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Antitumor progenitor exhausted CD8+ T cells are sustained by TCR engagement

Nature Immunology volume 25, pages 1046–1058 (2024) Cite this article

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Abstract

The durability of an antitumor immune response is mediated in part by the persistence of progenitor exhausted CD8+ T cells (Tpex). Tpex serve as a resource for replenishing effector T cells and preserve their quantity through self-renewal. However, it is unknown how T cell receptor (TCR) engagement affects the self-renewal capacity of Tpex in settings of continued antigen exposure. Here we use a Lewis lung carcinoma model that elicits either optimal or attenuated TCR signaling in CD8+ T cells to show that formation of Tpex in tumor-draining lymph nodes and their intratumoral persistence is dependent on optimal TCR engagement. Notably, attenuated TCR stimulation accelerates the terminal differentiation of optimally primed Tpex. This TCR-reinforced Tpex development and self-renewal is coupled to proximal positioning to dendritic cells and epigenetic imprinting involving increased chromatin accessibility at Egr2 and Tcf1 target loci. Collectively, this study highlights the critical function of TCR engagement in sustaining Tpex during tumor progression.

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Fig. 1: Establishment of the Tpex reservoir in tdLNs requires robust TCR stimulation.
The alternative text for this image may have been generated using AI.
Fig. 2: Suboptimal TCR signaling results in a greater decline in intratumoral Tpex.
The alternative text for this image may have been generated using AI.
Fig. 3: Robust TCR engagement reinforces Tpex self-renewal.
The alternative text for this image may have been generated using AI.
Fig. 4: Optimally engaged T cells are positioned in proximity to DC niches.
The alternative text for this image may have been generated using AI.
Fig. 5: Tpex-associated programs induced by robust TCR signaling are epigenetically imprinted.
The alternative text for this image may have been generated using AI.
Fig. 6: Suboptimal TCR engagement epigenetically predisposes Tpex to terminal differentiation.
The alternative text for this image may have been generated using AI.
Fig. 7: Tpex-specific Tcf1-bound element acts as a Slamf6 enhancer under optimal TCR stimulation.
The alternative text for this image may have been generated using AI.

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Data availability

Raw scRNA-seq, ATAC-seq, WGBS and CUT&Tag data have been deposited in the NCBI Gene Expression Omnibus database under the SuperSeries accession code GSE262845. All other data are available in the article, source data and Supplementary Information, or from the corresponding author upon request. Source data are provided with this paper.

Code availability

No new algorithms were developed for this paper. All analysis code is available upon request.

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Acknowledgements

We thank G. Lennon, R. Cross, K. Hays, S. Fatima and the Flow Cytometry and Cell Sorting Shared Resource at St. Jude Children’s Research Hospital for cell sorting assistance and flow instrument maintenance; S. Konduru, S. Trivedi and P. Kottapalli (St. Jude Children’s Research Hospital) for WGBS library preparation and Illumina sequencing assistance; T. Mori and Y. Wang (St. Jude Children’s Research Hospital) for statistical consulting; the Animal Resource Center at St. Jude Children’s Research Hospital for mouse husbandry; and A. Moustaki (Yale University) for providing the lentiviral vector backbone. This work was supported by the National Institutes of Health (R01AI114442 and R01CA237311 to B.Y. and F32CA250155 to S.B.) and the American Lebanese Syrian Associated Charities (ALSAC to B.Y.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Author information

Authors and Affiliations

  1. Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Xin Lan, Tian Mi, Shanta Alli, Cliff Guy, Shannon Boi, Partha Chowdhury, Minghong He, Yongqiang Feng & Ben Youngblood

  2. College of Graduate Health Sciences, University of Tennessee Health Science Center, Memphis, TN, USA

    Xin Lan

  3. Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, TN, USA

    Mohamed Nadhir Djekidel

  4. Department of Mathematics, University of New Orleans, New Orleans, LA, USA

    Xueyan Liu

  5. Division of Animal Physiology and Immunology, School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany

    Dietmar Zehn

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Contributions

X. Lan and B.Y. designed the experiments and conceptualized the study. X. Lan performed the experiments, and analyzed and visualized the data with help from the co-authors. T.M. and X. Lan analyzed the scRNA-seq, ATAC-seq, CUT&Tag and WGBS data. C.G. performed and analyzed imaging experiments with assistance from X. Liu. M.N.D. performed peak calling and comparison analysis for CUT&Tag. S.A., S.B. and P.C. helped with mouse harvests. M.H. provided technical guidance for CUT&Tag assay. D.Z. and Y.F. provided constructive feedback. X. Lan and B.Y. wrote the paper.

