Fig 2: Bach2 deficiency impaired the differentiation of CLPs and ILC2Ps.(A) Competitive mixed CLP chimera experimental design. (B) Quantification of the ratio of ILC2P and ILC2 differentiated from CD45.2 (Il7rCre/+) or (Bach2ΔIl7r) CLPs and CD45.1+ ILC2s after 10 to 12 weeks of reconstruction in recipients was performed by flow cytometry. (C) Competitive mixed ILC2P chimera experimental design. (D) Quantification of the ratio of ILC2 differentiation from CD45.2 (Il7rCre/+) or (Bach2ΔIl7r) ILC2s and CD45.1+ ILC2s after 10 to 12 weeks of reconstruction in recipients. (E) Competitive mixed BM chimera experimental design. Irradiated CD45.1/2 mice were intravenously inoculated with Bach2fl/+-CreERT2 (CD45.2) and Bach2fl/fl-CreERT2 (CD45.2) BM cells equally mixed with WT competitive BM cells (CD45.1) and analyzed 6 to 8 weeks after transplantation. (F) The recipients were sacrificed to analyze the contribution of ILC2s from Bach2fl/+-CreERT2 (and Bach2fl/fl-CreERT2) after 6 to 8 weeks of reconstitution. (G) Competitive BM chimeric experiments were performed. Lethal irradiated CD45.1 mice intravenously inoculated with Il5Cre/+ (CD45.2) and Bach2∆Il5 (CD45.2) BM cells equally mixed WT competitive BM cells (CD45.1/2) and analyzed 6 to 8 weeks after transplantation. (H) Representative flow plots and ratio of donor chimerism of lung ILC2 were determined by flow cytometry. The data were analyzed by two-way ANOVA followed by the Tukey-Kramer test for multiple comparisons [(B), (D), (F), and (H)] from two independent experiments, with n = 4 independent samples from each group. **P < 0.01, ***P < 0.001. The error bars represent the means ± SEM.

Fig 3: Bach2 regulates the development of ILC2.(A) Representative flow plots of CLP, α-LP, and ILC2P in BM from Bach2ΔIl7r and Il7rCre/+ mice. (B) Quantification of the proportion and counts of (A). (C and D) Representative histogram (C) and quantification (D) of CD25, ICOS, GATA3, and IL-18R1 in BM ILC2P from Bach2ΔIl7r and Il7rCre/+ mice. (E) Representative flow plots and quantification of lung ILC2 from Bach2ΔIl7r and Il7rCre/+ mice. ILC2s in lung were gated on CD45+Lin−CD90.2+CD25+. (F and G) Quantification of ILC2 proportions and counts in the mLN (F) and colon tissues (G) from Bach2ΔIl7r and Il7rCre/+ mice. ILC2s in mLN and colon were defined as CD45+Lin−CD127+KLRG1+ cells. (H to J) Competitive BM chimeric experiments were performed. NCG mice intravenously inoculated with Il7rCre/+ (CD45.2) or Bach2∆Il7r (CD45.2) BM cells equally mixed WT competitive BM cells (CD45.1) and analyzed 6 to 8 weeks after transplantation (H). Representative flow plots (I) and ratios of donor chimerism (J) of lung ILC2 and colonic ILC2 were determined by flow cytometry. Data were analyzed by two-way ANOVA followed by Tukey-Kramer test for multiple comparisons [(B), (D) to (G), and (J)] from three [(B) and (D) to (G)] or two [(H) to (J)] independent experiments and n = 4 to 5 independent samples of each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars are shown as mean ± SEM.

Fig 4: Bach2 regulates distinct type 2 immune responses.(A) 2D-UMAP plots showing the distribution from Bach2ΔIl7r and Il7rCre/+ mice after IL-33 challenge for 3 days. (B) UMAP plots showing the expression of the TH2 cytokine genes Il13, TF Gata3, and IL-33 receptor Il1rl1 in lymphocytes. (C) Volcano analysis indicating the differential peaks of Bach2 binding in ILC2 and TH2 cells. (D) Tracks of Irf4, Nfatc2, Rad50, and Cdk1 loci with signal densities of CUT&Tag-seq for Bach2 in lung ILC2 and TH2 during the steady-state condition. (E) Study design for ATAC-seq from Bach2ΔIl7r and Il7rCre/+ mice. (F) Heatmap displaying enriched motifs within differentially ACRs between ILC2s and TH2 cells. (G and H) Pie plots summarizing categories of differential ATAC peaks in ILC2s (G) and TH2 (H) between Bach2ΔIl7r and Il7rCre/+ mice. (I and J) Volcano plots showing the differentially accessible chromatin regions in lung TH2 (I) and ILC2 cells (J). (K) Heatmap comparing relative chromatin accessibilities of indicated loci in ILC2. (L and M) Tracks of Il5 (L), Gata3, and Il13 (M) gene loci with ATAC signal densities in pulmonary ILC2 and TH2. (N) Quantification of IL-5 and IL-13 from the supernatants in ILC2 from Bach2ΔIl7r mice during the vehicle, Irf4OE condition, and Bach2-sufficient ILC2 group, were determined by ELISA. Data were analyzed by unpaired t test (N) from two (N) independent experiments and n = 4 (N) independent samples of each group. **P < 0.01, ***P < 0.001. The error bars present the mean ± SEM.

Fig 5: Bach2 governs airway inflammation in an ILC2-dependent manner.(A) Study design for papain-induced airway inflammation. In Bach2fl/fl-CreERT2 and littermate mice, airway inflammation was induced by intranasal administration of papain for five consecutive days (20 μg per mouse per day). (B) Flow cytometric analysis and quantification of eosinophils gated as CD45+CD11c−Siglec-F+ in BALF from the indicated groups. (C) IL-5 and IL-13 concentrations in the BALF were determined by ELISA in Bach2fl/+-CreERT2 and Bach2fl/fl-CreERT2 mice. (D) Representative H&E staining and scores of lung sections from the indicated groups. Scale bars, 50 μm. (E and F) Quantification of the lung ILC2 proportions and counts (E) and cytokine production (F) from ILC2 were determined by flow cytometry in Bach2fl/+-CreERT2 and Bach2fl/fl-CreERT2 mice. (G to J) Bach2ΔIl5 and Il5Cre/+ mice were induced airway inflammation by papain administration. Study design (G). Representative flow plots and quantification of eosinophils (H), ILC2s (I), and representative flow plots and cytokine production (J) by ILC2s were determined by flow cytometry. (K to N) Purified ILC2s from Bach2ΔIl7r and Il7rCre/+ mice were transferred into NCG mice, and airway inflammation was subsequently induced by IL-33 challenge for 3 days (K). Quantification of ILC2 (L) and cytokine production from ILC2 (M) and eosinophils (N) were determined via flow cytometry. (O) Representative H&E staining and scores of lung sections from the indicated groups. Scale bars, 100 μm. The data were analyzed by two-way ANOVA followed by Tukey-Kramer test for multiple comparisons [(B), (C), (E), (F), (H) to (J), and (M) to (O)] from two [(A) to (O)] independent experiments and n = 5 [(A) to (J)] independent samples of each group. *P < 0.05, **P < 0.01, ***P < 0.001. The error bars present the mean ± SEM.