可溶型ULBP2蛋白对NK细胞的负调效应及其肝癌免疫逃逸机制探讨

Activating receptor NKG2D activate NK cells by interaction with its ligands expressed on tumor cells membrane, and tumor cells can escape immune attack from NK cells by secreting soluble NKG2D ligands, including MICA/B and ULBP1.However, there was few report about another NKG2D ligand, ULBP2, especially about soluble ULBP2(sULBP2) involvement in immune evasion mechanism of hepatocellular carcinoma. We have demonstrated that reduced expression of membrane NKG2D ligands on hepatocytes can inhibit NK cells activation significantly, but little is known about soluble NKG2D ligands relation with NK cells function. In our preliminary clinical study, we found that serum sULBP2 of HBV related HCC patients was closely related with HCC progression. The project intends to expand patient cases and analyze serum sULBP2 relation with HCC prognosis; and investigate recombinant sULBP2 regulation mechanism in human NK cell lines and fresh NK cells. Furtherly, we plan to carry out NK cells transfer therapy to nude mice with HCC xenografts, and validate sULBP2 negative effect on NK cells immune therapy in this model. We aim to confirm the feasibility of targeting ULBP2 to improve NK cells immunotherapy, which may provide a new target for immunotherapy of hepatocellular carcinoma.

活化性受体NKG2D通过识别肿瘤细胞膜配体活化NK细胞，而肿瘤细胞可通过分泌可溶型NKG2D配体(MICA/B及ULBP1)而逃逸NK细胞的免疫攻击。NKG2D配体ULBP2的研究较少，尤其分泌型sULBP2与肝癌的免疫逃逸机制研究仍为空白。我们曾证实阻断肝细胞NKG2D膜型配体表达可大幅减缓NK细胞活化（黄玫等.Hepatology 2013），但是可溶型配体对NK细胞的作用仍然不明。我们初步的临床研究表明，HBV肝癌患者血清sULBP2与肝癌发展密切相关。本项目拟扩大病人病例并动态分析血清sULBP2与患者预后的相关性；进一步研究的重组表达的sULBP2蛋白在体外对人NK细胞系或新鲜NK细胞负调的分子机制；在肝癌裸鼠模型进行NK细胞转输治疗，以此模型验证sULBP2对NK细胞免疫治疗的负调作用，并寻找靶向拮抗sULBP2以提高NK细胞免疫治疗效应的可行性，为肝癌免疫治疗提供新的靶点。

肝癌是一种预后极差的恶性肿瘤，尽管肝癌诊断和手术治疗技术不断提高，肝癌患者术后五年生存率仍然仅为15-40%，这主要是由于免疫逃逸引起的肿瘤复发与转移。由于肝脏富含天然免疫细胞，因此天然免疫受体 NKG2D 在肝癌的发生发展中起着至关重要的作用。本项目重点研究NKG2D及其配体ULBP2在肝癌的免疫逃逸中的作用机制：1，通过收集肝癌患者血清，ELISA检测ULBP2可溶型蛋白表达水平,结合患者临床病理资料，判断sULBP2与肝癌肿瘤大小、数目、分化程度、转移性、预后的相关性；2，建立肝癌细胞系与NK细胞共培养体系，研究可溶型sULBP2蛋白对NK细胞表型和功能的影响，并探索其分子机制；3，建立裸鼠肝癌模型，进一步确认sULBP2对NK细胞免疫治疗的负调作用。实验结果表明，肝癌患者血清中sULBP2的表达水平与患者的预后密切相关，sULBP2越高，患者预后越差；对肝癌细胞系进行ULBP2表达水平的筛选后，进一步通过体外共培养实验证明sULBP2可溶型蛋白显著抑制NK细胞对肝癌细胞的杀伤活性；siRNA干扰实验表明ULBP2的可溶性蛋白表达可能受HIF1-ADAM10调控，且与肝癌细胞的转移潜能密切相关；裸鼠成瘤实验结果表明ULBP2可溶型蛋白促进了SMMC7721 细胞对NK92细胞的免疫逃逸。以上数据表明，sULBP2在肝癌的NK细胞免疫治疗起负调作用，而缺氧环境通过HIF1-ADAM10通路促进ULBP2的蛋白切割，靶向HIF1-ADAM10通路可能为提高NK细胞免疫治疗效应，为肝癌免疫治疗提供新的靶点。