基础医学与临床2025，Vol.45Issue(5)：599-607,9.DOI:10.16352/j.issn.1001-6325.2025.05.0599
基因工程化iNKNKG2A KO细胞具有HLA-E特异的抗肿瘤活性
Engineered iNKNKG2A KO cells possess HLA-E specific anti-tumor activity
摘要
Abstract
Objective To target at the NKG2A-HLA-E inhibitory axis,a pluripotent stem cell(iPSC)-derived geneti-cally 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 expres-sion 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 pro-tein and three small-guide RNAs(sgRNAs)targeting at exons 1 and 2 of the KLRC1 gene(encoding NKG2A)suc-cessfully 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 CD 16 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 cytotox-icity 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 signif-icant difference between them and human myeloma cell line H929 cells with low HLA-E expression and human hepa-tocellular 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 disrupting the NKG2A-HLA-E inhibitory axis,NKG2A KO-iNK cells exhibit markedly enhanced in vitro cytotoxic-ity against HLA-E-high tumor cells.This result highlights their potential function as a novel adoptive cell therapy strategy for cancers reliant on HLA-E-mediated immune evasion.关键词
诱导多能干细胞(iPSCs)/NKG2A/自然杀伤(NK)细胞/免疫治疗Key words
induced pluripotent stem cells(iPSCs)/NKG2A/natural killer(NK)cell/immunotherapy分类
基础医学引用本文复制引用
乔雯华,许依,董鹏,何维,陈慧,张建民..基因工程化iNKNKG2A KO细胞具有HLA-E特异的抗肿瘤活性[J].基础医学与临床,2025,45(5):599-607,9.基金项目
国家自然科学基金(32270915,32300745) （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) （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) （2024-P-013）
