Flow cytometric and functional analyses confirm two distinct adult lung ILC subsets. (A) Tbet⁻Lin⁻YFP⁺Thy1⁺CD127⁺RORγt⁻ cells were sequentially gated and divided into IL-18Rα⁺ST2⁻ (green) and IL-18Rα⁻ST2⁺ (black) subsets in adult RORα-YFP mouse lungs. (B) The expression of GATA-3, CD25, KLRG1, IL-25R, and GITR by lung IL-18Rα⁺ cells (green) and ST2⁺ ILC2s (black) in A are shown. Lung NK cells (NKp46⁺Eomes⁺T-bet⁺; filled gray) and mesenteric lymph node (MLN) ILC2s (filled red) were used as controls. (C) Lin⁻YFP⁺Thy1⁺CD127⁺IL-18Rα⁺ST2⁻ (green) and IL-18Rα⁻ST2⁺ (black) were analyzed for the expression of RORγt and T-bet. (D) Lin⁻YFP⁺Thy1⁺CD127⁺ cells were sorted into IL-18Rα⁺ cells (green) and ST2⁺ ILC2s (black) and stimulated for 72 h with PMA and ionomycin. The amounts of cytokines and chemokines in the culture supernatants were determined. (E) Absolute numbers of IL-18Rα⁺ cells (green) and ST2⁺ ILC2s (black) gated as in A in naive, IL-18, and papain-treated lungs are shown. (F) Lin⁻YFP⁺Thy1⁺CD127⁺IL-18Rα⁺ cells (green) and ST2⁺ ILC2s (black) were analyzed for intracellular IL-5 and IL-13 in naive, IL-18, and papain-treated lungs. (G) Lin⁻Thy1⁺CD127⁺IL-18Rα⁺ cells (green) and ST2⁺ ILC2s (black) were analyzed for Tcf7^EGFP and Kit expression in naive and papain-treated Tcf7^EGFP adult mouse lungs. (H) Tbet⁻Lin⁻Thy1⁺CD127⁺RORγt⁻ cells were analyzed for TCF-1 and ST2 expression in naive and papain-treated lungs. Data in A–E are representative of three or more independent experiments with three or more mice per group in each experiment, and data in F and G are representative of two independent experiments with two or more mice per group in each experiment (mean ± SEM). *, P ≤ 0.05 by two-tailed Student’s t test.