Fig. 4: Single-cell (sc) RNA-seq analysis for assessment of ILC2 heterogeneity in lungs of HDM-sensitized mice.

A Schematic diagram of experimental study design for single-cell (sc) RNA-seq of lung tissues. B Uniform manifold approximation and projection (UMAP) plots of ILC2 subsets from HDM-challenged mice at day 45 and day 47. Each dot represents a single cell, with different ILC2 subsets shown in different colors. C UMAP plots displaying sorted ILC2 subsets at different time points. D Signature genes used for ILC2s subsets identification. E Heatmap displaying the differential gene expression among three ILC2 clusters. The right panel displays enriched pathways associated with the marker genes of each cluster. Enriched pathways of different ILC2 subsets are shown in different colors. P-values were calculated by right-tailed Fisher’s Exact Test with Benjamini-Hochberg correction. F. The scatter plot manifesting the differentially expressed genes (DEGs) between ILC2_c01 and ILC2_c02 subsets, colored by adjusted P-values. Adjusted P-values were calculated by a two-tailed Wilcoxon test with Bonferroni correction. G RNA velocity analysis of ILC2_c01, ILC2_c02 and ILC2_c03 subsets with velocity field projected onto the UMAP plot from Fig. 4B. Arrows show the local average velocity evaluated on a regular grid and indicate the extrapolated future states of cells. H UMAP showing the gene expression intensity of Tlr4 on ILC2 clusters. I Representative histogram showing the expression levels of IL-5, IL-13, CCR8, IL-17RB, and CD44 on TLR4⁺ILC2s and TLR4^⁻ILC2s detected by flow cytometry in lung tissues of HDM-sensitized mice at day 47. J Mean fluorescence intensity (MFI) of IL-5, IL-13, CCR8, IL-17RB, and CD44 on TLR4⁺ILC2s and TLR4^⁻ILC2s detected in the lung tissues of HDM-sensitized mice at day 47 (n = 6 mice per group). Data were pooled from two independent experiments. Each symbol indicates one mouse, and bars represent mean ± SEM. P-values were calculated by two-tailed Wilcoxon tests.