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

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© 2019 Clarke et al.

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Figure 7. — Single-cell transcriptome analysis of CTLs from anti-PD-1 responders. (A) Left: Contour plots show the expression of TIM-3 and IL-7R in CD14⁻CD19⁻CD20⁻CD4⁻CD45⁺CD3⁺CD8⁺CD103⁺ cells isolated from patients receiving anti-PD-1 treatment at the time point (TP) indicated above the plot; number in red indicates the percentage of tumor T_RM cells (CD8⁺CD103⁺) with TIM-3⁺IL-7R⁻ surface phenotype. Right: Quantification of the percentage of tumor-infiltrating TIM-3⁺IL-7R⁻ T_RM cells, isolated from the anti-PD-1 responding, nonresponding, and treatment-naive patients. Bars represent the mean, t-lines represents SEM, and symbols represent individual data points (*, P ≤ 0.05; **, P ≤ 0.01; n = 7, 8, and 12 biopsies for responders, treatment-naive patients, and nonresponders, respectively; Mann–Whitney U test). (B) Contour plots demonstrate the expression of TIM-3 and PD-1 in the T_RM cells isolated from preimmunotherapy biopsies (gated as per Fig. 7 A). (C) Single-cell RNA-seq analysis of transcripts (one per row) differentially expressed between CTLs pre– and post–anti-PD-1 (MAST analysis), with an adjusted P value of ≤0.05), presented as row-wise z-scores of TPM counts; each column represents a single cell (n = 127 and 151 cells, respectively). (D) Violin plot of expression of indicated transcripts differentially expressed between tumor-infiltrating CTLs isolated from pre– and post–anti-PD-1 treatment samples (as per C); shape represents the distribution of expression among cells, and color represents average expression, calculated from the TPM counts. (E) GSEA of the bulk tumor CD103⁺ versus CD103⁻ transcriptional signature (Fig. 2 A) and TIM-3⁺IL7R⁻ T_RM cell signature (Fig. 4 A) in tumor-infiltrating CTLs isolated from pre– and post–anti-PD-1 treatment samples. Top: RES for the gene set, from most enriched at the left to most underrepresented at the right. Middle: Positions of gene set members (blue vertical lines) in the ranked list of genes. Bottom: Value of the ranking metric. Values above the plot represent the NES and FDR-corrected significance. (F) Spearman coexpression analysis of transcripts enriched in tumor-infiltrating CTLs from post–anti-PD-1 treatment samples (C); matrix is clustered according to complete linkage. (G) Correlation analysis of all peaks identified in the OMNI-ATAC-seq libraries, pooled from nine donors across two experiments; cells were sorted on CD14⁻CD19⁻CD20⁻CD4⁻CD45⁺CD3⁺CD8⁺CD103⁺TIM-3⁺IL-7R⁻ and CD14⁻CD19⁻CD20⁻CD4⁻CD45⁺CD3⁺CD8⁺CD103⁻. The matrix is clustered according to complete linkage. (H) University of California Santa Cruz genome browser tracks for key T_RM-associated gene loci as indicated above the tracks. RNA-seq tracks are merged from all purified bulk RNA-seq data, presented as reads per kilobase million (RPKM; as per Fig. 1 A; tumor non-T_RM = 25, tumor T_RM = 19; OMNI-ATAC-seq as per Fig. 7 G).