Xbp1s regulates gut ILC2s in a cell-intrinsic manner. (A–D) Sorted large intestinal ILC2s (Lin⁻CD127⁺KLRG1⁺) were cultured for 5 days in the presence of IL-2, IL-7, IL-25, and IL-33 (10 ng/ml each), followed by treatment with Ceapin-A7 and ISRIB, respectively, at the indicated concentrations for 16 h. Flow cytometry analyses of Areg, IL-5, and IL-13 expression (Ceapin-A7, A; ISRIB, C) after gating on CD45.2⁺Lin⁻GATA3⁺ ILC2s. Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (Ceapin-A7, B; ISRIB, D). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (E and F) Flow cytometry analysis of Xbp1s expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) (E) following treatment with Toyo at indicated concentrations. MFI of Xbp1s in ILC2s (F). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (G–J) WT mice were injected i.p. with Toyo (0.7 mg/kg) every other day for four doses, followed by large intestine harvesting to analyze ILC2 function. Experimental design (G, top). Flow cytometry analysis of Areg, IL-5, and IL-13 expression in ILC2s (CD45.2⁺Lin⁻GATA3⁺) from the large intestine LPLs (G, bottom). Percentages of Areg⁺, IL-5⁺, and IL-13⁺ ILC2s (H). Flow cytometry analysis of Xbp1s expression in ILC2s (I). MFI of Xbp1s in ILC2s (J). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 5 per group). (K) Representative flow cytometry plots show frequencies of RFP⁺ (IL-5⁺) cells on the following colonic various immune cell subsets: ILC2s (Lin⁻KLRG1⁺), B cells (CD19⁺), CD11b⁺ myeloid cells (CD11b⁺), CD3e⁺ T cells (CD3e⁺), NK cells (NK1.1⁺). (L–P) Large intestine was harvested from Xbp1^f/fIl5^RFP-Cre and Xbp1^+/+Il5^RFP-Cre mice for subsequent analysis. Flow cytometry analysis of GATA3 expression after gating on CD45.2⁺Lin⁻ cells in large intestinal LPLs (L). Absolute numbers of ILC2s (CD45.2⁺Lin⁻GATA3⁺) (M). Flow cytometry analysis of Areg, IL-5, and IL-13 expression in ILC2s (N). Percentages (O) and absolute numbers (P) 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). (Q) Large intestines were harvested from Xbp1^+/+Il5^RFP-Cre or Xbp1^f/fIl5^RFP-Cre mice for analysis of ILC2 cell death at steady state. Flow cytometry analysis of Aqua⁺Annexin Ⅴ⁺ proportion after gating on CD45.2⁺Lin⁻CD127⁺KLRG1⁺ ILC2s in LPLs, and the percentages of dead ILC2s (left). 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 9 days and then detected cell death. Flow cytometry analysis of Aqua and Annexin Ⅴ expression in expanded ILC2s, and the percentages of dead ILC2s (right). Data were compiled from two independent experiments and are shown as the mean ± SD (n = 6 per group). (R–U) 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. Numbers of Thy1.1⁺ ILC2s in the indicated group (R). The protein levels of Xbp1s in the indicated group were detected by western blotting. β-Actin served as the internal control (S). Analysis of Xbp1s expression in ILC2s by flow cytometry (T). MFI of Xbp1s in ILC2s (U). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (V) 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) mice were mixed equally and transferred into Rag2^−/−Il2rg^−/− mice. Flow cytometry analyses of CD45.1 and CD45.2 expression ratio of mixed ILC2s. (W)Il9 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). (X) SMART-seq was performed on sorted large intestinal ILC2s from 3% DSS-treated (6 days) mice or control mice. The FPKM of Il9 was analyzed based on SMART-seq data. (Y and Z)Xbp1^f/f and Xbp1^f/fRorc-cre mice were fed with 3% DSS for 6 days to induce colitis. Flow cytometry analysis of IL-22 expression after gating on CD45.2⁺Lin⁻RORγt⁺ ILC3s in the large intestinal LPLs (Y). Percentages of IL-22⁺ ILC3s (Z). 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. Source data are available for this figure: SourceData FS2.