Enhanced susceptibility to IAV in TLR3-mutated iPSC-derived lung epithelial cells. Lung epithelial cells were derived from the ES cells of a healthy control (CTL-RUES2, shown as black filled circles in the figure), iPSCs from two healthy controls (Sendai virus–reprogrammed iPSC CTL-SViPS, and mRNA-reprogrammed iPSC CTL-mRNA, shown as dark or light blue triangles, respectively), P3 (three iPSC clones from the same patient, shown with pink squares, yellow triangles, or red circles, respectively), TLR3^−/−, IRF7^−/−, STAT1^−/−, and IL10RB^−/− patients. The cells were untreated or subjected to 16 h of pretreatment with IFN-α2b or IFN-λ1, then infected with IAV at a MOI of 1 or 10 for 24 h. The cells were immunostained for influenza NP (green) and Nkx2.1 (red), and their nuclei were stained with DAPI (blue). (A) The percentage of Nkx2.1-positive cells was determined for the DAPI-positive cells. (B) Representative images of CTL and patient PECs, showing the immunostaining of NP, Nkx2.1, and DAPI, 24 h after infection with IAV. hES, human embryonic stem. (C) The percentage of influenza NP-positive cells was then determined for the Nkx2.1-positive cells. We analyzed ∼60,000 Nkx2.1 cells per cell line. Without IFN pretreatment, higher proportions of PECs derived from the iPSCs of TLR3 P680L/WT, TLR3^−/−, IRF7^−/−, STAT1^−/−, and IL10RB^−/− patients were positive for IAV NP, than of PECs from CTL-RUES2, iPSC CTL-SViPS, and iPSC CTL-mRNA. The data shown represent the mean values ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.