Fig 1: 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.