EBV阳性胃癌免疫卡控点干预治疗意义及抗肿瘤作用研究

Analysis of the clinical significance of interventional treatment of EBV positive gastric carcinoma immune checkpoint blockade and its antitumor effects

目的本研究以EB病毒(EBV)相关胃癌为研究对象,探讨在EBV相关胃癌中介导免疫耐受的免疫卡控点基因,进一步建立靶向于EBV抗原的免疫卡控点基因敲除的细胞毒性T细胞(CTL)的方法,并通过联合低剂量放疗协同免疫细胞发挥抗肿瘤效应,由此建立一种新型的综合免疫治疗模式,评估该治疗模式在EBV相关胃癌治疗中的价值。方法通过GEO公共数据库分析EBV阳性胃癌相关的免疫调控基因的表达及免疫组化明确相关程序性死亡蛋白1(PD-1)/程序性死亡蛋白配体1(PD-L1)蛋白表达情况,进一步采用CRISPR-Cas9基因编辑系统,建立PD-1分子敲除的EBV-CTL,评价PD-1分子敲除对EBV-CTL功能的影响;并构建SNU-719荷瘤小鼠模型,联合小剂量放疗评价基因敲除后的细胞在动物体内抗肿瘤作用优势。结果 EBV相关胃癌患者胃癌组织中的免疫相关调控基因明显上调,PD-L1表达水平明显高于非EBV相关胃癌患者。在PD-1敲除的T细胞的基础上,采用EBV相关抗原潜伏膜蛋白2A(LMP2A)诱导的CTL具有更强的γ-干扰素(IFN-γ)分泌水平,在EBV相关胃癌的裸鼠模型中,联合低剂量放疗后该细胞的抗肿瘤效果明显增强。结论PD-1等免疫卡控点基因的阻断治疗联合细胞治疗有望为EBV阳性胃癌提供新的治疗策略。

Objective This study aims to explore the immune checkpoint genes which mediated immune tolerance on EBV associated gastric carcinoma and to generate immune checkpoint gene disrupted CTL targeted to EBV antigen. Moreover, this study is to evaluate the coordinate effect of this cellular therapy with low dose radiotherapy for the treatment of EBV associated gastric carcinoma. Method We analyze the up-regulated immunoregulatory genes in EBV positive gastric cancer by GEO data-analysis and their protein levels of PD-1/PD-L1 by immunohistochemistry. We further used gene knockout system CRISPR-Cas9 to generate PD-1 disrupted CTL and evaluate the synergy effect when combined with small dose radiotherapy through an immune-deficient mice tumor xenograft model. Result The immune related regulatory genes in gastric cancer tissues of EBV related gastric cancer patients were significantly up-regulated, and the expression level of PD-L1 was significantly higher than that of non EBV related gastric cancer patients. On the basis of PD-1 knockout T cells, CTL induced by EBV associated antigen LMP2A has a stronger IFN- gamma secretion level. In the nude mice model of EBV related gastric cancer, the antitumor effect of this cell therapy is obviously enhanced after combined low dose radiotherapy. Conclusion The blocking therapy of PD-1 and other immunosuppressive genes combined with cell therapy is expected to provide new strategies for the treatment of EBV positive gastric cancer.