Xbp1s targets DHFR to regulate folate-mediated 1C metabolism in gut ILC2s. (A) KEGG pathway enrichment analysis of the Xbp1s binding genes (Table S5). (B) Heatmap of overlapping genes between Xbp1s-binding and Toyo-changed genes described in Fig. 5 A; the overlapping genes are listed in Table S6. (C) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured for 5 days, followed by treatment with PH797804 at 10 μM for 16 h. The protein levels of DHFR were detected by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (D)Dhfr expression in ILC2s was analyzed by scRNA-seq of the colon from DSS-treated and control mice. (E)Dhfr expression in sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) from Xbp1^+/+Il5^RFP-Cre or Xbp1^f/fIl5^RFP-Cre mice was assessed by qRT-PCR. Data were compiled from two independent experiments and are shown as the mean ± SD (n = 6 per group). (F–H) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured in the presence of IL-2, IL-7, and IL-25 (10 ng/ml each), with or without MTX co-incubation for 3 days at the indicated concentrations. Flow cytometry analysis of viability in ILC2s (CD45.2⁺Lin⁻GATA3⁺) (F), and the corresponding statistical graphs (G). Data are representative of two independent experiments. Data are shown as the mean ± SD (n = 6 per group). Flow cytometry analyses of Areg, IL-5, and IL-13 expression in ILC2s (H). (I) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each) for 5 days, and then treated with Toyo at 4 μM for 16 h, followed by the targeted metabolomics analysis of 1C metabolism (n = 4 per group). Heatmap of folate metabolites in indicated ILC2s. (J) Schematic diagram of SQ22536 inhibiting cAMP generation. (K and L) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured in complete IMDM containing IL-2, IL-7, and IL-25 (K) or IL-33 (L) (10 ng/ml each), and then treated with SQ22536 at 1 mM for 48 h. Flow cytometry analyses of Areg, IL-5, and IL-13 expression after gating on CD45.2⁺Lin⁻GATA3⁺ ILC2s, and percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (M) Large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were sorted from Xbp1^f/fIl5^RFP-Cre or Xbp1^+/+Il5^RFP-Cre mice and underwent retroviral transduction of DHFR with IRES-controlled Thy1.1 expression to label transduced cells. Thy1.1⁺ cells after gating on Lin⁻CD45.2⁺ were sorted and cultured for 4 days. Levels of DHFR in indicated ILC2s were detected by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (N) Track views of Xbp1s CUT&Tag-seq peaks in colonic ILC2s of mice, and RNA-seq in ILC2s treated with Toyo at genes Srebf2. (O and P) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured for 5 days, followed by treatment with Toyo at 4 μM, with or without palmitic acid (50 μM) and cholesterol (30 μM) for 16 h. Flow cytometry analysis of Areg, IL-5, and IL-13 expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) (O). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (P). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData FS5.