Regulation of mTOR activation by amino acid transport controls magnitude of ILC2 response. (A and B) (A) Representative flow plots and (B) quantification of pS6 in sort-purified ILC2 cultured for 30 min in the presence of 20 ng/ml IL-7 alone or IL-7 in combination with 20 ng/ml IL-2, IL-25, IL-33, or 1 μg/ml NmU, with or without the mTOR inhibitor PP242 (500 nm). n = 3 technical replicates per condition, representative of two independent experiments. (C and D) Phosphorylation of S6 in sort-purified ILC2 cultured with either (C) IL-33 with or without a 2 h pre-incubation with 10 mM BCH (n = 3 technical replicates per condition, representative of two independent experiments), or (D) IL-33 in ILC2 cultured with either Leucine sufficient or deficient media (n = 3–6 technical replicates per condition, representative of two independent experiments). (E and F) (E) Representative flow plots and (F) quantification of RFP expression in IL-33 elicited lung ILC2 from Id2^ERT2Cre x Rosa26^tdRFP controls (Id2^mTOR+/+) or Id2^ERT2Cre x Rosa26^tdRFP x mTOR^fl/fl mice (Id2^mTORfl/fl; n = 9 per group, pooled from two independent experiments). (G–I) (G) Representative flow plots, (H) quantification of Ki-67 expression, and (I) cell numbers of RFP⁻ and RFP⁺ lung ILC2 from naïve and IL-33–treated Id2^mTOR+/+ and Id2^mTORfl/fl mice (n = 5–9 per group for naive mice and n = 11–13 for IL-33–treated mice, pooled from three independent experiments). (J and K) (J) Representative flow plots and (K) quantification of IL-5 and IL-13 expression in RFP⁺ ILC2 from naive and IL-33–treated Id2^mTOR+/+ and Id2^mTORfl/fl mice (n = 5–9 per group for naive mice and n = 11–13 for IL-33–treated mice, pooled from three independent experiments). Data shown as individual values or mean ± SEM. Statistical tests performed: (C and D) one-way ANOVA with multiple comparisons; (F, H, and I) Kruskal–Wallis Test with multiple comparisons. ** P < 0.01, *** P < 0.001, **** P < 0.0001.