靶向人类巨细胞病毒pp65495-503的T细胞受体工程化T细胞的构建及体外杀伤作用评价OA
Construction and in vitro cytotoxicity assessment of T cell receptor-engineered T cells targeting hu-man cytomegalovirus pp65495-503
目的 构建靶向人类巨细胞病毒(HCMV)表位肽pp65495-503(NLVPMVATV)的T细胞受体工程化T细胞(TCR-T细胞),并评价其对HCMV感染胶质母细胞瘤(GBM)细胞的体外杀伤作用.方法 通过流式细胞术分选获得HCMV pp65特异性T细胞,克隆其TCR序列并构建慢病毒载体,转导CD8+T细胞后,在多种细胞因子刺激下扩增,制备得到HCMV TCR-T细胞,并通过流式细胞术检测HCMV TCR-T细胞中CD8+NLVPM-VATV+细胞占比.将U-87 MG细胞分为A、B两组,A组细胞不做任何处理,B组细胞加入HCMV AD169病毒株进行感染,48 h后收集细胞并通过Western Blot(WB)实验检测两组细胞中pp65蛋白相对表达水平.将HCMV TCR-T细胞按照不同效应细胞∶靶细胞(E∶T)比例(50∶1、30∶1、20∶1、10∶1、5∶1和1∶1)分别与 A组和B组靶细胞共同孵育12 h和24 h,并通过乳酸脱氢酶(LDH)法检测HCMV TCR-T细胞对两组靶细胞的体外杀伤作用.结果 流式细胞术检测结果显示,扩增培养13 d的HCMV TCR-T细胞中CD3+CD8+细胞比例为86.15％,CD8+NLVPMVATV+细胞比例为74.65％.WB实验结果显示,与 A组相比,B组细胞中pp65蛋白相对表达水平显著升高(t=19.01,P＜0.01).LDH法检测结果显示,在培养第12、24小时时,不同E∶T比例下,HCMV TCR-T细胞对A组和B组细胞的杀伤率均有显著差异(F=3.37～112.30,P＜0.05);与 A组相比,各E∶T比例下HC-MV TCR-T细胞对B组细胞的杀伤率均显著升高(F=19.54～428.97,P＜0.05).结论 本研究成功构建了靶向HCMV pp65495-503的TCR-T细胞,该细胞对HCMV感染GBM细胞具有高效且特异性体外杀伤作用.
Objective To construct T cell receptor-engineered T(TCR-T)cells targeting human cytomegalovirus(HC-MV)pp65495-5o3,and to evaluate their in vitro cytotoxicity against HCMV-infected glioblastoma(GBM)cells.Methods Flow cytometry sorting was used to obtain HCMV pp65 antigen-specific T cells,and HCMV-pp65-specific TCRα/β sequences were cloned and constructed into lentiviral expression vectors.Lentivirus was packaged in 293T cells and were used to transfect CD8+T cells,which were then expanded under stimulation with various cytokines.HCMV TCR-T cells were prepared,and flow cytometry was used to measure the proportion of CD8+NLVPMVATV+cells within HCMV TCR-T cells.U-87 MG cells were divided into group A(no treatment)and group B(infected with HCMV AD169 strain),and after 48 hours,Western Blot was used to measure the relative protein expression level of pp65 in both groups.HCMV TCR-T cells were co-cultured with the target cells from groups A and B at different effector-to-target(E/T)ratios(50∶1,30∶1,20∶1,10∶1,5∶1,and 1∶1)for 12 and 24 hours,and lac-tate dehydrogenase(LDH)assay was used to observe the cytotoxicity of HCMV TCR-T cells against the two groups of target cells.Results Flow cytometry showed that after 13 days of expansion,CD3+CD8+cells accounted for 86.15％in HCMV TCR-T cells,and CD8+NLVPMVATV+cells accounted for 74.65％.Western Blot showed that compared with group A,group B had a signifi-cant increase in the relative protein expression level of pp65(t=19.01,P＜0.01).LDH assay showed that after co-culture for 12 and 24 hours,there were significant differences in the cytotoxicity rate of HCMV TCR-T cells against the cells in groups A and B at different E/T ratios(F=3.37-112.30,P＜0.05).The cytotoxicity rate of HCMV TCR-T cells against the cells in group B was significantly increased compared with that in group A at all E/T ratios(F=19.54-428.97,P＜0.05).Conclusion TCR-T cells targeting HCMV pp65495-503 are successfully constructed in this study,and these cells exhibit efficient and specific in vitro cytoto-xicity against HCMV-infected GBM cells.
王晨;王斌;沈文;江莎莎;钱冬萌
青岛大学基础医学院病原生物学系,山东青岛 266071青岛大学基础医学院病原生物学系,山东青岛 266071青岛大学基础医学院病原生物学系,山东青岛 266071西安交通大学附属红会医院检验科青岛大学基础医学院病原生物学系,山东青岛 266071
医药卫生
胶质母细胞瘤巨细胞病毒感染T淋巴细胞受体,抗原,T细胞细胞和组织疗法
GlioblastomaCytomegalovirus infectionsT-lymphocytesReceptors,antigen,T-cellCell-and tissue-based therapy
《精准医学杂志》 2026 (2)
113-117,5
青岛市自主创新重大专项任务项目(20-3-2-4-nsh)
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