Fig. 4

CD27 editing reduces IFNγ-producing CD4 T cells. (A) The CD27 gene was efficiently “knocked out” in CD4 T cells by using CRISPR-Cas9-single-guide RNA ribonucleoproteins (Cas9-RNP). CRISPR-cas9 ribonucleoproteins (crRNPs) were synthesized in vitro and delivered to activated CD4 T cells by nucleofection for editing. The expression of CD27 on the surface of CD4 T cells was investigated by flow cytometry and immunoblotting. (B) Flow cytometry histograms showing CD27 levels on the cell surface of purified CD4 T cells treated with non-targeting crRNPs (non-targeting, in grey) or CD27-targeting crRNP (KO, in dark) in 4 independent experiments. (C) Immunoblots for CD27 and granzyme B in the whole cell lysate of CD4 KO and CD4 non-targeting T cells. Quantification of CD27 (D) and (E) granzyme B as a percentage of control by western blotting. Data represent mean ± SD of 4 independent biological experiments. Statistical analyses were performed using one-tailed t and Wilcoxon test. *p < 0.05, ***p < 0.001. (F) Levels of sCD27 (pg/ml) from stimulated CD4 KO and CD4 non-targeting T cells were compared at 24 h and 48 h as measured by ELISA. Statistical analyses were performed using ratio paired t-test. *p < 0.05. Histograms reporting intracellular levels of T-bet (G) and RoR-γT (H) in CD4 KO and CD4 non-targeting T cells as measured by flow cytometry. T-bet and RoR-γT were reported as mean fluorescence intensity (MFI). Data represent mean ± SD. Statistical analyses were performed using paired t-tests of 4 independent experiments. NS not significant, *p < 0.05