Xbp1s governs colonic ILC2s by targeting DHFR. (A) Venn diagram showed the numbers of overlapping genes between Xbp1s bindings in CUT&Tag-seq (FDR < 0.01) and DEGs in RNA-seq (log₂[fold change] >1 or less-than −1, FDR < 0.01) of ILC2s after treated with Toyo at 4 μM. (B) Volcano plot of overlapping genes described in Fig. 5 A. (C) Recruitment of Xbp1s to the Dhfr locus in ILC2s by analyzing CUT&Tag-seq data (upper). Representative RNA-seq tracks at the Dhfr locus in indicated ILC2s (bottom). (D and E) Putative Xbp1s-responsive element in the Dhfr promoter (D). Luciferase activity of a Dhfr wild-type promoter reporter (Dhfr-WT-Luc) or of a Dhfr mutant reporter (Dhfr-Mut-Luc) containing a deletion in the Xbp1s-responsive element in HEK293T cells transiently expressing either vector (pcDNA3.1) or Xbp1s. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (F) Sorted ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured for 5 days in the presence of IL-2, IL-7, and IL-25 (10 ng/ml each), and IL-25 was removed for 2 days, followed by restimulation with or without IL-25 (10 ng/ml) for 1 day. The level of DHFR in ILC2s was detected by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (G) Sorted ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured for 5 days in the presence of IL-2, IL-7, and IL-25 (10 ng/ml each), followed by treatment with Toyo at 4 μM for 16 h. The level of DHFR in indicated ILC2s was detected by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (H) Sorted ILC2s (Lin⁻CD127⁺KLRG1⁺) were stimulated with the IFN-γ (500 ng/ml) for 3 days in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each). The level of DHFR in indicated ILC2s was detected by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (I) Sorted 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. Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (CD45.2⁺Lin⁻GATA3⁺) were analyzed by flow cytometry. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (J) Purified human peripheral blood ILC2s were cultured for 3 wk in the presence of hIL-2, hIL-7, hIL-25, and hIL-33 (20 ng/ml each), followed by treatment with Toyo at 25 nM for 16 h. DHFR mRNA expression in ILC2s was detected by qRT-PCR. Data are shown as the mean ± SD (n = 10 per group). (K and L) Human colonic LPMNCs were stimulated with the MTX at 20 μM for 16 h in complete RPMI 1640 medium. AREG expression in ILC2s (CD45⁺Lin⁻CD127⁺CRTH2⁺) was analyzed by flow cytometry (K). Percentage of AREG⁺ ILC2s (L). Connected lines are samples from the same clinical samples (n = 10). P value was determined by two-tailed paired Student’s t test. (M–P) Large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were sorted from WT 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 3 days, followed by treatment with Toyo at 4 μM for 16 h. Experimental design (M). Levels of DHFR protein in indicated ILC2s were detected by western blotting (N). β-Actin served as the internal control. Flow cytometry analyses of Areg, IL-5, and IL-13 expression in the ILC2s (CD45⁺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). (Q) Schematic diagram of folate-dependent 1C metabolic pathways, including representative enzymes and metabolic products. (R) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured for 5 days, and then incubated with or without Toyo at 4 μM for 16 h. Levels of folate-derived metabolites AMP, GMP, dTMP, and SAM were detected by HPLC-MS/MS (n = 5 per group). **P < 0.01, ***P < 0.001. FDR, false discovery rate. Source data are available for this figure: SourceData F5.