion can be reversed simultaneously. In conclusion, the hypothesis that CD8+T cell exhaustion is derived from pre-exhausted NK cell in this study is important for the design of targeted NKG2A and early reversal of NK cell exhaustion to prevent subsequent exhaustion of CD8+T cells, and thus has important anti-viral and anti-tumor significance.

逆转CD8+T细胞免疫耗竭是阻止慢性病毒感染的关键。前期研究表明CHB（慢乙肝）患者NK细胞高表达NKG2A并伴有CD8+T细胞耗竭，阻断NKG2A可恢复CD8+T细胞抗HBV效能，提示NK细胞耗竭可能是CD8+T细胞耗竭的前奏。本课题拟利用HBV患者、小鼠HBV、LCMV模型及缺陷小鼠进一步阐释NKG2A+NK细胞参与诱导CD8+T细胞耗竭；深入揭示NKG2A高表达导致NK细胞耗竭的机制及耗竭NK的表型与功能特性；进而观察NK细胞耗竭与CD8+T细胞耗竭的时间动力学顺序，以及正常NK与耗竭NK细胞对CD8+T细胞调控特征；探究阻断NKG2A可否抑制NK细胞分泌IL-10或增强分泌IFN-g，并观察可否同时逆转CD8+T细胞耗竭。该研究提出的CD8+T细胞耗竭来源于NK细胞预先耗竭的假设，对于设计靶向NKG2A并早期逆转NK细胞耗竭以阻止后续CD8+T细胞耗竭有重要的抗病毒及抗肿瘤意义。

乙型肝炎病毒（hepatitis B virus, HBV）是引起慢性乙型肝炎（chronic HBV, CHB）及其进一步进展为肝硬化和肝癌的重要原因。研究表明CHB患者抗原特异性CD8+ T细胞免疫耗竭是病毒持续存在的重要原因。然而目前对于慢性感染过程中CD8+T细胞耗竭产生的根本原因及其耗竭的“前奏”尚不清楚。自然杀伤细胞（natural killer cells，NK cells）被誉为天然免疫的核心细胞，其表型、功能与CD8+T细胞的免疫应答状态具有相关性。.本课题发现CHB患者外周血NK细胞表面NKG2A表达比例增高，与HBV-DNA载量呈正相关。通过阻断NKG2A明显提高CHB患者NK细胞功能。此外，CHB患者CD4+ Treg表达比例及其分泌IL-10明显增高，且CHB患者外周血NKG2A+ NK细胞与IL-10+ Treg呈明显正相关。体外实验发现CHB患者血清及HBeAg可通过IL-10诱导NKG2A+ NK细胞表达，阻断IL-10可恢复NK细胞功能耗竭。因此，我们的研究结果表明CHB患者中NK细胞高表达NKG2A导致免疫耗竭，HBeAg通过诱导CD4+ Treg细胞分泌IL-10介导NK细胞高表达NKG2A 及NK细胞功能降低。进一步扩大分析CHB、 LC 和 HCC 患者，发现IFN-gamma+ NK 细胞比例及其与 Treg 比例、 血清 IL-10水平呈负相关，提示NK 细胞分泌IFN-gamma功能减低可能是慢性 HBV 感染及其进展至HCC的重要因素之一。此外，在SARS-CoV-2感染导致的新型冠状病毒肺炎（COVID-19）患者中也发现外周血中NKG2A+ NK细胞和NKG2A+ CD8+ T细胞的比例明显升高并伴有功能耗竭。而恢复期COVID-19感染者经治疗后NKG2A在NK和CD8+ T细胞表达减低，揭示COVID-19患者早期感染中免疫检查点NKG2A介导NK和CD8+ T细胞功能耗竭及其可能参与患者病情恶化及后续发生炎症风暴，表明NKG2A靶点控制COVID-19的炎症风暴也具有重要意义。.上述研究揭示了COVID-19患者NK细胞免疫耗竭以及CHB患者NKG2A+ NK细胞免疫耗竭的新机制，提出靶向NK细胞及其免疫调节功能逆转病毒感染所致免疫耐受的新思路，也为阐明重大疾病的发病机制和利用NK细胞治疗疾病提供科学依据。