精准医学杂志2026，Vol.41Issue(2)：113-117,5.DOI:10.13362/j.jpmed.202641021
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
Construction and in vitro cytotoxicity assessment of T cell receptor-engineered T cells targeting hu-man cytomegalovirus pp65495-503
摘要
Abstract
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.关键词
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Key words
Glioblastoma/Cytomegalovirus infections/T-lymphocytes/Receptors,antigen,T-cell/Cell-and tissue-based therapy分类
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王晨,王斌,沈文,江莎莎,钱冬萌..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[J].精准医学杂志,2026,41(2):113-117,5.基金项目
青岛市自主创新重大专项任务项目(20-3-2-4-nsh) （20-3-2-4-nsh）
