Fig 2: 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 3: Human ILC2s express CD200R and this expression is inducible by IL-33.a CD200 mRNA expression among 22 cell types in lungs of healthy donors and asthmatic patients. b The gating strategy of human ILC2s identified as Lin−CD45+CD127+CRTH2+ cells. c Human peripheral-blood ILC2s were freshly sorted and cultured with 10 ng of recombinant human (rh)IL-2 and rhIL-7 in presence or absence of rhIL-33 (10 ng) for 24, 48, and 72 h. Freshly isolated ILC2 at 0 h and ex vivo activated ILC2s were analyzed by flow cytometry as indicated in the scheme. d kinetics of CD200R induction by IL-33. e The levels of IL-4, IL-5, IL-6, IL-9, IL-13, and GM-CSF in the culture supernatants were measured by Luminex after 48 h of stimulation with CD200-Fc (10 µg/mL). Data representative of five individual blood donors, n = 5. Error bars are the mean ± SEM. Statistical analysis, one-way ANOVA and two-tailed student’s t-test. Human and cell images are sourced through an open access license from Servier Medical Art.

Fig 4: CD200R signaling inhibits the canonical/non-canonical NF-κB pathways.In vivo activated lung-derived mouse ILC2s were cultured in presence of recombinant mouse (rm)IL-2, rmIL-7, and rmIL-33 and stimulated CD200-Fc (10 µg/mL) or isotype control for 24 h. Total RNA was isolated and sequenced. a Principal Component Analysis (PCA) of activated WT ILC2s treated with CD200-Fc (red) or isotype control (blue). b Volcano plot comparison representing whole transcriptome gene expression of sorted WT ILC2s treated with either isotype control or CD200-Fc for 24 h ex vivo. Differentially expressed genes (described as statistically significant adjusted p-value ≤ 0.05; GSA statistics using lognormal with shrinkage) with changes of at least 1.5-fold change (FC) are shown in yellow (upregulated) and blue (downregulated). Relevant differentially expressed genes are identified. c Heat plot of all differentially expressed genes, d selected cytokine, and cytokine receptor genes, and e transcription factors. f, g scRNAseq profiles of 4428 IL-33-activated ILC2s from WT mice. Cells are colored based on expression levels of Cd200r1, Nfkb1, and Nfkb2. Dot size is indicative of the total gene expression level of each cell. Dashed lines enclose cells with highest CD200R expression. Representative protein expression of pIKKα/β (h), mRNA expression of RelA/p65 and RelB (i, j). The representative protein expression of NF-κB p65 (k), and NF-κB p52 (l) in isolated ILC2s from WT mice challenged with IL-33 and cultured ex vivo for 24 h with CD200-Fc (red) or isotype control (blue), n = 6 mice. The staining FMO controls are shown as gray. The corresponding quantifications are presented as mean fluorescence intensity (MFI), and the error bars denote the mean ± SEM and are representative of three individual experiments. Statistical analysis, two-tailed student’s t-test.

Fig 5: 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.