Fig 2: 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 3: DR3 signaling induces non-canonical NF-κB pathway in activated VAT-derived ILC2s.In vivo activated VAT-derived murine ILC2s (aILC2s) were cultured in presence of recombinant mouse (rm) IL-2, rmIL-7 and rmIL-33 and stimulated with DR3 agonist (5 µg/mL) or isotype control for 24 h. Total RNA was isolated and sequenced. a Volcano plot comparison representing whole transcriptome gene expression of sorted WT ILC2s treated with either isotype control or DR3 agonist (5 µg/mL) for 24 h ex vivo. Differentially expressed genes (described as statistically significant adjusted p-value < 0.05) with changes of at least 1.5-fold change (FC) are shown in yellow (upregulated) and blue (downregulated). Relevant differentially expressed genes are identified. b Heat plot of all differentially expressed genes. c Selected cytokine and cytokine receptor genes and transcription factors (d) plotted as the normalized counts in isotype-treated cohort compared to DR3 stimulated cohort. Notable ILC2 related genes are labeled and highlighted in red. Gray area represents region of 1.5-fold change in gene expression. e Upregulated (red) and downregulated (green) genes in the canonical and non-canonical NF-κB pathways. f Representative expression of NF-κB p52 (f) and NF-κB p65 (g) in isolated ILC2s from mice challenged with IL-33 and cultured ex vivo for 24 hrs with DR3 agonist (red) or isotype control (blue), n = 6 mice. The staining FMO control is shown as grey. The corresponding quantification are presented as Mean Fluorescence Intensity (MFI), and the error bars denote the mean ± SD. Statistical analysis, two-tailed student’s t-test (f, g); n.s.: p-value of <0.05 was considered as non-significant.

Fig 4: TNFR2 Engagement on ILC2s Induces the Non-canonical NF-?B Pathway(A) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33. On day 4, aILC2s were FACS sorted as lineage-, CD45+ ST2+ CD127+ and cultured in vitro for 24 h with rmIL-2 (10 ng/mL) and rmIL-7 (10 ng/mL) with or without rmTNFa (40 ng/mL).(B) Volcano plot comparison of whole-transcriptome gene expression on ILC2s from PBS-versus rmTNF-a—stimulated ILC2s. Differentially downregulated or upregulated genes (p < 0.05 and 2-fold change cutoff) are presented in blue and yellow, respectively.(C) Heatmap representation of statistically differentially regulated cytokines, chemokines, and their receptors in ILC2s from PBS-versus rmTNF-a-stimulated ILC2s.(D) Upregulated (red) and downregulated (green) genes in the non-canonical NF-?B pathway and corresponding quantitation, presented as normalized counts ± SEM.(E and F) Representative flow cytometry plots of NF-?B p65 (E) and NF-?B p52 (F) and corresponding quantitation, presented as MFI ± SEM in rmTNF-a-versus PBS-treated ILC2s.Data are representative of 3 individual experiments (n = 3–5). *p < 0.05, **p < 0.01, ***p < 0.001.