Xbp1s directs DHFR expression, orchestrating 1C metabolism to sustain gut ILC2s. (A and B) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured in the presence of IL-2, IL-7, IL-25 (10 ng/ml each) for 5 days, followed by treatment with Toyo at 4 μM, with or without AMP, GMP, dTMP, and SAM (1 mM each) for 16 h. Flow cytometry analysis of Areg, IL-5, and IL-13 expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) (A). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (B). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (C and D) Human colonic LPMNCs were stimulated with Toyo at 25 μM, supplemented with or without AMP for 16 h in complete RPMI 1640 media. AREG expression was analyzed after gating on CD45⁺Lin⁻CD127⁺CRTH2⁺ ILC2s (C). Percentage of AREG⁺ ILC2s (D). Connected lines are samples from the same clinical samples (n = 10). P value was determined by two-tailed paired Student’s t test. (E and F) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured in the presence of IL-2, IL-7, IL-25 (10 ng/ml each) for 5 days, followed by treatment with Toyo at 4 μM, and then supplemented with or without AMP (1 mM), and SQ22536 (1 mM), a potent inhibitor of AC. Flow cytometry analysis of Areg, IL-5, and IL-13 expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) (E). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (F). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (G and H) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) from Xbp1^+/+Il5^RFP-Cre and Xbp1^f/fIl5^RFP-Cre mice were cultured in the presence of IL-2, IL-7, IL-25 (10 ng/ml each) for 5 days; the ILC2s from Xbp1^f/fIl5^RFP-Cre mice were incubated with AMP (1 mM) for 16 h. Flow cytometry analyses of Areg, IL-5, and IL-13 expression after gating on CD45.2⁺Lin⁻GATA3⁺ ILC2s (G). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (H). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (I–K) Large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were sorted from Xbp1^f/fIl5^RFP-Cre or littermate 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. Experimental design (I). Flow cytometry analysis of Areg, IL-5, and IL-13 expression in ILC2s (CD45⁺Lin⁻GATA3⁺) (J). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (K). 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.