GSDMD–deficient, iPSC–derived human neutrophils resist CC–induced pyroptosis and β₂-integrin activation. (A) Immunoblot analysis of GSDMD in iPSC-derived neutrophils, performed on control and 2 distinct GSDMD-knockout clones. β-actin was used as a loading control. (B) Immunoblot analysis of GSDMD in control iPSC-derived neutrophils cultured at day 18 (undifferentiated time point) and day 38 (differentiated time point). (C-D) iPSC-derived neutrophils were primed with LPS for 4 hours, followed by stimulation with CC (1.5 mg/mL) for 3 hours under shaking conditions. Cell-free supernatants were collected for IL-1β ELISA (C) and LDH assay (D). (E-F) iPSC-derived neutrophils were stimulated with or without CXCL8 (100 ng/mL) for 10 minutes. Surface expression levels of β₂ integrins lymphocyte function–associated antigen 1 (E) and MAC-1 (F) were quantified as the MFI using flow cytometry. (G) Representative immunofluorescent and live images of NETs. iPSC-derived neutrophils were incubated on CC-precoated slides for 4 hours. NETs were visualized by immunofluorescent images, stained with CitH3 (green), MPO (red), and DAPI (blue), and by live cell images stained with SG (green). (H) iPSC-derived neutrophils were primed with CXCL8 and then stimulated with CC (1.0 mg/mL) for 3 hours under shaking conditions. The cells were stained with SG for flow cytometric analysis. (I) Representative histogram of SG-positive iPSC-derived neutrophils. Scale bars: for panel G, 10 μm (immunofluorescent images) and 100 μm (live images). The data represent mean ± SEM. ∗∗∗P < .001 using 2-way ANOVA with Dunnett multiple comparisons test. Data are representative of 4 to 5 independent experiments.