Fig 2: The nature and longevity of allergic responses depend on the type of adjuvant used during sensitization.(A and B) Mice were sensitized to OVA using either Aspergillus oryzae (ASP) or LPS as the adjuvant, then challenged on a single occasion with aerosolized OVA. (A) Timeline of allergic sensitizations, single challenge, and harvest. (B) Percentages of Th2 (Gata3+) cells, Th17 (ROR?t+) cells, and Tregs (Foxp3+CD25+) in CD4+ T cells from lungs of mice 2 days after a single challenge. (n = 6 mice/group). (C) Timeline of sensitizations, daily challenges, and harvest. (D) Airway inflammation 1 day after the last of the indicated number of OVA challenges (n = 5 mice/group). (E) Airway hyperresponsiveness (AHR) measured 2 days after a single challenge or 1 day after the last of 7 daily challenges (n = 6 mice/group). Data shown represent mean values ± SEM from a single experiment, representative of 2, except for B, a single experiment. *P < 0.05; **P < 0.01; ***P < 0.001; unchallenged (0) group vs. allergen-challenged groups; Kruskal-Wallis 1-way ANOVA with Dunn’s multiple-comparison test. R, resistance; MCH, methacholine.

Fig 3: The Early IL-4 Wave Is Localized at the Periphery of B Cell Follicles(A–C) Confocal microscopy analysis of (A and B) wild-type and CD1d-/- and (C) IL4-GFP mice on day 3 of influenza infection. Lymph nodes were labeled with CD1d tetramer (magenta) and anti-B220 antibody (green in A and B and white in C). The arrows in (C) indicate CD1d tetramer+ cells expressing IL-4 (IL-4 GFP, green). Scale bars, 300 µm (lymph node) and 60 µm (section).(D) Flow cytometry analysis of IL-4 GFP+ cells in mediastinal lymph nodes on day 3 of influenza infection, showing CD1d-tet- and CD1d-tet+ cells.(E) t-SNE plots of CD1d-tet- and CD1d-tet+ subsets. The CD1d-tet+ population separates into two clusters (1, dark purple; 2, light purple), as does the CD1d-tet- population (3, light green; 4, dark green).(F and G) Expression distribution (violin plots) in each population (horizontal axes) for (F) CD3e, Zbtb16, CXCR6, and CXCR3 and (G) Gata3, Tbx21, and Rorc. Expression levels are log2(tpm+1).(H) Flow cytometry analysis of CXCR3 levels in IL-4-GFP- (red) and GFP+ (blue) NKT cells on day 3 of influenza infection. Lines connect cells from the same mouse.(I) Flow cytometry analysis of IL-4 production by CXCR3- (red) and CXCR3+ (blue) NKT cells on day 3 of influenza infection. Lines connect cells from the same mouse.(J) ELISPOT analysis of IL-4-producing CXCR3- (red) and CXCR3+ (blue) NKT cells sorted on day 3 of influenza infection. Each dot represents pooled NKT cells from 6 mice.In graphs showing statistical analysis, data are representative of at least two independent experiments. Horizontal bars, mean; error bars, SEM. Student’s t test, *p < 0.05, **p < 0.01.

Fig 4: Single-Cell RNA-Seq Analysis of IL-4+ Lymph Node Cells, Related to Figure 4(A) Confocal microscopy of mediastinal lymph nodes sections at day 3 of influenza infection. Lymph nodes were labeled with PBS-57 loaded CD1d tetramer (magenta) and antibodies to B220 (white) and CD169 (green). IF, interfollicular. Scale bars, 300 µm (left); 60 µm (right). Chart on the right shows the quantification of NKT cells-macrophages contacts observed in the different areas of the lymph node at day 0, 1, 2 and 3 of infection in mediastinal lymph nodes. Data are representative from three experiments.(B) Flow cytometry analysis of IL4-GFP+ CD1d-tet- cells in mediastinal lymph nodes at day 3 of influenza infection showing TCRß- and TCRß + cells.(C) Single-cell RNA-seq heatmap showing the expression of 25 significant marker genes (Table S1) for the CD1d-tet+ clusters (columns 1 and 2) and CD1d-tet- clusters (columns 3 and 4). Single cells and genes were determined on the basis of unsupervised hierarchical clustering (Table S1).(D) Expression distribution (violin plots) in each population (horizontal axes) for Ccr7, Sell, S1pr1, CD4, CD8a and Bcl6. Expression levels are Log2(tpm+1).(E) Flow cytometry analysis of IL4-GFP+ TCRß+ CD1d-tet- cells in mediastinal lymph nodes at day 3 of influenza infection showing CD4+ and CD8a + cells.(F) Representative contour plot show the expression of GATA-3 by NKT cells, ILC2s (Live/Dead- B220- CD3- CD5- CD11b- CD11c- CD127+ CD45+ GATA-3+) and CD4+ T cells at day 3 of infection. Quantification on the right chart shows the mean fluorescence intensity for GATA-3.(G) Flow cytometry analysis of ROR?t+ and T-bet+ NKT cells in mediastinal lymph nodes at day 3 of influenza infection. Histogram shows the levels of CXCR3 in ROR?t+ and T-bet+ populations. Dot graphs show the quantification of CXCR3+ NKT cells in the ROR?t+ (red dots) and T-bet+ populations (blue dots), with lines connecting NKT cells from the same mouse.In graphs showing statistical analysis, data are representative of at least two independent experiments. Statistical analysis two-tailed Student’s t test; **p < 0.01, ***p < 0.001 and ****p < 0.0001.