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. 2019 Jun 21;216(9):2128–2149. doi: 10.1084/jem.20190249

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© 2019 Clarke et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/).

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Figure 3. — Single-cell transcriptomic analysis reveals previously uncharacterized T_RM subsets. (A) tSNE visualization of ∼12,000 live and singlet-gated CD14⁻CD19⁻CD20⁻CD4⁻CD56⁻CD45⁺CD3⁺CD8⁺ single-cell transcriptomes obtained from 12 tumors and 6 matched normal lung samples. Each symbol represents a cell; color indicates protein expression of CD103 detected by flow cytometry. (B) Seurat clustering of cells in A, identifying nine clusters. (C) Cells from tumor and lung were randomly down-sampled to equivalent numbers of cells. Left: Distribution of T_RM-enriched clusters in tumor and lung. Right: Pie chart representing the relative proportions of cells in each T_RM cluster. (D) Expression of transcripts previously identified as up-regulated in the bulk tumor T_RM population (Fig. 2 A) by each cluster; each column represents the average expression in a particular cluster. (E) Breakdown of cell type and tissue localization of cells defined as being in cluster 1 (shown in light purple in B and D). (F) Violin plots of expression of example tumor T_RM genes in each T_RM-enriched cluster (square below indicates the cluster type) and non-T_RM cells; shape represents the distribution of expression among cells and color represents the Seurat-normalized average expression counts. (G) Cell-state hierarchy maps generated by Monocle2 bioinformatic modeling of the T_RM clusters. In the center plot, each dot represents a cell colored according to Seurat-assigned cluster. Surrounding panels show relative Seurat-normalized expression of the indicated genes. (H) Cluster analysis of the location in PCA space for cells. Each cluster was randomly down sampled to the equivalent size of the smallest cluster (n = 135 cells per cluster, 675 total). The correlation method was Spearman, and the dataset was clustered with average linkage.