Flow cytometric and functional analysis shows distinct effector ILC2 subsets in neonatal mouse lungs. (A) Lin⁻ST2⁺Thy1⁺CD127⁺ ILC2s from 12-d-old B6 mouse lungs were analyzed for KLRG1 and ICOS expression. The expression of ST2 and CD127 by the KLRG1⁺ICOS⁻ (red) and KLRG1⁻ICOS⁺ (purple) subsets were overlapped in dot plot. (B) Neonatal ILC2s were divided into four subsets based on KLRG1 and ICOS expression, purified, and cultured for the indicated number of hours with IL-33 and IL-7 (1 ng/ml each for cytokine and 10 ng/ml for amphiregulin analysis). The amounts of IL-5, IL-13, and amphiregulin in the culture supernatants were determined by ELISA. (C) ILC2s from neonatal, naive, and papain-treated WT and Il33^−/− mouse lungs were analyzed for KLRG1 and ICOS expression. The frequencies of KLRG1⁺, ICOS⁺, and KLRG1⁻ICOS⁻ subsets are shown in bar graphs. Data are representative of three to five independent experiments with ≥12 mice per group (mean ± SEM). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 by two-tailed Student’s t test.