Application of TARP luciferase reporter system in function identification of CAR-T cells.
- Author:
Sixin LIANG
1
,
2
;
Rui ZHENG
3
;
Xiaojuan ZHAO
3
;
Yiting ZHANG
3
;
Pengju WANG
3
;
Ruotong MENG
4
;
Bo YAN
5
;
Angang YANG
6
Author Information
1. College of Medical Technology, Xinxiang Medical University, Xinxiang 453003
2. Department of Biochemistry and Molecular Biology, Air Force Medical University, Xi'an 710032, China.
3. Department of Biochemistry and Molecular Biology, Air Force Medical University, Xi'an 710032, China.
4. College of Life Science, Yanan University, Yan'an 716000, China.
5. Department of Biochemistry and Molecular Biology, Air Force Medical University, Xi'an 710032, China. *Corresponding authors, E-mail: yb5870@163.com.
6. Department of Immunology, Air Force Medical University, Xi'an 710032, China. *Corresponding authors, E-mail: agyang@fmmu.edu.cn.
- Publication Type:Journal Article
- MeSH:
Humans;
Animals;
Mice;
T-Lymphocytes;
Cell Line, Tumor;
Receptors, Chimeric Antigen/genetics*;
Promoter Regions, Genetic;
Immunotherapy, Adoptive/methods*
- From:
Chinese Journal of Cellular and Molecular Immunology
2023;39(5):397-403
- CountryChina
- Language:Chinese
-
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.
