融合蛋白Tandab(CD3/CD19/4-1BBL)介导的T细胞对B细胞淋巴瘤细胞杀伤作用观察  

Killing effect of Tandab(CD3/CD19/4-1BBL)-mediated T cells on B-cell lymphoma cells

作　　者：张晓龙 孟帅 吴春暖 熊冬生 张洁 ZHANG Xiaolong;MENG Shuai;WU Chunnuan;XIONG Dongsheng;ZHANG Jie(Tianjin Medical University Cancer Institute and Hospital,Tianjin 300060,China;不详)

机构地区：[1]天津医科大学肿瘤医院,国家肿瘤临床医学研究中心,天津市肿瘤防治重点实验室,天津300060 [2]中国医学科学院北京协和医学院血液病医院

出　　处：《山东医药》2021年第8期1-5,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(82000197);吴阶平医学基金会临床科研专项资助基金项目(320.6750.2020-10-24)。

摘　　要：目的观察融合蛋白Tandab(CD3/CD19/4-1BBL)介导的T细胞对人B细胞淋巴瘤细胞Raji的杀伤作用。方法采用常规酶切、连接等分子克隆方法构建慢病毒表达载体pLentiR.SV40-Tandab(CD3/CD19/4-1BBL),将其瞬时转染至293T细胞,收集培养上清并纯化融合蛋白Tandab(CD3/CD19/4-1BBL)。采用流式细胞术分析Tandab(CD3/CD19/4-1BBL)与CD19^(+)细胞(Raji细胞)、CD3^(+)细胞(人急性T淋巴细胞白血病细胞Jurkat)、4-1BB^(+)细胞(Jurkat细胞)的结合活性。收集Raji细胞,按照效靶比20∶1加入T细胞,分别加入0.08、0.8、8 pmol/mL的Tandab(CD3/CD19/4-1BBL)、Tandab(CD3/CD19)、4-1BBL,采用LDH释放实验测算T细胞对Raji细胞的杀伤效率,采用ELISA法检测8 pmol/mL融合蛋白作用后细胞培养液上清中的IL-2、采用流式细胞术检测CD3^(+)CD69^(+)细胞、CD3^(+)CD25^(+)细胞比例。结果pLentiR.SV40-Tandab(CD3/CD19/4-1BBL)构建成功,表达的融合蛋白Tandab(CD3/CD19/4-1BBL)可与CD19^(+)、CD3^(+)、4-1BB^(+)细胞结合。Tandab(CD3/CD19/4-1BBL)组、Tandab(CD3/CD19)组T细胞对Raji细胞杀伤百分比均高于4-1BBL组(P均<0.01)。在效靶比一定时,随着融合蛋白Tandab浓度增高,T细胞对Raji细胞的杀伤百分比逐渐增高(P均<0.01)。Tandab(CD3/CD19/4-1BBL)组、Tandab(CD3/CD19)组、4-1BBL组细胞培养液上清中的IL-2水平均高于PBS组(P均<0.05)。Tandab(CD3/CD19/4-1BBL)组、Tandab(CD3/CD19)组CD3^(+)CD69^(+)细胞、CD3^(+)CD25^(+)细胞比例高于4-1BBL组、PBS组(P均<0.01)。结论融合蛋白Tandab(CD3/CD19/4-1BBL)可有效介导T细胞对B细胞淋巴瘤细胞的杀伤作用。Objective To investigate the killing effect of Tandab(CD3/CD19/4-1BBL)-mediated T cells on human B-cell lymphoma Raji cells.Methods The methods of restriction enzyme cleavage and linkage were employed to construct the lentiviral expression vector pLentiR.SV40-Tandab(CD3/CD19/4-1BBL),which was then transiently transfected into 293T cells.The Tandab(CD3/CD19/4-1BBL)was extracted and purified from the culture medium.The binding abilities of Tandab(CD3/CD19/4-1BBL)to CD19^(+)(Raji cells),CD3^(+)(human acute T-lymphoblastic leukemia cell line Jurkat cells),and 4-1BB^(+)(Jurkat cells)cells were determined by flow cytometry,respectively.T cells were co-cultured with Raji cells(E∶T=20∶1)in the presence of Tandab(CD3/CD19/4-1BBL),Tandab(CD3/CD19),and 4-1BBL with the indicated concentrations of 0.08,0.8,and 8 pmol/mL.Lactic dehydrogenase(LDH)release assay was applied to detect the killing efficiency of T cells mediated by Tandab(CD3/CD19/4-1BBL)on Raji cells.Expression of IL-2 in the culture medium treated with 8 pmol/mL Tandab(CD3/CD19/4-1BBL)was determined by enzyme-linked immunosorbent assay(ELISA).Flow cytometry was used to measure the percentages of CD3^(+)CD69^(+)and CD3^(+)CD25^(+)cells.Results The pLentiR.SV40-Tandab(CD3/CD19/4-1BBL)was successfully constructed.Tandab(CD3/CD19/4-1BBL)could specifically bind to CD19^(+),CD3^(+),and 4-1BB^(+)cells.The Tandab(CD3/CD19/4-1BBL)group and Tandab(CD3/CD19)group had significantly higher percentages of cell lysis than the 4-1BBL group(both P<0.01).With the increased concentrations of Tandab,the percentages of cell lysis increased in a dose-dependent manner at the same E/T ratio(both P<0.01).The expression of IL-2 in the Tandab(CD3/CD19/4-1BBL)group,Tandab(CD3/CD19)group,and 4-1BBL group was higher than that of the PBS group(all P<0.05).The percentages of CD3^(+)CD69^(+)cells and CD3^(+)CD25^(+)cells in the Tandab(CD3/CD19/4-1BBL)group and Tandab(CD3/CD19)group were higher than those in the 4-1BBL group and PBS group(all P<0.01).Conclusion Tandab(CD3/CD19/4-1BBL)could eff

关 键 词：融合蛋白Tandab(CD3/CD19/4-1BBL) 串联四价双特异性抗体 B细胞淋巴瘤 细胞免疫 

分 类 号：R318[医药卫生—生物医学工程] R733[医药卫生—基础医学]