人外周血TIM3基因敲除T淋巴细胞的制备及其抗肿瘤作用

Preparation of human peripheral blood T lymphocytes with TIM3 gene knockout and its anti-tumor efficacy

目的:通过CRISPR-Cas9基因编辑技术及质粒电转染技术制备T细胞免疫球蛋白黏液素3(TIM3)基因敲除的人外周血T淋巴细胞,探讨敲除TIM3基因后能否增强T细胞免疫功能及其抗肿瘤作用。方法:通过电转染法将hTIM3 sgRNA/Cas9双质粒共转染EBV阳性胃癌患者外周血T淋巴细胞,电转24 h后流式细胞术检测转染效率并利用荧光显微镜观察;体外培养过程中观察基因敲除后T细胞增殖活性,流式细胞术验证TIM3基因敲除效率以及细胞表型的变化。用肿瘤抗原肽活化T细胞检测TIM3基因敲除后T细胞分泌细胞因子水平及体外杀伤胃癌AGS-EBV细胞的能力。结果:电转染法能够有效地将hTIM3 sgRNA/Cas9双质粒敲除体系转入人外周血T淋巴细胞,其转染效率平均达(41.5±3.6)%,基因敲除效率波动在40.0%～50.0%(均P<0.01);经抗原活化后的基因敲除组T细胞的增殖活性和免疫表型未见明显变化,表面活化分子中仅见HLA-DR较对照组明显提高(P<0.05);TIM3基因敲除T细胞分泌TNF-α、IFN-γ的水平显著增高(P<0.01或P<0.05),体外杀伤胃癌AGS-EBV细胞的能力也明显增强(均P<0.05)。结论:用CRISPR-Cas9基因编辑及质粒电转染技术制备人外周血TIM3基因敲除T淋巴细胞的方法简易可行,在体外的扩增活化中保持TIM3基因水平下调并具有更高的免疫应答水平以及更强的抗肿瘤作用。这一新技术的研发为基因工程化细胞免疫治疗提供了新的思路。

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 24h 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 h TIM3 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 antigenic peptide;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-αand 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.