Effect of CRISPR/Cas9-mediated PD-1 gene knockout on proliferation, phenotype,IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys / 中国肿瘤生物治疗杂志

ABSTRACT

@#[Abstract] Objective: To investigate the effects of CRISPR/Cas9 gene editing mediated PD-1 knockdown on the proliferation, phenotype, IFN-γ and IL-2 secretion of T cells in Cynomolgus monkeys. Methods: gRNA targeting PD-1 gene of Cynomolgus monkey was designed, and the corresponding plasmid was constructed and extracted. peripheral blood mononuclear cells (PBMCs) of Cynomolgus monkeys were isolated, and plasmid DNAs were added for transfection by using Lonza 4D electrorotometer. FACS analysis and fluorescence microscopy were used to detect transfection efficiency at 48 h after transfection. Genomic DNA of T cells was extracted for PCR amplification and T7E1 digestion identification. The proliferation of T cells was induced under the stimulation of human CD3 antibody and IL-2, and the cell growth curve was drawn. PI staining flow cytometry was used to detect cell cycle and the expression levels of CD4 and CD8, and ELISA was used to detect the secretion of IFN- γ and IL-2. Results: At 48 h after transfection, the cells with green fluorescent protein expression in experimental group were observed under fluorescence microscopy with a transfection efficiency of (21.6±3.2)%. T7E1 enzyme digestion results showed that the PCR product of genomic DNA of cells in experimental group showed 3 bands after digestion, including the target cleavage bands(243，197 bp). Compared with non-transfected cells, the cells in experimental group exhibited slow proliferation, delayed colony formation, with small volume and weak refraction; the number of T cells at G0/G1 phase of the experimental group was significantly increased (P<0.05), while the number of cells at G2/M phase was significantly reduced (P<0.05); and the secretion levels of IFN-γ and IL-2 in the cells of the experimental group increased significantly (both P<0.05). However, the difference in the expression levels of CD4 and CD8 was not statistically significant between the two groups (both P>0.05). Conclusion: PD-1 gene knockout can arrest T cells in Cynomolgus monkey at G0/G1 phase, thereby inhibiting its proliferation and increasing the secretion of IFN-γ and IL-2 in the meanwhile.