NK细胞抑制Treg细胞介导的HBV免疫耐受机制

Regulatory T cells contribute to the persistant hepatitis B virus (HBV) infecion. Reversal of Treg cell-induced immune tolerance during chronic hepatitis B virus infection is a promising stategy in clinic but remains to be explored. In previous studies, patients with acute hepatitis B (AHB) have a decreased proportion of regulatory T (Treg) cells but have significantly activated natural killer (NK) cells in peripheral blood compared with patients suffering from chronic hepatitis B (CHB). Moreover, by using a mouse model of acute hepatitis B virus infection mimicking patients with AHB, depletion of NK cells in vivo results in HBV persistence in mice,with higher absolute number of Treg cells and higher IL-10 secretion in liver draining lymph nodes. Thus,we hypothesize that Treg cells may be negatively regulated by NK cells during acute hepatitis B virus infection. Therefore, the aim of this present project is to define the regulatory role of NK cells on Treg cells by analysis of asymptomatic HBV-carrier patients,AHB patients, CHB patients (immune tolerant phase and immune activation phase) and mouse models mimicking AHB and CHB patients. Firstly, the number, phenotype and function of Treg and NK cells in HBV patients will be investigated and the underlying molecular mechanisms involved in this regulation will be revealed priliminarily. Secondly, we will evaluate the regulatory effects of NK cells on Treg cells in mouse models of HBV clearance and HBV tolerance. At last, we will confirm the inhibitory role of NK cells on Treg cell-mediated immune tolerance.In conclusion,this project will provide an important sight for the understanding of the regulatory role of NK cells in immunology,and the mechanisms of reversal of Treg cell-mediated HBV tolerance by NK cells, contributing to the immunological basis for the treatment of CHB patients.

调节性T（Treg）细胞是导致HBV免疫耐受的主要原因，打破Treg细胞介导的HBV免疫耐受是提高HBV治愈率的关键。研究表明NK细胞显著活化的急性HBV患者（AHB）体内调节性T（Treg）细胞比例低于慢性HBV（CHB）患者，且清除小鼠HBV感染模型中NK细胞则导致Treg细胞数目和IL-10分泌显著升高，但其机制不明。推测急性HBV患者体内NK细胞负向调控Treg细胞。本课题拟比较AHB、CHB患者以及无症状HBV携带者体内NK细胞和Treg细胞的表型和功能，阐明NK细胞对Treg细胞的抑制效应，寻找NK细胞对Treg细胞行使负向调节的关键分子；利用小鼠HBV清除模型探讨NK细胞对Treg的负向调控及其与HBV清除之间的关系；进一步采用小鼠HBV耐受模型验证NK细胞抑制Treg细胞所介导的免疫耐受，得以率先了解NK细胞在免疫耐受中的作用，为筛选新的药物靶标治疗CHB患者提供理论依据。

慢性乙型肝炎（chronic hepatitis B, CHB）的主要特点是免疫耐受，而调节性T（regulatory T cells,Treg）是导致HBV免疫耐受形成的主要原因。本研究旨在探索负向调控Treg细胞的分子机制，以期干预HBV免疫耐受和提高CHB患者治愈率。通过对C57BL/6小鼠尾静脉高压注射HBV质粒构建模拟人HBV急性感染的小鼠模型，血清HBsAg和HBeAg在3-4 周内清除。NK细胞分泌IFN-g明显增加， NK细胞清除则导致HBV持续感染及HBcAg-特异性CD8+ T细胞明显减低。过继实验发现NK-IFN-g可促进CD8+ T细胞的增殖和活化。此外， NK细胞介导抗-HBV CD8+ T细胞的再次免疫应答。肝脏DX5+CD49a- NK细胞促进抗-HBV CD8+ T细胞的免疫应答。该研究成果揭示了肝脏NK细胞通过其分泌的IFN-g促进抗-HBV CD8+ T 细胞的免疫应答，为HBV的免疫治疗提供理论基础。接下来，我们清除HBV感染小鼠体内的NK细胞，发现脾脏及肝脏引流淋巴结中Treg细胞的比例及数目显著增加，且肝脏淋巴结细胞IL-10分泌显著升高。因此， NK细胞清除导致HBV持续感染可能与Treg细胞和IL-10的分泌增加有关，提示急性HBV感染过程中可能存在NK细胞对Treg细胞的负向调控效应。.进一步研究表明CHB患者HBV DNA高水平组患者ALT明显高于HBV DNA低水平组。并且HBV DNA高水平组NKG2A+NK细胞明显高于HBV DNA低水平组。同时HBV DNA高水平组Treg亦明显高于HBV DNA低水平组和对照组。此外，NKG2A+ NK细胞与HBV DNA载量及Treg均呈正相关性，提示CHB患者NK细胞高表达抑制性受体NKG2A与Treg密切相关，为以NK细胞为靶标抑制Treg细胞介导的免疫耐受提供理论依据。.上述研究表明，NK细胞作为一个异质性群体，DX5+NK细胞亚群参与抑制HBV免疫逃逸，而NKG2A+NK细胞可能参与诱导HBV免疫耐受，该研究更新了已有NK细胞在HBV感染中的认识，提出不同NK细胞亚群参与不同的HBV感染状态，升华了NK细胞在HBV免疫治疗中的应用价值。