CRISPR/Cas9 Mediated Efficient TIM3 Gene Knockout On Human Peripheral Blood T-cells And Its Anti-tumor Efficacy

Purpose:T cell immune globulin and mucin-3(TIM3)as one of the most important novel immune checkpoint protein,plays a significant role in the immunomodulation of tumor microenvironment.Previous studies showed that the compensatory upregulation of TIM3 molecule was particularly obvious in the tumor microenvironment where PD-1 blocking antibody treatment was resistant,and its overexpression led to the blocked proliferation and cytokine secretion of T cells.Therefore,in order to confirm the feasibility of TIM3 molecule as a novel target for tumor immunotherapy,CRISPR/Cas9 gene editing technique was used to knock out TIM3 molecule expressed on human peripheral blood T cells in this study,and preliminarily studied whether TIM3 gene knockout could enhance anti-tumor efficacy of T cells in vitro.Methods:First,hTIM3 sgRNA/Cas9 double plasmids were transfected into human peripheral blood T lymphocytes of EBV positive gastric cancer patients by using electrotransfection system.Then,the transfection efficiency was examined 24h later by flow cytometry and fluorescence microscope.Simultaneously,the proliferation,expression level of TIM3 gene and cell phenotype of the gene modified T cells were monitored and evaluated in vitro.Furthermore,EBV positive gastric cancer AGS-EBV cells were taken as the target cells,and TIM3 gene knockout antigen-specific T lymphocytes were stimulated by tumor associated antigen peptide.To detect whether TIM3 gene knockout would affect the anti-tumor ability of antigen-specific T cells,the level of cytokines production and cytotoxicity of these gene modified cytotoxic T lymphocytes(CTLs)were then collected to evaluate its immune function and cytotoxicity.Results:CRISPR/Cas9 mediated efficient TIM3 gene knockout on human peripheral blood T cells by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible with an average transfection efficiency of(41.5±3.6)%,and the gene knockout efficiency fluctuated between 40.0%and 50.0%(all P<0.001).The phenotype and proliferation of the modified T cells were 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 elevation as compared with control group(P<0.05).Remarkably,cellular immune response of these gene modified CTLs was characterized by enhancing cytokine production of TNF-α and IFN-γ(P<0.05 or P<0.01),and in vitro cytotoxic activity of these effector T cells against human AGS-EBV was shown better in the gene knockout group rather than the control group(all P<0.05).Conclusion:These results suggested that the engineered TIM3 gene knockout cells established by CRISPR/Cas9 gene editing technique was simple and feasible with low cost.The expression level of TIM3 was down-regulated when culturing in vitro without affecting the proliferation and the phenotype of T cells.More importantly,the gene modified CTLs performed superior in immune response and cytotoxicity.Collectively,these results provided validation for the utilization of TIM3 gene as a target of cellular adoptive therapy,which could also provide a new idea for gene engineering cell immunotherapy.