Figure 4.
scRNA-seqof CD4 TRM reveals differential cluster distribution between organs. (A) Schematic diagram showing the preparation of sorted antigen-specific cells from mice infected with a sublethal dose (25 μl) of PR8 IAV i.n. for generation of GEM using 10x chromium controller. (B) UMAP plot of unsupervised clustering for 15,301 CD4 TRM and other non-CD4 T cells isolated from lungs and NT of naïve and PR8 IAV-infected mice. (C) Dot plot representing mean expression of selected marker genes for each T cell cluster. Color intensity from blue to red indicates average expression of genes, and size of the dot depicts percentage of cells expressing the gene within the clusters. Only T cell clusters were included in the analysis. Selected gene signatures are as follows: Naïve CD4 T signature (Wang et al., 2022), Th1 signature (Andreatta et al., 2022), Tfh signature (Andreatta et al., 2022), Th17 signature (Szabo et al., 2019a), Treg signature (Yang et al., 2021), cytotoxic CD4 signature (Hashimoto et al., 2019), IFN response signature (Szabo et al., 2019a), NKT cells signature (Cohen et al., 2013; Hu et al., 2022), Acutely activated signature (Kurd et al., 2020; Woodring et al., 2022), Quiescent memory signature (Plasek and Valadkhan, 2021; Dey et al., 2023), early activated signature (Yan et al., 2012; Li et al., 2016). (D) Density plot showing the expression of selected marker genes or gene signatures, projected on the same UMAP as in Fig. 4 B. Signatures were the same as defined in Fig. 4 C. (E) Bar graph showing proportion of each UMAP cluster divided by organ and antigen specificity. Only antigen-specific CD4 TRM from infected mice were included in the analysis. Refer to the image caption for details. Panel A shows schematic diagram of single cell R N A sequencing workflow from infection to cell sorting and library preparation. Panel B shows U M A P plots of clustered C D 4 T R M populations across nasal tissue and lungs. Panel C shows dot plots of marker gene expression across different C D 4 T cell clusters and signatures. Panel D shows density plots of gene expression and signature distribution projected onto UMAP clusters. Panel E shows bar plots of cluster proportions across tissues and antigen specific C D 4 T R M populations.

scRNA-seq of CD4 TRM reveals differential cluster distribution between organs. (A) Schematic diagram showing the preparation of sorted antigen-specific cells from mice infected with a sublethal dose (25 μl) of PR8 IAV i.n. for generation of GEM using 10x chromium controller. (B) UMAP plot of unsupervised clustering for 15,301 CD4 TRM and other non-CD4 T cells isolated from lungs and NT of naïve and PR8 IAV-infected mice. (C) Dot plot representing mean expression of selected marker genes for each T cell cluster. Color intensity from blue to red indicates average expression of genes, and size of the dot depicts percentage of cells expressing the gene within the clusters. Only T cell clusters were included in the analysis. Selected gene signatures are as follows: Naïve CD4 T signature (Wang et al., 2022), Th1 signature (Andreatta et al., 2022), Tfh signature (Andreatta et al., 2022), Th17 signature (Szabo et al., 2019a), Treg signature (Yang et al., 2021), cytotoxic CD4 signature (Hashimoto et al., 2019), IFN response signature (Szabo et al., 2019a), NKT cells signature (Cohen et al., 2013; Hu et al., 2022), Acutely activated signature (Kurd et al., 2020; Woodring et al., 2022), Quiescent memory signature (Plasek and Valadkhan, 2021; Dey et al., 2023), early activated signature (Yan et al., 2012; Li et al., 2016). (D) Density plot showing the expression of selected marker genes or gene signatures, projected on the same UMAP as in Fig. 4 B. Signatures were the same as defined in Fig. 4 C. (E) Bar graph showing proportion of each UMAP cluster divided by organ and antigen specificity. Only antigen-specific CD4 TRM from infected mice were included in the analysis.

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