Fig 2: ILC3s are enriched in lung-draining mediastinal lymph nodes (MedLNs) and have high expression of MHC class II(A and B) Representative plots and quantitative graphs of frequencies (A) or total numbers of ILC2s and ILC3s (B) in mouse lung parenchyma and MedLNs (pooled from 2 independent assays, n = 8–10). Cells are gated on live CD45+ Lin- CD127+ (lineage markers: CD3e, CD5, CD8a, NK1.1, CD11c, CD11b, B220, and F4/80) as in Figure S1A.(C) Representative histograms of specific markers on gated ILC2s and ILC3s in mouse MedLNs (representative of 6 mice).(D) Representative plots and quantitative graphs of ratios of ILC2s and ILC3s in human lung and MedLN tissues. Cells were gated on live CD45+ Lin- (CD3, CD4, CD5, CD11b, CD11c, CD14, CD19, FceRI) CD127+ populations from matched lung and MedLN tissues pooled from 5 donors.(E) Representative histograms of specific markers on ILC2s and ILC3s from human MedLN tissues (representative from 5 donors). Data are represented as mean ± SEM. Statistics are calculated by Student’s t test. See also Figure S1.

Fig 3: MHC class II+ ILC3s limit Th2 cell responses and allergic airway inflammation(A) Representative plots and quantitative graphs of the frequency and number of Th2 cells in lung parenchyma and MedLN from H2-Ab1fl/fl control mice and MHC class II?ILC3 mice after exposure to HDM or PBS. Cells were gated on live CD45+ CD19- CD3/CD5+ CD4+ (data pooled from 4 independent assays).(B) Quantitative graphs of the numbers of eosinophils in lung parenchyma and BAL from mice in (A). Eosinophils were identified as live CD45+ SSC-Ahi, Siglec-F+ (data pooled from 4 independent assays, n = 8).(C) ELISA analysis of sera from mice in (A) for HDM-specific IgG1 and total IgE (pooled from 2 independent assays, n = 4).(D) Representative periodic acid-Schiff staining sections from lung of mice in (A). The scale bars represent 100 µm.(E) Analysis of airway resistance on mice from (A) (pooled from 2 independent assays, n = 8). Data are represented as mean ± SEM. Statistics are calculated by two-way ANOVA (A and B) and AUC followed by one-way ANOVA (C and E). See also Figure S3.

Fig 4: MHC class II+ ILC3s limit airway Th17 cell responses to microbes upon HDM exposure(A) Representative plots and quantitative graphs of the frequency and number of Th17 cells in lung parenchyma and MedLN from H2-Ab1fl/fl control mice and MHC class II?ILC3 mice after exposure to HDM or PBS. Cells were gated on live CD45+ CD19- CD3/CD5+ CD4+ (data pooled from 4 independent assays, n = 6).(B) Quantitative graph of the number of neutrophils in lung parenchyma from mice in (A). Neutrophils were identified as live CD45+ CD19- TCRß- CD11b+ Ly6G+ (data pooled from 4 independent assays, n = 7).(C) ELISA analysis for IL-5 and IL-17A levels from supernatants of microbiota-extract-restimulated MedLN cells from HDM-exposed MHC class II?ILC3 mice (data pooled from 2 independent assays).(D) Quantitative graphs of the number of Th17 cells in lung parenchyma and MedLNs from H2-Ab1fl/fl control mice and MHC class II?ILC3 mice after exposure to sterile papain (data pooled from 2 independent assays, n = 4).(E) Bar graphs of major bacterial composition in HDM from four different vendors. Data are represented as mean ± SEM. Statistics are calculated by two-way ANOVA. See also Figure S4.