Figure 2.
Phenotype and molecular signature of iC9/CAR19/IL-15 CISH KO NK cells. (A) Comparative heatmap of mass cytometry data showing the expression of NK cell surface markers, transcription factors, and cytotoxicity markers in iC9/CAR19/IL-15 CISH KO compared with iC9/CAR19/IL-15 control NK cells. Each column represents a separate cluster identified by FlowSOM analysis, and each row reflects the expression of a certain marker for each annotation. Color scale shows the expression level for each marker, with red representing higher expression and blue lower expression in iC9/CAR19/IL-15 CISH KO NK cells. The t-SNE map generated from FlowSOM analysis in the right panel shows the 20 NK cell metaclusters (MCs) represented in the mass cytometry heatmap in the left panel. (B) Individual t-SNE maps show the expression of selected NK cell markers for iC9/CAR19/IL-15 CISH KO compared with iC9/CAR19/IL-15 control NK cells. Color scale indicates signal intensity, ranging from low (blue) to high (red) after arcsine transformation. (C) Global gene expression analysis by RNA sequencing. Heatmap displays the genes that were differentially expressed in purified iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control NK cells (n = 4). Color scale shows the expression level of each marker, with red representing higher expression and blue lower expression in iC9/CAR19/IL-15 control (CAR) or iC9/CAR19/IL-15 CISH KO (CAR KO) NK cells (q < 0.1 and absolute log2foldchange > 0.8). (D) GSEA showing enrichment in IFN-γ response, TNF-α signaling via NF-κB, IL-2/STAT5 signaling, IL-6/JAK/STAT3 signaling and inflammatory response in iC9/CAR19/IL-15 CISH KO compared with iC9/CAR19/IL-15 control NK cells. (E) Representative histogram showing enhanced phosphorylation of STAT5 (p-STAT5), STAT3 (p-STAT3) and phospholipase C γ 1 (p-PLCγ1) in iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control NK cells after coculture with Raji cells for 30 minutes. Blue histograms represent CAR control, and red histograms represent CAR CISH KO. (F) Bar graphs showing mean fluorescence intensity (MFI) of p-STAT5, p-STAT3, and p-PLCγ1 in iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control NK cells (n = 3). Blue bars represent CAR control and red bars represent CAR CISH KO. Bars represent mean values with standard deviation. **P ≤ .01.

Phenotype and molecular signature of iC9/CAR19/IL-15 CISH KO NK cells. (A) Comparative heatmap of mass cytometry data showing the expression of NK cell surface markers, transcription factors, and cytotoxicity markers in iC9/CAR19/IL-15 CISH KO compared with iC9/CAR19/IL-15 control NK cells. Each column represents a separate cluster identified by FlowSOM analysis, and each row reflects the expression of a certain marker for each annotation. Color scale shows the expression level for each marker, with red representing higher expression and blue lower expression in iC9/CAR19/IL-15 CISH KO NK cells. The t-SNE map generated from FlowSOM analysis in the right panel shows the 20 NK cell metaclusters (MCs) represented in the mass cytometry heatmap in the left panel. (B) Individual t-SNE maps show the expression of selected NK cell markers for iC9/CAR19/IL-15 CISH KO compared with iC9/CAR19/IL-15 control NK cells. Color scale indicates signal intensity, ranging from low (blue) to high (red) after arcsine transformation. (C) Global gene expression analysis by RNA sequencing. Heatmap displays the genes that were differentially expressed in purified iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control NK cells (n = 4). Color scale shows the expression level of each marker, with red representing higher expression and blue lower expression in iC9/CAR19/IL-15 control (CAR) or iC9/CAR19/IL-15 CISH KO (CAR KO) NK cells (q < 0.1 and absolute log2foldchange > 0.8). (D) GSEA showing enrichment in IFN-γ response, TNF-α signaling via NF-κB, IL-2/STAT5 signaling, IL-6/JAK/STAT3 signaling and inflammatory response in iC9/CAR19/IL-15 CISH KO compared with iC9/CAR19/IL-15 control NK cells. (E) Representative histogram showing enhanced phosphorylation of STAT5 (p-STAT5), STAT3 (p-STAT3) and phospholipase C γ 1 (p-PLCγ1) in iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control NK cells after coculture with Raji cells for 30 minutes. Blue histograms represent CAR control, and red histograms represent CAR CISH KO. (F) Bar graphs showing mean fluorescence intensity (MFI) of p-STAT5, p-STAT3, and p-PLCγ1 in iC9/CAR19/IL-15 CISH KO vs iC9/CAR19/IL-15 control NK cells (n = 3). Blue bars represent CAR control and red bars represent CAR CISH KO. Bars represent mean values with standard deviation. **P ≤ .01.

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