Fig. 3: Lung Tγδ17 cells have a distinct transcriptome indicative of functional maturity.

a Sorting-purified single γδ T cells from lungs at 4 dpi were subjected to scRNA-seq. UMAP clustering following dimension reduction based on highly variable genes across the 7863 single cells recovered revealed the formation of 4 primary clusters of distinct cell types that were then assigned identifiers based on their expression profiles. b, c Visualization of the expression profiles for key genes previously reported to be associated with Tγδ17 in a. d Representative flow cytometric plots showing expression of AQP3 and IL-17A in gated CD3⁺TCRγδ⁺ cells from pdmH1N1-infected lungs at 4 dpi. e Heatmap visualization of the expression patterns of the most varied genes in each cluster across each cell in the cluster. These highly varied genes were used to help annotate the clusters. f Representative GSEA results generated using distinct gene list databases (KEGG, Hallmark, Reactome) as visualized through UMAP confirmed that the substantial heterogeneity observed between the γδ T clusters was driven by significant differences in gene expression along unique biological pathways/processes. Eight significantly variant pathways between the activated and Tγδ17 clusters are shown here as these 2 clusters are highly separated within the UMAP space and in GSEA analysis. g, h UMAP plots show the expression profiles of genes associated with Tγδ17 in a. i Correlation analysis of scRNA-seq data with previously published sequencing of Tγδ17 cells (GSE123400) as shown visually through UMAP following canonical correlation analysis. j UMAP visualization of the expression levels of prominent genes associated with Tγδ17 cells. k, l Violin plots of the expression profiles of markers associated with Tγδ17 in i.