Fig 2: CD200R engagement ameliorates human ILC2-mediated AHR.a Human peripheral ILC2s from healthy donors were purified via FACS and cultured with rhIL-2 (20 ng/ml) and rhIL-7 (20 ng/ml) for 48 h, and then adoptively transferred into Rag2−/−Il2rg−/− mice. Additionally, the mice were challenged with either rhIL-33 (1 µg) or PBS intranasally (i.n.) and treated with CD200-Fc or isotype control on days 1, 2, and 3. Measurement of lung function and analysis of BAL fluid followed on day 4, as shown in the timeline, n = 6 mice. b, c Line graphs show lung resistance and dynamic compliance in response to increasing doses of methacholine. d The numbers of eosinophils in the BAL. e The numbers of human ILC2s in the lungs. f Histological images of the lungs, and g corresponding quantifications of cell numbers, and h thickness of epithelium. Scale bars = 50 μm. i Human peripheral ILC2s from healthy donors were purified via FACS and cultured with rhIL-2 (20 ng/ml) and rhIL-7 (20 ng/ml) for 48 h, and then adoptively transferred into Rag2−/−Il2rg−/− mice. The mice were then challenged with either rhIL-33 (1 µg) or PBS intranasally (i.n.) on days 1, 2, and 3. Subsequently, the mice were treated with CD200-Fc or vehicle control on days 4, 5, and 6. The lung function, BAL fluid and lung tissue were analyzed on day 7, n = 6 mice. j Lung resistance, and k dynamic compliance. l BAL eosinophilia and m pulmonary ILC2s are shown as bar graphs. n Histological images of the lungs, and o, p corresponding quantifications. Scale bars = 50 μm. Data are shown as means ± SEMs and are representative of three individual experiments. Statistical analysis, one-way ANOVA followed by Tukey post-hoc tests. Human, mouse and lung images are sourced through an open access license from Servier Medical Art.

Fig 3: CD200R engagement ameliorates IL-33-induced AHR and pulmonary inflammation.a A cohort of WT were challenged with rmIL-33 (0.5 µg) or PBS intranasally (i.n.) and treated with CD200-Fc or isotype control on days 1, 2, and 3, n = 6 mice. On day 4, we assessed the lung function and acquired the samples, as shown in the timeline. b, c Line graph show lung resistance and dynamic compliance (cDyn) in response to increasing doses of methacholine. Total numbers of CD45+ cells (d), CD3+ T cells (e), neutrophils (f), and eosinophils (g) in BAL fluid have been demonstrated in the bar graphs. h the numbers of ILC2s in the lungs. i representative images, and j, k quantification of hematoxylin and eosin (H&E) and alcian blue/periodic acid–schiff (AB-PAS) stained histologic sections of the lungs of mice. Scale bars = 50 μm. l A cohort of WT were challenged with rmIL-33 (0.5 µg) or PBS intranasally (i.n.) on days 1–3, n = 6 mice. Subsequently, the mice treated intranasally with CD200-Fc or isotype control for three days. On day 7 the lung function was assessed, and samples were collected, as shown in the timeline. m lung resistance. n dynamic compliance. Bar graphs displaying the total numbers of CD45+ cells (o), CD3+ T cells (p), neutrophils (q), and eosinophils (r) in BAL fluid. s Number of ILC2s in lungs. t Representative images and quantification (u, v) of H&E and AB-PAS stained histologic sections of the lungs of mice. Scale bars = 50 μm. Data are shown as means ± SEMs and are representative of three individual experiments. Statistical analysis, one-way ANOVA and two-tailed student’s t-test. Mouse and lung images are sourced through an open access license from Servier Medical Art.

Fig 4: CD200R engagement inhibits ILC2 activation and effector function.a A cohort of WT mice were challenged with recombinant mouse (rm) IL-33 (0.5 µg in 50 µL) for three consecutive days, and in vivo pulmonary ILC2s isolated on the fourth day, n = 6 mice. Activated ILC2s were subsequently cultured in the presence of recombinant mouse rmIL-2 and rmIL-7, and stimulated with CD200-Fc (10 µg/mL) or isotype control for 24 h. a scRNAseq profiles of 3390 IL-33-activated ILC2s from WT mice. Cells are colored based on expression levels of Cd200r1 and Il5. Dot size is indicative of the total gene expression level of each cell. Dashed lines enclose cells with highest CD200R expression. b Representative flow cytometry plots of intracellular IL-5 protein expression levels in ILC2s cultured with CD200-Fc or isotype control. c t-Distributed stochastic neighbor embedding (tSNE) of 3390 ILC2s colored based on expression levels of Il13 and Cd200r1. d intracellular protein expression levels of IL-13 in cultured ILC2s. e tSNE plots colored based on expression levels of Cd200r1 and Gata3 in single ILC2s. f Intranuclear protein expression level of transcription factor GATA3 in cultured ILC2s. In vivo-activated ILC2s were cultured in presence of rmIL-2, rmIL-7, and rmIL-33 and stimulated with CD200-Fc (10 µg/mL) or isotype control for 24 h. Total RNA was isolated and sequenced. g Network analysis of upregulated (red) and downregulated (green) genes predicted to influence cell proliferation in cultured ILC2s. h Representative flow cytometry plots of intranuclear Ki67 levels in cultured ILC2s. Quantitation of protein expression levels are presented as MFI +/− SEM, n = 6 mice. Statistical analysis, two-tailed student’s t-test (b, d, f, and h).

Fig 5: CD200R engagement ameliorates ILC2-dependent AHR.a A cohort of Rag2−/− were challenged with rmIL-33 (0.5 µg) or PBS intranasally (i.n.) and treated with CD200-Fc or isotype control on for three days, n = 6 mice. On day 4, we assessed the lung function and acquired the samples, as shown in the timeline. Lung resistance (b) and dynamic compliance (c) in response to increasing doses of methacholine. Total numbers of eosinophils (d) and neutrophils (e) in BAL fluid and ILC2s in the lungs (f) are demonstrated in the bar graphs. g representative histological images, h, i quantification of H&E and AB-PAS stained histologic sections of the lungs. Scale bars = 50 μm. j A cohort of Rag2−/− mice were challenged with rmIL-33 (0.5 µg) or PBS intranasally (i.n.) for three days, n = 6 mice. Subsequently, the mice were treated intranasally with CD200-Fc or isotype control for an additional three days. On day 7 the lung function was assessed, and samples were acquired. k Lung resistance. l dynamic compliance. Bar graphs displaying the numbers of BAL eosinophils (m), neutrophils (n), and pulmonary ILC2s (o). p Representative images and quantification (q, r) of H&E and AB-PAS stained histologic sections of the lungs of mice. Scale bars = 50 μm. Data are shown as means ± SEMs and are representative of three individual experiments. Statistical analysis, one-way ANOVA and two-tailed student’s t-test. Mouse and lung images are sourced through an open access license from Servier Medical Art.