NK细胞输注联合甲基化抑制剂、组蛋白去乙酰化酶抑制剂清除残留恶性B细胞的研究

Natural killer (NK) cell is the main component of immune surveillance system whose responsibility is eliminating malignant or infected cells. However, the cytotoxicity induced by NK cells is miminal when targeting on malignant B cells. Therefore, enhancement the sensitivity of malignant B cells to NK cells would increase the therapeutic effect toward B cell malignancies dramatically, and improve the long term survival of patients. We have demonstrated previously,that aberrant epigenetic mechanisms including hypermethylation in the promoter associated region and histone deacetylation repress the transcription of PVR and Nectin-2 genes in malignant B cell lines. The two genes are function as activating ligands which could promote NK cells to kill target cells after combining with their corresponding receptors in NK cells. We have also shown that the hypermethylation inhibitor and histone deacetylase (HDAC) inhibitor could reverse the aberrant epigenetic modulation, therefore enhance the transcription of the two genes. As a result, the sensitivity of malignant B cell lines to NK cell lines has increased significantly. In this study, we are going to continue the research project with primary malignant B cells being isolated from patients in both in vitro and in vivo experiments. We want to clarify that aberrant epigenetic mechanisms repress the transcription of PVR and Nectin-2,while hypermethylation inhibitor and HDAC inhibitor could restore the transcription of the two genes, therefore enhance the sensitivity of primary malignant B cells to NK cells. We want to provide basic data in order to improve the therapeutic effect in B cell malignancies and increase the immunotherapeutic effect by combining chemotherapy and NK cell-transfusion.

NK细胞发挥重要的抗肿瘤免疫监视作用。但NK细胞对恶性B细胞杀伤作用微弱。若能解决这一难题，将极大提高B细胞肿瘤的治疗效果，改善患者的长期生存。我们发现：基因启动子区异常高甲基化、组蛋白去乙酰化，抑制了恶性B细胞系中活化型NK细胞配体PVR、Nectin-2的转录；甲基化抑制剂、组蛋白去乙酰化酶（HDAC）抑制剂能促进这两种基因转录，进而增加其在细胞表面的表达，最终显著提高NK细胞杀伤恶性B细胞的效率。本研究拟用患者原代恶性B细胞进行体外细胞实验及荷瘤小鼠实验。希望揭示：启动子区异常高甲基化、组蛋白去乙酰化同样抑制了原代恶性B细胞中PVR、Nectin-2的转录；药物治疗能逆转异常机制、促进基因转录、进而增加这两种基因在原代恶性B细胞的表达，最终提高NK细胞杀伤原代恶性B细胞的效率；为将甲基化抑制剂、HDAC抑制剂与NK细胞输注联合使用，清除残留恶性B细胞的I期临床提供实验支持。

1、研究背景：B淋巴细胞来源的恶性肿瘤是一大类成人高发的血液系统恶性肿瘤。治疗的核心是在化疗、放疗降低肿瘤负荷基础上，依靠机体的抗肿瘤免疫反应来清除残留恶性B细胞，达到治愈。自然杀伤（NK）细胞在机体的抗肿瘤免疫反应中发挥重要作用，是抗肿瘤免疫治疗的重要工具细胞。但是由于恶性B细胞低表达活化型NK细胞配体，高表达抑制型NK细胞配体，因此减弱了NK细胞对于恶性B细胞的杀伤作用。若能提高NK细胞对于恶性B细胞的杀伤效率，则能减少、甚至避免疾病复发。PVR、Nectin-2是目前已知的、分布在靶细胞表面的、活化型NK细胞配体。但在恶性B细胞表面这两种配体表达水平显著降低。本研究旨在回答下述问题：异常表观遗传学是否导致了恶性B细胞表面PVR、Nectin-2的低表达？用药物处理原代恶性B细胞，能否提高这两种基因的表达，进而促进NK细胞杀伤原代恶性B细胞？在荷瘤小鼠模型中，联合5AZA、SAHA治疗与NK细胞输注，是否能促进NK细胞清除恶性B细胞？.2、研究内容：用流式细胞术检测从患者体内分离的原代恶性B细胞表面PVR、Nectin-2表达水平；用实时定量PCR检测纯化原代恶性B细胞中这两种配体在mRNA表达水平；用重硫酸盐PCR测序方法检测PVR、Nectin-2基因启动子区CpG甲基化水平；对比药物处理前后这两种配体在mRNA、蛋白水平的变化；体外用NK细胞系与药物处理前后的原代恶性B细胞共同培养，对比细胞毒作用的强弱；恶性B细胞荷瘤小鼠（NOD/SCID）实验。.3、重要结果：恶性B细胞在蛋白及mRNA水平均低表达PVR、Nectin-2。恶性B细胞中PVR、Nectin-2基因启动子区异常高甲基化抑制了这两种基因的转录，进而导致两种蛋白表达降低。用甲基化抑制剂5AZA可以部分逆转高甲基化状态，进而部分恢复PVR、Nectin-2的表达。组蛋白去乙酰化酶抑制剂SAHA也能单独或与5AZA联合，上调PVR、Nectin-2在恶性B细胞的表达。用5AZA、SAHA单独或者联合处理原代恶性B细胞后，能部分纠正异常的表观遗传学（包括异常高甲基化、组蛋白去乙酰化），从而增加恶性B细胞对于NK细胞介导细胞毒作用的敏感性。用RAJI、DAUDI两种恶性B细胞系建立NOD/SCID荷瘤小鼠模型，之后降NK细胞与5AZA和或SAHA联合使用，能显著提高荷瘤小鼠的生存时间。