FIGURE 6 from p35 is a Crucial Player in NK-cell Cytotoxicity and TGFβ-mediated NK-cell Dysfunction

NK92 cells with p35 knockdown or CDK5-K33T overexpression mitigate TGFβ-induced inhibition of cytotoxicity. A, NK92 cells (shGFP or shp35) were cultured for 48 hours in the presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). They were then cocultured with fluorescently-labeled Jeko-1 cells at 5:1 E:T ratio for 16 hours in the continued presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). Cells were stained with PI and gated on labeled cells to measure cancer cell death. Average cytotoxicity percentage is overlaid on the corresponding bar. ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. B, Supernatant from NK92 cell culture (shGFP or shp35) with or without Jeko cells was collected after 16 hours. Cytokine concentration was determined using a flow-based, multiplex human cytokine release assay. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Graphs display mean ± SD, n = 3 biological cocultures, multiple unpaired two-tailed t tests with correction for multiple comparisons using Holm-Šídák method. C, NK92 cells (OE ctrl, OE CDK5, OE CDK5-K33T, or OE p35) were cultured for 48 hours in the presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). They were then cocultured with fluorescently-labeled Jeko-1 cells at 5:1 E:T ratio for 16 hours in the continued presence of 2.5 ng/mL TGFβ or PBS (−TGFβ). Cells were stained with PI and gated on labeled cells to measure cancer cell death. Average cytotoxicity percentage is overlaid on the corresponding bar. **, P < 0.01; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Tukey multiple comparisons test. D, Supernatant from NK92 cell culture (OE ctrl, OE CDK5, OE CDK5-K33T, or OE p35) with or without Jeko cells was collected after 16 hours. Cytokine concentration was determined using a flow-based, multiplex human cytokine release assay. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Graphs display mean ± SD, n = 3 biological cocultures, two-way ANOVA with Tukey multiple comparisons test.