Loss of G9a in iILC2s results in an ILC3 phenotype. (A) RNA-Seq analysis of IL-2–, IL-7­–, and IL-25–expanded BM iILC2s showing differential of expression down-regulated genes (green) and up-regulated (red) in Vav.G9a^−/− cells relative to control G9a^fl/fl cells. Data are from n = 3 mice per genotype. FPKM, fragments per kilobase of transcript per million mapped reads. (B) Heat maps of the expression of ILC2 and ILC3 core genes obtained from the RNA-Seq data in A. (C) Intracellular expression of IL-17F and IL-5 from sorted and expanded BM iILC2s treated with PMA, ionomycin, and brefeldin A for 4 h. The numbers in the quadrants indicate percentage of cells in each. Data are representative of three independent experiments. (D) ST2 and RORγt expression in BM iILC2s from G9a^fl/fl or Vav.G9a^−/− mice. Data are representative of two independent experiments (n = 2–3 per experiment). ns, not significant. *, P < 0.05; Student’s t test. Errors bars indicate SEM. (E) RORγt and KLRG1 expression on CD45.2⁺ Lin⁻ CD90.2⁺ Sca1⁺ CD127⁺ cells isolated from of the small intestine (SI) and CD45.2⁺ Lin⁻ CD90.2⁺ CD127⁺ cells isolated from the lungs of NSG mice 3–4 wk after transfer of iILC2s from G9a^fl/fl or Vav.G9a^−/− mice. Data are representative of two independent experiments (n = 1–2 per experiment). (F) Frequencies of ILC3s (live CD45⁺ Lin⁻ CD90.2⁺ RORγt⁺) in Peyer’s patches (PP) and small intestinal lamina propria of G9a^fl/fl or Vav.G9a^−/− mice. Data are representative of n = 4 mice per genotype. LPL, lamina propria lymphocyte. (G) Intracellular expression of IL-17A and IL-13 from G9a^fl/fl- or Vav.G9a^−/−-gated lung ILC2s. After a short course of papain, cells were treated with cell stimulation cocktail (plus protein transport inhibitors) for 4 h. The numbers in the quadrants indicate the percentage of cells in each. The data are concatenated plots from three individual mice and are representative of two independent experiments (n = 3 per experiment).