[Application of TARP luciferase reporter system in function identification of CAR-T cells].

Sixin Liang, Rui Zheng, Xiaojuan Zhao, Yiting Zhang, Pengju Wang, Ruotong Meng, Bo Yan, Angang Yang
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Abstract

Objective To investigate a convenient and quantitative solution to activation levels and functional characterization of CAR-T cells by inserting T cell activity-responsive promoter (TARP) nanoluciferase reporter gene system into a lentiviral plasmid containing the gene encoding the chimeric antigen receptor (CAR). Methods The recombinant plasmid was constructed by using whole gene synthesis and molecular cloning techniques. The lentivirus was packaged and was infected with human primary T lymphocytes. Flow cytometry was used to detected the positive rate of lentivirus-infected T cells. The functional characterization of CAR-T cells was identified by luciferase reporter gene system, Western blot, flow cytometry, and small animal live imaging techniques. Results The results of enzyme digestion identification and the plasmid sequencing showed that the recombinant plasmids were constructed, and flow cytometry displayed the normal preparation of CAR-T cells. This system could dynamically respond to the activation of CAR-T cells by luciferase reporter gene system. The functional assay in vitro confirmed that the system could reflect the exhaustion of CAR-T cells, and the small animal live imaging results demonstrated that the system can be used as a tracer of CAR-T cells in mice. Conclusion TARP nanoluciferase reporter gene system provides a more convenient, sensitive and quantitative method for evaluating CAR-T cells activation level, exhaustion phenotype and tracing.

[TARP荧光素酶报告系统在CAR-T细胞功能鉴定中的应用]。
目的将T细胞活性响应启动子(TARP)纳米荧光素酶报告基因系统插入含有嵌合抗原受体(CAR)编码基因的慢病毒质粒中,探索一种简便、定量测定CAR-T细胞活化水平和功能表征的方法。方法采用全基因合成和分子克隆技术构建重组质粒。将慢病毒包装,用人原代T淋巴细胞感染。采用流式细胞术检测慢病毒感染T细胞的阳性率。通过荧光素酶报告基因系统、Western blot、流式细胞术和小动物活体成像技术鉴定CAR-T细胞的功能特征。结果酶切鉴定和质粒测序结果显示重组质粒构建成功,流式细胞术显示CAR-T细胞制备正常。该系统能够动态响应荧光素酶报告基因系统对CAR-T细胞的激活。体外功能实验证实该系统能反映CAR-T细胞的衰竭,小动物活体成像结果证实该系统可作为小鼠CAR-T细胞的示踪剂。结论TARP纳米荧光素酶报告基因系统为评价CAR-T细胞活化水平、衰竭表型和追踪提供了更为方便、灵敏和定量的方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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