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Correspondence to Ben Youngblood.

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Competing interests

B.Y. declares patents related to epigenetic biomarkers and methods for enhancing T cell function (US11020430B2). The other authors declare no competing interests.

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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: N. Bernard, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 TCR stimulation strength controls the magnitude of T cell activation during priming.

a, Amino acid sequences of indicated Gp33 variants with alterations highlighted75. b,c, Percentages of GFP (Nr4a)+ P14s in vitro stimulated by indicated concentrations of Gp33 peptides (b) or MC38 tumor cells stably expressing indicated Gp33 variants (c). d, LLC-Gp33 tumor growth in C57BL/6 mice, n = 7–12. e, Percentages of mCherry+ LLC-Gp33 at D18 post tumor inoculation, n = 5 per group. f, LLC-Gp33 tumor growth in Rag-KO mice, n(M9) = 5, n(C6M9) = 6. g, Representative flow plot of P14 percentages in tdLN, related to Fig. 1b. h,i, Percentages of CD44+(h, n = 6–8) and PD1+ (i, n = 3–8) P14s in tdLN. j,m, GMFI of GFP, n = 5. Gated on total (j) or CD44+ (m) P14-Nur77-GFP CD8+ T cells. P value 0.0022 (j). k, GMFI of Tox (left) and Tpex percentages (right) in Rag-KO, n = 4–6. Gated on CD44+ tdLN-P14s. P value 0.0029 (left). l, Percentages of CD62L+ P14s in tdLN, n = 4–6. Gated on CD44+ P14s. n, Percentages of PD1+Tim3+ P14s in tdLN, n = 5–9. d–f,j,k,m, Representative data of two independent experiments, two-tailed unpaired Student’s t test (mean ± s.e.m.), P value ** < 0.01, **** < 0.0001, ns represents not significant. h,i,l,n, Representative data of more than three independent experiments, n represents numbers of mice per group, Multiple two-tailed unpaired t test (mean ± s.e.m.), FDR = 0.05, q value indicated in each graph.

Source data

Extended Data Fig. 2 Tpex-associated programs are minimal in suboptimally primed T cells in tdLNs.

a–f Related to scRNA-seq in Fig. 1f, naïve T cell cluster 1 and cluster 9 (low cell count) excluded from analysis. a, Cell number in each cell cluster. b, UMAP colored by cell cycle phases (left) and percentage of cells in each phase (right). c, Barplot displaying percentage of cells in each cell cluster. d, Pathway enrichment analysis of differentially expressed genes (DEGs) in each cluster. Bubble graph displays the top enriched pathways determined by log(q-value). e, Heatmap of the top 10 DEGs for each cluster. f, Relative mRNA expression of representative genes.

Source data

Extended Data Fig. 3 Suboptimal TCR stimulation results in a heightened decline in intratumoral Tpex.

a–g Related to Fig. 1a. a, Representative flow plot of P14 percentages in tumors, related to Fig. 2a. b,c, Percentages of Ki67+ (b, n = 4–7) and Gzmb+ (c, n = 3–7) P14s in tumors. d, GMFI of PD1 in tumors, n = 3–5. b–d Gated on P14s, representative data of more than two independent experiments. Multiple two-tailed unpaired t test (mean ± s.e.m.), FDR = 0.05, q value indicated in each graph. e,f,h, GMFI and percentages of CX3CR1+ P14s in tumors, n = 5 (e,h), n = 6–8 (f). P value 0.0434 (e, left) and 0.0198 (h, left). g, GMFI of GFP. Gated on P14-Nur77-GFP CD8+ T cells in LLC-M9 tumors, n = 5 per group. e–h, Representative data of two independent experiments. i, Representative flow plot of T cell percentage in peripheral blood with PBS (Ctr) or FTY720 treatment. j,k, Percentages of P14s in tumors (j, n = 8–13) and tdLN (k, n = 11–13), related to Fig. 2d. l, Percentages of P14s in tdLN, related to Fig. 2fn = 4. m, Percentages and absolute cell numbers of P14s in tdLN, related to Fig. 3a, n(M9) = 9, n(C6M9) = 10. P value 0.0254 (left) and 0.0216 (right). n, Percentages of CX3CR1+ P14s in tumors, related to Fig. 3d. Gated on Tim3+ P14s. n(M9) = 8, n(C6M9) = 6. j–n, Representative data of three independent experiments. e–h,j–n, Two-tailed Student’s t test (mean ± s.e.m.). P value * < 0.05, **** < 0.0001, ns represents not significant. n represents numbers of mice per group.

