肝窦内皮细胞上TLR3/CXCL10信号通路活化对改善肝癌免疫治疗的作用及机制研究

The therapeutic choices for advanced liver cancer are limited, and immune checkpoint blockade (ICBs) have been developed to be a new kind of therapies. However, tumors always develop resistance to ICBs. And combined immunotherapy has become a trend. My previous research, which was published in Hepatology, has shown that polyinosinic-polycytidylic acid (PolyIC), a TLR3 agonist, could enhance the effect of PD-L1 Ab on the treatment of hepatocellular carcinoma in mice, which was dependent on PolyIC-triggered recruitment, proliferation, activation and cytotoxicity of CTLs. However, the mechanism remains to be unclear. In our pre-experiment, we found that PolyIC could also enhance the anti-tumor effect of another immune checkpoint inhibitor, CTLA-4 Ab. We further found that TLR3 is highly expressed in the liver sinusoidal endothelial cells (LSECs) and in LSECs, TLR3 activation by PolyIC lead to the high expression of a chemokine, CXCL10. So, we propose the scientific hypothesis: TLR3 agonist PolyIC activates the TLR3/CXCL10 signaling in LSECs to regulate the aggregation and activation of CTLs in liver, and finally enhance the therapeutic effects of ICBs on hepatocellular carcinoma in mice. By using the hydrodynamic transfection-induced mouse HCC model, this study aims to clarify the detailed mechanism of TLR3 activation to regulate CTLs in liver microenvironment, and provide the new strategy to improve the immunotherapeutic effect of hepatocellular carcinoma.

晚期肝癌的治疗手段有限，免疫检查点抑制剂（ICB）成为一种新疗法，但肿瘤常产生抗性。联合免疫治疗成为一种趋势。申请人发表在Hepatology的研究发现TLR3激动剂聚肌胞（PolyIC）能够增强PD-L1 Ab对小鼠肝癌的治疗作用，依赖于PolyIC调控的细胞毒性T细胞（CTLs）的聚集和活化，但机制尚未明确。进一步预实验发现，PolyIC也能够增强CTLA-4 Ab的抗肝癌效果。而TLR3在小鼠肝窦内皮细胞上高表达，且PolyIC诱导肝窦内皮细胞高表达趋化因子CXCL10。因此，我们提出科学假设：TLR3激动剂PolyIC通过激活肝窦内皮细胞上的TLR3/CXCL10信号通路，调控肝脏中CTLs的聚集和活化，增强免疫检查点抑制剂治疗小鼠肝癌的疗效。利用水动力转染技术诱导小鼠肝癌模型，本课题旨在明确肝脏微环境中TLR3激活后调控CTLs的作用机制，为提高肝癌的免疫治疗效果提供新的策略。

原发性肝癌是一种预后较差的恶性肿瘤，是全球第七大常见癌症，也是癌症相关死亡的第三大原因，具有极强的组织病理学和分子生物学异质性，近年来，免疫疗法成为肝癌治疗的重要方法，PolyIC作为一种免疫佐剂，在肝癌免疫治疗领域拥有深厚的潜力，其通过激活Toll样受体3从而提升肿瘤微环境中固有免疫反应和特异性免疫反应。然而既往的临床研究发现，poly(I:C)的治疗效果并不理想，表明其可能存在其他免疫作用机制。本研究发现，在小鼠肝癌模型中不同时期PolyIC治疗效果均不佳，流式分析发现PolyIC能够上调肿瘤微环境中的IL10(+)与GR1(+)免疫细胞，而NK细胞等效应细胞的水平无变化，这表明PolyIC已失去免疫激活功能。因此我们构建了不同免疫细胞上的Tlr3特异性敲除转基因小鼠来研究其对肝癌的作用，发现了树突状细胞上Tlr3可促进肝癌的发展。对Tlr3特异性敲除转基因小鼠研究发现，肝癌组织中的IL10水平显著下降，NK细胞显著上调，MDSC细胞显著下调，我们进行了NK细胞中和实验证明了NK细胞为主要变化的效应细胞。最后我们联合IL10单抗和PolyIC给药发现与单独给药相比，联合给药显著降低了肿瘤负荷。以上结果表明，树突状细胞上Tlr3的激活可上调肝癌免疫微环境中的IL10和MDSC水平，进一步抑制NK细胞从而促进癌症的发展，这一现象揭示了PolyIC治疗效果不佳的机制，为免疫治疗提供了全新的靶点。