CRISPR-Cas9 Mediated Efficient PD-1 Disruption With Primary T Cells In Macaca Fascicularis

Objective:To establish a method for in vitro isolation and culture of T lymphocytes from peripheral blood of cynomolgus monkeys that induced by human CD3 antibody based on the foundation of protein homology of CD3 from human,cynomolgus monkey and porcine.Using the CRISPR/Cas9 Gene Editing Technique to knock out PD-1 Gene of Cynomolgus Monkey T cell and Explore the Effect of PD-1 Gene Knockout on T Cells,To establish a strategy for using CRISPR/Cas9-targeted PD-1 gene of T cell for tumor cell immunotherapy,and to provide experimental evidence for the research and application of T cell immunotherapy.Methods:The amino acid sequences of human,cynomolgus monkeys and porcine CD3 proteins were obtained from NCBI,and the Sequence,homology and phylogenetic tree were analyzed by DNAMAN software.Western blotting was used to detect the expression of CD3 protein on T cell membranes from the three species.PBMCs of healthy cynomolgus were isolated and divided in-to three groups:group A was stimulated with anti-human CD3Ab alone,group B was stimulated with IL-2 alone,and group C was co-stimulated with anti-human CD3Ab and IL-2.Cell morphology and growth status were observed under inverted microscope and the cell growth curve was plotted.Cell viability was detected by trypan blue staining and the expressions of CD3,CD4 and CD8 on T cell sur-face were detected by flow cytometry.CRISPR/Cas9-Mediated Knockout of PD-1 Gene in Cynomolgus Monkey T Cells:(1)Plasmid construction:Using the gRNA online design tool(http://crispr.mit.edu),a specific sgRNA targeting the PD-1 gene was designed and screened and sent to the company(Kunming Shuo Qing Bio)for synthesis.After being connected to the target vector,the cells were transformed into Trans-5a competent cells,plated,and cultured overnight.The clones were picked and digested and identified by sequencing.The correct sgRNA-expressing monoclonal colonies were obtained.Plasmids were extracted using the endotoxin plasmid kit(QIAGEN,12162)for cell transfection.(2)Cell transfection:PBMCs of cynomolgus monkeys were isolated,and the number of cells was adjusted to 15 million.Plasmid DNAs were added and electroporated with ProgramEH-100 using a Lonza 4D electrorotometer and inoculated into 12-well plates.FACS analysis and fluorescence microscopy were performed 48 h after transfection to detect transfection efficiency.On the 7th day after transfection,some cells were collected and genomic DNA was extracted for PCR amplification and T7E1 digestion.(3)Culture and detection of T cells after transfection:After 24 hours of transfection,change the liquid,add PHA,anti-human CD3Ab to induce proliferation of T cells under sustained stimulation of IL-2,and observe cell morphology and growth status,count cell number under inverted microscope,the cell growth curve was drawn,PI staining was used to detect the cell cycle,flow cytometry was used to detect the expression of T cell surface markers CD4 and CD8,and ELISA was used to detect the secretion of cytokines IFN-γ and IL-2.Results:1.CD3 protein homology analysis:The homology of the amino acid sequence of human CD3 protein to cynomolgus monkey and porcine were 86.9%and 65.6%respectively.Western blot results showed that The expression levels of CD3 protein on cynomolgus and porcine T cell membrane were 0.79times,0.17 times contrast to human.2.Cynomolgus T cell culture:Group A(CD3)cells did not proliferate.Cell proliferation,cell viability and CD3 expression in group C(CD3 + IL-2)were significantly higher than those in groupB[(93.8 ± 3.6)%vs(70.3 ± 4.7)%,P<0.01].And growth curve was S-shaped,consistent with Logistic growth curve.The cell growth cycle was 28 days and the growth curve was S-shaped,consistent with the Logistic growth curve.3.Transfection of cynomolgus monkey T cells:48h after transfection,observation with an inverted fluorescence microscope revealed cells with green fluorescent protein expression in the experimental group.The transfection efficiency was 18.7%by flow cytometry.The extracted cell genomic DNA was amplified by PCR and identified by T7E1 enzyme digestion.The results showed that the obtained PCR product was digested with the target cleavage band(243 bp,197 bp).4.The effect of PD-1 knockout on T cells of cynomolgus monkeys:(1)Cellular morphology:Compared with the non-transfected group,the cells in the transfection group were round,with small volume and weak refraction.(2)Cell proliferation:Compared with non-transfected cells,cells in the transfection group exhibited slow proliferation,delayed colony formation,and reduced cell number.(3)Cell cycle:The number of cells in the G0/G1 phase of the transfection group increased,and the number of cells in the G2/M phase decreased.Compared with the non-transfected group,the difference was statistically significant(P<0.05).There was no significant change in the number of cells in the S phase compared with the non-transfected group(P>0.05).(4)Cell phenotype:There was no significant difference between the expression of cell surface molecular markers CD4 and CD8 in the transfection group and the non-transfection group.(5)Secretion of cytokines:The secretion of IFN-y and IL-2 in transfected cells was higher than that in non-transfected cells,and the difference was statistically significant(P<0.05).Conclusion:1.The expression of CD3 protein on the surface of T lymphocytes of cynomolgus have high homology with human.2.In the anti-human CD3Ab、IL-2 and 1%PHA co-stimulation can induce the proliferation and differentiation of cynomolgus ’ s T lymphocytes,and obtain T lymphocytes with good growth status,high proliferation ability and high purity.3.The growth cycle of cynomolgus T lymphocytes cultured in vitro was 28 days.The growth rate of cynomolgus monkey T lymphocytes decreased with the increase of in vitro culture time,and part of the proliferation was gradually lost.4.PD-1 gene knockout can make cynomolgus monkey T cell arrest in G0/G1 phase,thereby inhibiting its proliferation;but no significant effect on the expression of T cell surface markers CD4,CD8.5.The cynomolgus monkey T cells with PD-1 knockout increased the secretion of IFN-γ and IL-2.