Source data

Extended Data Fig. 4 Optimally primed T cells colocalize with DCs and B cells in tdLN.

a, Representative immunofluorescence images of CD90.1+ (red), CD44+ (green) and CD11c+ (pink) cells in M9- and C6M9-tdLN. Scale bars, 100 μm. b, Boxplot displaying relative proportion (Proportion of P14s – Proportion of DCs) of CD44+ and CD44− P14s in DC clusters in M9- and C6M9-tdLN, n(M9) = 22, n(C6M9) = 14. c, Representative immunofluorescence images of CD90.1+ (red), CD44+ (green) and B220+ (blue) cells in M9- and C6M9-tdLN. Scale bars, 100 μm. d, Boxplot displaying correlation of CD44+ and CD44− P14s with B220+ cells, n(M9) = 22, n(C6M9) = 14. b,d, Representative data of two independent experiments, left-tailed Wilcox signed rank test, Bonferroni adjusted significance level, alpha = 0.0015625. n represents total image numbers per group. For all box-and-whisker plots, the center line denotes the median, the box range denotes the 25th and 75th percentiles and whiskers denote the minimum and maximum values.

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Extended Data Fig. 5 DNA methylation programming adapts to TCR engagement strength.

a, Cell sorting strategy for ATAC-seq and WGBS. b, PCA plot based on top 3000 significant CpGs, n = 3 for tdLN samples or n = 1 for tumor samples per group, cells pooled from 3 ~ 5 (M9) or 20 ~ 30 mice (C6M9) for each sample. c, Pie chart displaying distributions of cis-element categories of DMRs in M9-tdLN and C6M9-tdLN. d, Volcano plots of differentially methylated genes between M9-tdLN (orange) and C6M9-tdLN (grey) with the top hypomethylated genes displayed. P value ≤ 0.01, two-tailed Wald test. e, Barplot of top pathways enriched in M9-tdLN-DMRs. f, Heatmap of representative DMRs in M9-tdLN and C6M9-tdLN. g, TF motifs enriched in M9-tdLN-DMRs. h,i, Representative genome tracks displaying chromatin accessibility and DNA methylation at Gzmk (h) and Ptpn5 (i) loci. Red: methylated, Blue: unmethylated.

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Extended Data Fig. 6 Subset-specific Tcf1 binding patterns in tumor-reactive CD8 T cells.

a, Summary table of Tcf1 differentially binding regions (DBRs) numbers among naïve P14 (Tn), Tpex from tdLN (LN-Tpex) and Tpex from tumors (TU-Tpex). Two-sided empirical Bayes moderated t-test, FC > 2, P < 0.05. b,f, Signal enrichment heatmap of Tcf1 DBRs between LN-Tpex versus Tn (FC > 2, FDR < 0.05) (b) and TU-Tpex versus LN-Tpex (FC > 2, P < 0.05) (f). c,g, Spearman’s correlation of differential chromatin accessibility with differential Tcf1 binding between indicated groups. Gene region counts in each quadrant (n) and correlation coefficient (r) indicated in each graph. d,h, Top TF motifs enriched among the DBRs of indicated subsets. e,i, Heatmap of Tcf1 binding intensity at representative gene loci.

Extended Data Fig. 7 CRISPR-Cas9 editing of TCR-induced Tcf1 binding site at Slamf6.

Sequence of TCR-induced Tcf1 binding site at Slamf6 (171965904–171966678). Blue, Tcf1 motifs; Green and Red, CRSIPR gRNA.

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Lan, X., Mi, T., Alli, S. et al. Antitumor progenitor exhausted CD8+ T cells are sustained by TCR engagement. Nat Immunol 25, 1046–1058 (2024). https://doi.org/10.1038/s41590-024-01843-8

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