Upregulation of programmed cell death‐ligand 1 (PD‐L1) expression on C4‐2 cells cocultured with chimeric antigen receptor NK‐92 (CAR NK‐92) cells was dependent on interferon‐γ (IFN‐γ). (A) C4‐2 cells were cocultured with CAR NK‐92 cells for 2, 6, 12 and 24 h, and the expression of PD‐L1 on C4‐2 cells was detected by flow cytometry; the representative images and summary data (n = 3) are shown. The median fluorescence intensity (MFI) for PD‐L1 and the percentages of PD‐L1+ C4‐2 cells were significantly increased in a time‐dependent manner. (B) IFN‐γ concentrations in the culture supernatant of CAR NK‐92 cells cocultured with or without C4‐2 cells were measured by ELISA at 2, 6, 12 and 24 h. The experiments were repeated three times. (C) Representative flow cytometry plots and summary data (n = 3) of PD‐L1 expression on C4‐2 cells in response to stimulation with different concentrations of IFN‐γ. The MFI for PD‐L1 and PD‐L1+ percentages increased in a concentration‐dependent manner. (D) Representative flow cytometry images and summary data (n = 3) of PD‐L1 expression on C4‐2 cells cocultured with CAR NK‐92 cells for 24 h with or without an IFN‐γ blocker. IFN‐γ blocker completely reversed PD‐L1 upregulation in C4‐2 cells cocultured with CAR NK‐92 cells. Bars represent the means ± standard deviation (SD), one‐way analysis of variance (ANOVA) followed by a Tukey post hoc test was used for multiple group comparisons (A, C, D), and Student's t‐test was used for two‐group comparisons (B). *p < .05; Median fluorescence intensity; NS, not significant