RORα regulates ILC2 survival, cytokine production, and heterogeneity in the SI. (A) Experimental schematic outlining tamoxifen treatment and analysis timeline. (B) Quantification of the absolute number of CD45⁺Lineage⁻IL-25R⁺Il17rb⁺ cells in the SI (n = 3 mice). (C) Quantification of the frequency of CD45⁺CD3^–GATA3⁺Il17rb⁺ cells in the SI (n = 3 mice). (D) IL-25R expression levels in CD45⁺Lineage⁻IL-25R⁺ ILC2s (n = 3 mice). (E) Flow cytometry plots of CD3^-Il17rb⁺ ILC2s and their subsequent IL-5 and IL-13 production. (F–G) Quantification of the frequency of IL-5⁺ and IL-13⁺Il17rb⁺ cells among ILC2s (n = 3 mice). (H) Masson’s trichrome staining of the cecum in naïve tamoxifen-treated Il17rb-CreERT2-eGFP and Rora^iIl17rb (Rorα^iIl17rb refers to Il17rb-CreERT2-eGFP × Rora^fl/fl mice). Scale bar = 100 μm. Data are representative of two independent experiments and are presented as the mean ± SEM. For intracellular cytokine detection, isolated cells were stimulated with 50 ng/ml PMA (Sigma-Aldrich) and 750 ng/ml ionomycin (Sigma-Aldrich) for 4–6 h in the presence of 10 μg/ml brefeldin A (Sigma-Aldrich) in complete RPMI-1640 medium (containing 10% FBS, 50 mM 2-mercaptoethanol, 1 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) to evaluate cytokine production. Statistical significance was determined using an unpaired two-tailed t test (*P < 0.05, **P < 0.01). PMA, phorbol 12-myristate 13-acetate.