基因工程化iNK^(NKG2A KO)细胞具有HLA-E特异的抗肿瘤活性  

Engineered iNK^(NKG2A KO)cells possess HLA-E specific anti-tumor activity

作　　者：乔雯华 许依 董鹏 何维 陈慧 张建民 QIAO Wenhua;XU Yi;DONG Peng;HE Wei;CHEN Hui;ZHANG Jianmin(Department of Immunology,Key Laboratory for T Cell and Immunotherapy,State Key Laboratory of Common Mechanism Research for Major Diseases,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005;Changzhou Xitaihu Institute for Frontier Technology of Cell Therapy,Changzhou 213000,China)

机构地区：[1]中国医学科学院基础医学研究所,北京协和医学院基础医学院,免疫学系T细胞与免疫治疗重点实验室,重大疾病共性机制研究全国重点实验室,北京100005 [2]常州西太湖细胞治疗前沿技术研究院,江苏常州213000

出　　处：《基础医学与临床》2025年第5期599-607,共9页Basic and Clinical Medicine

基　　金：国家自然科学基金(32270915,32300745);中国医学科学院医学与健康科技创新工程(2021-I2M-1-035,2021-I2M-1-005);常州西太湖细胞治疗前沿技术发展基金会项目(2024-P-013)。

摘　　要：目的针对NKG2A-HLA-E负调控通路,构建诱导多能干细胞(iPSC)来源的基因工程NKG2A敲除的NK细胞(NKG2A KO-iNK),在体外初步评价其肿瘤杀伤功能。方法利用基因编辑技术在iPSC中敲除NKG2A,将其在体外诱导分化为NKG2A KO-iNK细胞,并在不同分化阶段利用流式细胞测量术检测特异性的表面标志物;利用Western blot验证NKG2A KO-iNK细胞NKG2A的敲除情况,并用流式细胞测量术检测NKG2A KO-iNK细胞常见的NK细胞活化性受体及天然细胞毒性受体的表达水平;乳酸脱氢酶检测法(LDH)检测NKG2A KO-iNK细胞对不同人白细胞抗原E(HLA-E)表达水平肿瘤细胞的体外细胞毒活性。结果将Cas9蛋白和靶向编码NKG2A的KLRC1基因第1、2外显子的3条向导RNA(sgRNA)共转染iPSC对其进行基因敲除,测序结果显示成功获得在第1外显子插入1个T碱基的单克隆NKG2A敲除的iPSC(NKG2A KO-iPSC)。iPSC向NK细胞诱导分化的过程中,第8天类胚体(EBs)阶段检测CD34表达水平在30%~50%;第28天NK诱导分化阶段CD56和CD16的表达均达80%以上。Western blot结果显示,NKG2A KO-iNK细胞NKG2A被成功敲除;流式结果显示NKG2A KO-iNK细胞活化性受体NKG2D及细胞毒性受体NKp30、NKp44、NKp46的表达水平和野生型iNK(WT-iNK)细胞基本一致。LDH实验结果显示,NKG2A KO-iNK细胞对HLA-E高表达的B细胞前体白血病细胞系Nalm6的细胞毒活性显著高于WT-iNK细胞,而对HLA-E低表达的人骨髓瘤细胞系H929和HLA-E几乎不表达的人肝癌细胞系HepG2,NKH2A KO-iNK细胞和WT-iNK细胞两者之间的细胞毒活性差异无统计学意义;γ干扰素(IFN-γ)处理能显著上调Nalm6细胞的HLA-E表达水平,进而进一步增强NKG2A KO-iNK细胞对Nalm6细胞的细胞毒活性。结论由于阻断了NKG2A-HLA-E负调控通路,基因工程NKG2A KO-iNK细胞对HLA-E高表达的肿瘤细胞系在体外显示出明显增强的细胞毒活性,提示其作为一种新型细胞治疗的可能性,为肿瘤的细胞过继免疫治疗提供一Objective To target at the NKG2A-HLA-E inhibitory axis,a pluripotent stem cell(iPSC)-derived genetically engineered natural killer cells(NK cells)with NKG2A knockout(NKG2A KO-iNK)were prepared and then their tumor-killing efficacy was evaluated in vitro.Methods NKG2A was knocked out in iPSCs using gene-editing technology.These cells were then differentiated into NKG2A KO-iNK cells.Surface markers at each differentiation stage were analyzed by flow cytometry.Western blot confirmed NKG2A knockout,and flow cytometry assessed expression of activating receptors(NKG2D)and natural cytotoxicity receptors(NKp30,NKp44,NKp46)in NKG2A KO-iNK cells.Cytotoxic activity against tumor cell lines with varying human leukocyte antigen E(HLA-E)expression level was evaluated via lactate dehydrogenase(LDH)release assay.Results Co-transfection of iPSCs with Cas9 protein and three small-guide RNAs(sgRNAs)targeting at exons 1 and 2 of the KLRC1 gene(encoding NKG2A)successfully generated monoclonal NKG2A-knockout iPSCs(NKG2A KO-iPSCs)with a single T-base insertion in exon 1.During iPSC differentiation into NK cells,CD34 expression reached 30%-50%at the embryoid body(EB)stage(day 8),while CD56 and CD16 expression exceeded 80%by day 28.Western blot confirmed complete NKG2A knockout in NKG2A KO-iNK cells.Flow cytometry revealed comparable expression level of activating receptor NKG2D and cytotoxicity receptors(NKp30,NKp44,NKp46)between NKG2A KO-iNK and wild-type iNK(WT-iNK)cells.The LDH assay results indicated that the cytotoxic activity of NKG2A KO-iNK cells against the HLA-E highly-expressed B-cell precursor leukemia cell line Nalm6 cells was significantly higher than that of WT-iNK cells,while there was no significant difference between them and human myeloma cell line H929 cells with low HLA-E expression and human hepatocellular carcinoma cell line HepG2 cells with almost no HLA-E expression.Interferon-γ(IFN-γ)pretreatment up regulated HLA-E expression in Nalm6 cells,further amplifying NKG2A KO-iNK-mediated cytotoxicity.Conclusions By d

关 键 词：诱导多能干细胞(iPSCs) NKG2A 自然杀伤(NK)细胞 免疫治疗 

分 类 号：R392.4[医药卫生—免疫学]