ILC2s regulate the homeostatic frequency of immune cell subsets in tissues. (A) Representative flow cytometry plots of live CD45⁺Lineage⁻IL-25R⁺ ILC2s in the SI after tamoxifen treatment (5 consecutive days) followed by a 7–14-day washout period. (B and C) Quantification of total ILC2 numbers and frequency (n = 3–4 mice). (D and E) Quantification of live CD45⁺Lineage⁻CD3⁻Il17rb⁺IL-4⁺ and IL-13⁺ ILC2s (n = 3 mice per group). (F) IL-25R expression levels in ILC2s (n = 3 mice). (G) Gating strategy for sorting ILC2s from the SI of Il17rb-CreERT2-eGFP × R26-RFP × Rora^fl/fl mice (n = 2–3 mice pooled per experiment). (H) Genomic PCR analysis of Rora exon 3 and 4 rearrangement in small intestinal ILC2s. Gapdh was used as a loading control, and splenocytes from C57BL/6J mice served as a reference. (I) Representative flow cytometry plots of IL-10 expression in Lineage⁻IL-13⁺ ILC2s from the SI. (J and K) Quantification of Lineage⁻IL-13⁺IL-10⁺ ILC2s and frequency of IL-10⁺ ILC2s (n = 3 mice). (L)Il10 transcript levels in the cecum (n = 3–4 mice). (M) Representative flow cytometry plots of IL-17A production by CD4⁺ Th17 cells. (N) Frequency of IL-17A⁺ Th17 cells in the SI (n = 5–7 mice). (O) Representative flow cytometry plots of IFNγ production by CD4⁺ Th1 cells. (P and Q) Frequency and quantification of IFNγ⁺ Th1 cells in the SI (n = 4–7 and n = 3 mice, respectively). (R) Representative flow cytometry plots of MHCII⁺CD64⁺ macrophages in the SI. (S) Frequency of macrophages (n = 3 mice). (T) MHCII expression levels in MHCII⁺CD64⁺ macrophages (n = 3 mice). (U) Quantification of Lineage⁻RORγt⁺ ILC3s (n = 3 mice). WT mice refer to Il17rb-CreERT2-eGFP mice (both groups were tamoxifen-treated at the same time). Data are representative of two to three independent experiments. Data are presented as the mean ± SEM. Statistical significance was determined using an unpaired two-tailed t test. *P < 0.05, **P < 0.01; ns, not significant. Lineage markers: CD3ε, CD3, CD5, CD8α, TCRβ, Cd11b, CD11c, CD19, B220, Ly6C, Ly6G, Ter119. For the detection of intracellular cytokines, 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 their cytokine production. PMA, phorbol 12-myristate 13-acetate. Source data are available for this figure: SourceData F4.