CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients
Su S, Hu B, Shao J, Shen B, Du J, Du Y, Zhou J, Yu L, Zhang L, Chen F, Sha H, Cheng L, Meng F, Zou Z, Huang X, Liu B
Cancer Research/Cell Biology
Cells used in publication:
T cell, human peripheral blood unstim.
Tissue Origin: blood
A mixture of 5?µg of pST1374-Cas9-GFP and 10?µg of pGL3-U6-hPD-1-sgRNA plasmids was used, and different programs (Y-001, T-007, T-023, X-001, U-014, V-024) were applied. GFP expression was determined 24?h later by fluorescence microscope or flow cytometry. We found that higher transfection efficacy was obtained with program U-014 than with V-024 and T-007. Notably, during the following days, significant improved cell viability was observed with program T-007 than with U-014 and V-024 shown by Trypan blue exclusion assay. Therefore, T-007 was used as the optimal program for the following experiments.
Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn't affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-? production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentially be useful to improve the efficacy of T-cell based adoptive therapies.
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