Mobile website
Archive > Volume 26 Issue 4 > 2019,26(4):374-380. DOI:10.3872/j.issn.1007-385X.2019.04.002 Prev Next
Prompt Report
Preparation of human peripheral blood T lymphocytes with TIM3 gene knockout and its anti-tumor efficacy
- Article
- Figures
- Metrics
- Preview PDF
- Reference
- Related
- Cited by
- Materials
Abstract:
[Abstract] Objective: To prepare human peripheral blood T lymphocytes with TIM3 (T cell immunologlobulin and mucin-3) gene knockout by using CRISPR-Cas9 gene editing technique and plasmid electrotransfection system, and to discuss whether the knockout of TIM3 gene could enhance the immune response and anti-tumor efficacy of T cells. Methods: Double plasmids hTIM3 sgRNA/Cas9 were transfected into human peripheral blood T lymphocytes of EBV positive gastric cancer patients by using electrotransfection system.The transfection efficiency was examined 24 h later by flow cytometry and fluorescence microscope. The proliferation activity of the T cells after gene knockout was observed during in vitro culture, and the knockout efficiency and phenotypes of the modified T cells were evaluated by flow cytometry. Furthermore, tumor antigen peptide was used to activate T cells, and the level of modified T cells secreting cytokines and its cytotoxicity against gastric cancer AGS-EBV cells were evaluated. Results: Electrotransfection system could successfully transfect hTIM3 sgRNA/Cas9 double plasmids into human peripheral blood T lymphocytes with an average transfection efficiency of (41.5±3.6)%, and the gene knockout efficiency fluctuated between 40.0% and 50.0% (all P<0.01). The proliferation of the modified T cells was not significantly changed in the TIM3 gene knockout group even after the prolonged co-culturing with tumor antigenicpeptide; and for the activated molecules, only HLA-DR exhibited significant elevation as compared with control group (P<0.05).Remarkably, T cells with TIM3 gene knockout showed significantly elevated secretion of TNF-α and IFN-γ (P<0.01 or P<0.05), as well as obviously enhanced in vitro cytotoxicity against gastric cancer AGS-EBV cells (P<0.05). Conclusion: It’s simple and feasible of CRISPR-Cas9 gene editing technique and plasmid electrotransfection system to prepare T lymphocytes with engineered TIM3 gene knockout. The expression level of TIM3 was down-regulated in in vitro culture. More importantly, the modified T cells performed superior immune response and cytotoxicity, which may provide a new idea for gene engineering cell immunotherapy.
Keywords:
Project Supported:
Project supported by the Special Funding Project of National Key Research and Development Plans(No.2017YFC1308900), the National Natural Science Foundation of China (No.81702811, No.81803093), and the Natural Science Foundation of Jiangsu Province (No. SBK2018040809)
Clc Number:
