NK细胞通过CXCR3/TNF-α/p53通路调控肝窦内皮细胞表型促进HBV特异性CTL应答的机制

Liver sinusoidal endothelial cells (LSEC) play an important role in maintaining immune tolerance by induction of specific T cells inability. It is still unclear how to reverse the tolerogenic effect of LSEC and mediate the clearance of HBV. It has been reported that natural killer cells (NK cells) could enhance virus specific cytotoxic T lymphocyte response through activation of antigen presenting cells. Our previous studies also indicated that activation of Pattern recognition receptor (for example NOD1) signaling in the liver could enhance T cell response in a LSEC dependent manner. The recruitment and activation of NK cells in the liver and the following TNF-α secretion facilitated the activation of LSEC, the mechanisms involved need to be clarified. Our hypothesis is that NK cells could induce the activation phenotype of LSEC, which are associated with the outcome of HBV infection. In this study, we will use NK-LSEC co-culture system, neutralization antibody, gene knockout mice, HBV infection animal model, as well as blood and liver sample of chronic hepatitis B patient to determine activating effect of NK cells on LSEC and intrahepatic CTL responses and to explore its mechanisms. Besides, we try to regulate the balance between co-stimulatory molecules and co-inhibitory molecules of LSEC by blocking or stimulating associated molecules, which may activate CTL responses and control HBV infection. Our study will be helpful to provide theoretical basis and novel targets for the development of new anti-HBV immunotherapies.

肝窦内皮细胞（LSEC）诱导特异性T细胞产生耐受，在维持肝脏免疫耐受中发挥重要作用。迄今尚不清楚，机体通过何种机制逆转LSEC的致耐作用而清除肝内HBV。文献报道：NK细胞可通过调控抗原提呈细胞功能而增强特异性CTL应答。我们前期研究也发现：模式识别受体NOD1信号通路可以LSEC依赖性方式增强T细胞应答；NK细胞在肝内募集、活化及分泌TNF-α，可促进LSEC活化，其机制有待阐明。申报者推测：HBV感染过程中，NK细胞可诱导LSEC活化表型表达，并与HBV感染转归相关。为此，本项目拟借助NK-LSEC孵育体系、抗体阻断或基因敲除小鼠、HBV感染动物模型、乙肝患者肝组织标本，开展如下研究：①探讨NK细胞对LSEC表型的调控作用，及其对肝内CTL应答的影响；②通过阻断和激活相关分子，调控LSEC表面共刺激分子/共抑制分子间平衡。本项目可望为探寻防治慢性HBV感染的新策略提供理论依据和新靶点。

实验背景与目的：肝窦内皮细胞（LSEC）的功能成熟在肝内T细胞活化和病毒感染控制中起着重要作用。据研究报道，自然杀伤（NK）细胞可促进抗原呈递细胞（APC）的功能成熟，特别是对于树突状细胞（DC）。然而，NK细胞与LSEC之间的相互作用却鲜有报道。本研究中，我们主要探讨了NK细胞是否参与调节LSEC成熟以及如何参与，并进一步在小鼠模型中研究该调控对控制HBV感染的实际作用。.实验方法和研究结果：通过高压尾静脉注射6μg pAAV/ HBV 1.2质粒建立慢性HBV复制小鼠模型。在HBV质粒注射后第14天，同样以高压尾静脉注射的方式将NOD1配体（二氨基庚二酸[DAP]）导入肝脏。我们发现高压尾静脉注射DAP可将外周NK细胞（cNK）募集到肝脏中，并以CXCR3依赖性方式促进NK细胞产生TNF-α和IFN-γ。重要的是，在CXCR3-/-小鼠中LSEC的成熟和DAP诱导的抗HBV效应均受到抑制，这可能与肝内cNK细胞数量的减少有关。与此一致地是，敲除cNK细胞而非肝脏驻留rNK细胞，也抑制LSEC的成熟和活化T细胞功能，从而降低了肝内HBV特异性T细胞反应，进而抑制了野生型和Rag1-/-小鼠肝内HBV的清除。此外，TNF-α或IFN-γ的刺激以及与肝内NK细胞共培养在一定程度上促进了LSECs的表型和功能成熟。.结论：NOD1诱导的NK细胞活化可能通过可溶性细胞因子和直接接触方式促进LSEC的成熟，从而增强肝内T细胞的免疫反应，从而控制HBV的复制和表达。