Figure 3.
CISH deletion improves function and cytotoxicity of NT and iC9/CAR19/IL-15 NK cells. (A) Representative FACS plots of cytokine production (IFN-γ and TNF-α) and CD107a degranulation by NT control, NT CISH KO, iC9/CAR19/IL-15 control, or iC9/CAR19/IL-15 CISH KO NK cells after coculture with Raji target cells for 6 hours. Inset values indicate the frequency of IFN-γ–, TNF-α–, and CD107a-positive cells from each group. (B) Bar plots summarize the flow cytometry data on cytokine production (IFN-γ and TNF-α) and CD107a degranulation by NT control (blue bars), NT CISH KO (green bars), iC9/CAR19/IL-15 control (purple bars) or iC9/CAR19/IL-15 CISH KO NK cells (red bars) after coculture with Raji target cells for 6 hours (n = 3). Statistical significance is indicated as *P ≤ .05, **P ≤ .01, and ****P ≤ .0001; bars represent mean values with standard deviation. (C) Cytotoxicity of NT control, NT CISH KO, iC9/CAR19/IL-15 control, or iC9/CAR19/IL-15 CISH KO NK cells against Raji targets at different effector-to-target (E:T) ratios, as measured by 51Cr-release assay (n = 3). The bars represent mean values with standard deviation. The red asterisks represent the statistical significance between iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control NK cells (*P ≤ .05). The green asterisks represent the statistical significance between NT CISH KO vs NT control NK cells (***P ≤ .001; **P ≤ .01). (D) Cytotoxicity of NT control, NT CISH KO, iC9/CAR19/IL-15 control, or iC9/CAR19/IL-15 CISH KO NK cells against Raji targets over 24 hours at 1:1 E:T ratio as measured by Incucyte live-imaging cell killing assay (n = 3). Bars represent mean values with standard deviation. At 10 hours, NT CISH KO vs NT control (P = .04), iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control (P = .007). (E) Confocal microscopy showing representative synapse images of NT control, NT CISH KO, iC9/CAR19/IL-15 control or iC9/CAR19/IL-15 CISH KO NK cells conjugated with Raji cells. Images show conjugates in bright field (BF) or stained with anti-CD19 (green), anti-perforin (red), phalloidin-F-actin (blue), and anti-CAR (gray), and an overlay of fluorescence channels are also shown. (F) NT control, NT CISH KO, iC9/CAR19/IL-15 control, or iC9/CAR19/IL-15 CISH KO NK cells were assessed for their ability to polarize lytic granules to Raji cells as measured by distance from the MTOC to the IS. Results from 3 independent donors are shown. Each data point represents a single IS. Statistical significance is indicated as *P ≤ .05 and ***P ≤ .001.

CISH deletion improves function and cytotoxicity of NT and iC9/CAR19/IL-15 NK cells. (A) Representative FACS plots of cytokine production (IFN-γ and TNF-α) and CD107a degranulation by NT control, NT CISH KO, iC9/CAR19/IL-15 control, or iC9/CAR19/IL-15 CISH KO NK cells after coculture with Raji target cells for 6 hours. Inset values indicate the frequency of IFN-γ–, TNF-α–, and CD107a-positive cells from each group. (B) Bar plots summarize the flow cytometry data on cytokine production (IFN-γ and TNF-α) and CD107a degranulation by NT control (blue bars), NT CISH KO (green bars), iC9/CAR19/IL-15 control (purple bars) or iC9/CAR19/IL-15 CISH KO NK cells (red bars) after coculture with Raji target cells for 6 hours (n = 3). Statistical significance is indicated as *P ≤ .05, **P ≤ .01, and ****P ≤ .0001; bars represent mean values with standard deviation. (C) Cytotoxicity of NT control, NT CISH KO, iC9/CAR19/IL-15 control, or iC9/CAR19/IL-15 CISH KO NK cells against Raji targets at different effector-to-target (E:T) ratios, as measured by 51Cr-release assay (n = 3). The bars represent mean values with standard deviation. The red asterisks represent the statistical significance between iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control NK cells (*P ≤ .05). The green asterisks represent the statistical significance between NT CISH KO vs NT control NK cells (***P ≤ .001; **P ≤ .01). (D) Cytotoxicity of NT control, NT CISH KO, iC9/CAR19/IL-15 control, or iC9/CAR19/IL-15 CISH KO NK cells against Raji targets over 24 hours at 1:1 E:T ratio as measured by Incucyte live-imaging cell killing assay (n = 3). Bars represent mean values with standard deviation. At 10 hours, NT CISH KO vs NT control (P = .04), iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control (P = .007). (E) Confocal microscopy showing representative synapse images of NT control, NT CISH KO, iC9/CAR19/IL-15 control or iC9/CAR19/IL-15 CISH KO NK cells conjugated with Raji cells. Images show conjugates in bright field (BF) or stained with anti-CD19 (green), anti-perforin (red), phalloidin-F-actin (blue), and anti-CAR (gray), and an overlay of fluorescence channels are also shown. (F) NT control, NT CISH KO, iC9/CAR19/IL-15 control, or iC9/CAR19/IL-15 CISH KO NK cells were assessed for their ability to polarize lytic granules to Raji cells as measured by distance from the MTOC to the IS. Results from 3 independent donors are shown. Each data point represents a single IS. Statistical significance is indicated as *P ≤ .05 and ***P ≤ .001.

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