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. 2023 Nov 29;14:7825. doi: 10.1038/s41467-023-43381-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a. UMAP representation of intra-tumoural CD8 T cells (n = 8989). Left: depicting three distinct trajectories: CD8 T_RM, CD8 T_EX and CD8 T_EMRA. Right: depicting shared T cells (i.e. intra-tumoural T cells characterized by a TCR found in PBMC week 0) versus non-shared T cells. b. Bar plot showing the shared TCR weight for CD8 T_EM with other CD8 phenotypes in the TME. c. T cell density, TCR richness and Gini-index along each CD8 trajectory in atezo/bev-treated patients (n = 20), stratified for response (12 Resp versus 8 NonResp). The density plots reflect the relative number of T cells separately for Resp versus NonResp along each CD8 trajectory. P-values reflect the difference in distributions, calculated using the two-sided Kolmogorov-Smirnov test. d. Density of shared T cells along each CD8 trajectory in atezo/bev-treated patients (n = 17) stratified for response (10 Resp versus 7 NonResp). The density plots reflect the relative number of shared T cells separately for Resp versus NonResp along each CD8 trajectory. Shared intra-tumoural T cells are characterized by a TCR found in peripheral blood prior to treatment. P-values reflect the difference in distributions, calculated using the two-sided Kolmogorov-Smirnov test. e. Top 15 pathways identified using fGSEA on differentially expressed genes along CD8 T_EX versus CD8 T_EMRA trajectory (using diffEnd test; TradeSeq³⁵) for the REACTOME and GO: Biological processes gene sets. Significantly enriched pathways (adjusted p-value < 0.01) were identified and ranked based on enrichment score for each gene set separately. Only the top 15 pathways of each gene set, enriched in each trajectory were retained. f. Top pathways identified using fGSEA on differentially expressed genes identified at the point where the three trajectories diverge (using earlyDEG test; TradeSeq³⁵) for the REACTOME and GO: Biological processes gene sets. Adjusted p-value calculated using the Benjamini-Hochberg method. g. UMAP representation of peripheral T cells at week 0 (W0), week 3 (W3) and week 6 (W6) characterized by a TCR shared with CD8 T_EMRA (left) and CD8 T_EX (right) in the pre-treatment TME, stratified for response to atezo/bev (top: Resp (n = 10), bottom: NonResp (n = 17)). (PBMC, peripheral blood mononuclear cells; TCR, T cell receptor; TME, tumour microenvironment; UMAP, Uniform Manifold Approximation and Projection; Resp, responder; NonResp, non-responder).