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. 2022 Jan 19;10(1):e004034. doi: 10.1136/jitc-2021-004034

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© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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Single-cell RNA-seq analysis defines distinct subsets of CD8 TILs. (A) UMAP of scRNA-seq results of total tumor infiltrating CD45 +cells pooled from different treatment groups. Tumor infiltrating CD45 +cells from 5 mice in the same treatment group were pooled and subjected to single cell sequencing. (B) UMAP of total T cells in MOC1esc1 tumors colored by indicated major subsets. T cells from all three conditions were pooled for clustering analysis. (C) Heatmap illustrating the relative gene expression levels of genes in major T cell subsets in MOC1esc1 tumors. (D) Tcf7 +Pd1−, Tcf7 +Pd1+, and Tcf7-Pd1+CD8+T cells in MOC1esc1 tumors were detected by flow cytometry. Dot plot was pre-gated on live CD8 +T cells. Percentages of CD8 +subsets in MOC1esc1 tumors at indicated treatment condition are shown. MOC1esc1 tumors treated with isotype control, anti-PD1, or anti-CTLA4 were harvested at day 12 postinoculation and analyzed by flow cytometry for CD8 +T cell subset distribution (*p<0.05, ***p<0.001). Significance was calculated by one-way ANOVA. Data are shown as mean±SEM, n=5 mice per group.) (E) Pseudo-bulk differential expression analysis was performed in total CD8 +T cells between responder (anti-CTLA4) and control MOC1esc1 tumors. Colors of dots represent the anti-CTLA4 treated MOC1esc1 infiltrating CD8 +T cell upregulated genes (red) and downregulated genes (blue) compared with control. The statistical significance (log10 FDR) was plotted against the log2 fold-change of gene expression levels. ANOVA, analysis of variance; FDR, false discovery rates; MOC1, murine oral carcinoma; TILs, tumor infiltrating lymphocytes; UMAP, uniform manifold approximation and projection.