Xbp1s regulates colonic ILC2s intrinsically and is essential for restraining colitis progression. (A–C) 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 Xbp1s 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 (A). Areg, IL-5, and IL-13 expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) was analyzed by flow cytometry (B). Percentages of Areg⁺, IL5⁺, and IL-13⁺ cells in ILC2s (C). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (D–F) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) from WT (Xbp1^+/+Il5^RFP-Cre) (CD45.1/CD45.1) or cKO (Xbp1^f/fIl5^RFP-Cre) (CD45.2/CD45.2) age- and sex-matched mice were mixed equally (2 × 10⁵ cells in total) and transferred into Rag2^−/−Il2rg^−/− littermate recipient mice. Experimental design (D). Flow cytometry analyses of CD45.1 and GATA3 expression (upper), Areg, IL-5, and IL-13 expression (bottom) after gating on indicated populations (E). Proportion of ILC2s (Lin⁻GATA3⁺) from different donor, and percentages of Areg⁺, IL-5⁺, and IL-13⁺ cells in recovered WT or cKO ILC2s (F). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 5 per group). (G–Q)Xbp1^+/+Il5^RFP-Cre and Xbp1^f/fIl5^RFP-Cre mice were fed with 3% DSS in drinking water for 6 days to induce colitis model. Experimental design (G) and DAI scores (H) were monitored at the indicated time points. Representative large intestine images (I). Lengths of colons (J). Representative H&E staining of the colon. Scale bar, 500 μm (K). Histological severity scores (L). Flow cytometry analysis of GATA3 expression after gating on CD45.2⁺Lin⁻ cells in large intestinal LPLs (M), absolute numbers of ILC2s (CD45.2⁺Lin⁻GATA3⁺) (N). Flow cytometry analysis of Areg, IL-5, and IL-13 expression in ILC2s (O). Percentages (P) and absolute numbers (Q) of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s. Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 5 per group). (R–W)Xbp1^f/f and Xbp1^f/fRorc-cre mice were fed with 3% DSS for 6 days to induce colitis model. Experimental design (R). DAI scores (S). Representative large intestine images (T). Lengths of colons (U). Representative H&E staining of the colon. Scale bar, 500 μm (V). Histological severity scores (W). Data were compiled from two independent experiments and are shown as the mean ± SD (n = 7 per group). *P < 0.05, **P < 0.01, ***P < 0.001.