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Hallmarks of CD8+ T cell dysfunction are established within hours of tumor antigen encounter before cell division

Nature Immunology volume 24, pages 1527–1539 (2023)Cite this article

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Abstract

Tumor-specific CD8+ T cells (TST) in patients with cancer are dysfunctional and unable to halt cancer progression. TST dysfunction, also known as exhaustion, is thought to be driven by chronic T cell antigen receptor (TCR) stimulation over days to weeks. However, we know little about the interplay between CD8+ T cell function, cell division and epigenetic remodeling within hours of activation. Here, we assessed early CD8+ T cell differentiation, cell division, chromatin accessibility and transcription in tumor-bearing mice and acutely infected mice. Surprisingly, despite robust activation and proliferation, TST had near complete effector function impairment even before undergoing cell division and had acquired hallmark chromatin accessibility features previously associated with later dysfunction/exhaustion. Moreover, continued tumor/antigen exposure drove progressive epigenetic remodeling, ‘imprinting’ the dysfunctional state. Our study reveals the rapid divergence of T cell fate choice before cell division in the context of tumors versus infection.

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Fig. 1: Tumor-specific T cells undergo robust activation and proliferation but do not gain effector function.
Fig. 2: Tumor-induced effector function impairment begins before cell division.
Fig. 3: Committed effector CD8+ T cells rapidly lose function in tumor-bearing hosts.
Fig. 4: Naive and effector tumor-specific T cells robustly proliferate in mice with metastatic melanoma but lack effector function.
Fig. 5: Dysfunction-associated epigenetic programming begins before cell division.
Fig. 6: Pre-division tumor-induced tumor-specific T cell chromatin remodeling is reinforced with time and tumor antigen exposure.
Fig. 7: Duration of tumor antigen exposure determines dysfunction stability and imprinting.

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Data availability

RNA-seq and ATAC-seq data have been deposited in the Gene Expression Omnibus under super-series accession number GSE209712. KEGG, Hallmark and Reactome gene sets were retrieved from the Broad Institute’s MSigDB collections using msigbr v7.5.177 (https://www.gsea-msigdb.org/gsea/msigdb/index.jsp). Source data are provided with this paper. All other data that support the findings of this study are present in the article or are available from the corresponding author upon request.

Code availability

Code has been deposited in GitHub: https://github.com/abcwcm/Rudloff2022.

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Acknowledgements

We thank A. Schietinger and members of the laboratory of M.P. for helpful discussions. We thank E. Chiu, A. Dave, G. Anzarova and T. Bryson for technical assistance. We thank the Vanderbilt Division of Animal Care, D. Flaherty and the VUMC Flow Cytometry Shared Resource Core, A. Jones and the Vanderbilt Technologies for Advanced Genomics (VANTAGE) Core, and A. Viale and the Sloan Kettering Integrated Genomics Operation Core (IGO). We thank P. Lauer and Aduro Biotech for providing attenuated Listeria strains. We thank A. Schietinger, J. C. Rathmell and J. M. Balko for critical review of the manuscript. This work was supported by the following funding sources: V Foundation Scholar Award (to M.P.), National Institutes of Health (NIH) R37CA263614 (to M.P.), Serodino Family Adventure Allee Fund (to M.P.), Vanderbilt-Ingram Cancer Center (VICC) SPORE Career Enhancement Program (to M.P.) NIH P50CA098131, Vanderbilt Digestive Disease Research Center (VDDRC) Young Investigator and Pilot Award (to M.P.) NIH P30DK058404, Medical Scientist Training Program (MSTP) NIH T32GM007347 (to M.W.R.), NIH T32GM008554 (to N.R.F.), NIH T32CA009592 (to C.R.D.R) and NIH T32AR059039 (to M.M.E.). The VUMC Flow Cytometry Shared Resource is supported by the VICC (NIH P30CA68485) and the VDDRC (NIH P30DK058404). VANTAGE is supported by the VICC (NIH P30CA68485), the Vanderbilt Vision Center (NIH P30EY08126) and the NIH G20RR030956. IGO is supported by NIH P30CA08748, Cycle for Survival and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology.

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Authors and Affiliations

  1. Department of Medicine, Division of Hematology and Oncology, Department of Pathology, Microbiology, and Immunology, Vanderbilt School of Medicine, Nashville, TN, USA

    Michael W. Rudloff, Natalie R. Favret, Jessica J. Roetman, Carlos R. Detrés Román, Megan M. Erwin, Kristen A. Murray, Sriya T. Jonnakuti & Mary Philip

  2. Department of Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA

    Paul Zumbo & Friederike Dündar

  3. Applied Bioinformatics Core, Weill Cornell Medicine, New York, NY, USA

    Paul Zumbo, Friederike Dündar & Doron Betel

  4. Institute for Computational Biomedicine, Weill Cornell Medicine, New York, NY, USA

    Doron Betel

  5. Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA

    Doron Betel

  6. Vanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN, USA

    Mary Philip

  7. Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, TN, USA

    Mary Philip

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Contributions

M.W.R. and M.P. conceived and designed the study and analyzed and interpreted data. M.W.R. carried out experiments, assisted by N.R.F., J.J.R., C.R.D.R., M.M.E., K.A.M. and S.T.J. P.Z., F.D. and D.B. designed and performed computational analyses of RNA-seq and ATAC-seq data. M.W.R. and M.P. wrote the manuscript, with all authors contributing to the writing and providing feedback.

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Correspondence to Mary Philip.

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Nature Immunology thanks Mohamed Abdel-Hakeem, Ansuman Satpathy, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: L. A. Dempsey, in collaboration with the Nature Immunology team.

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Extended data

Extended Data Fig. 1 TST undergo robust activation and proliferation but do not gain effector function.

a, Live CD8+ Thy1.1+ TCRTAG CFSE dilution and expression of surface markers at each time point from LMTAG-infected mice (spleens; green) or from tumor-bearing mice (blue), from liver tumors, tumor draining lymph nodes (tdLN), and spleens, shown together with naive in vivo control (N; grey). Each time point shows data concatenated from 3-4 biologic replicates. Three independent experiments were carried out with 3-4 mice/group. b, Counts of TCRTAG per organ at 12h, 36h, 48h, and 60h as well as later time points 5 and 21+ days. Each symbol represents an individual mouse with n = 3 per group. ****P <0.0001 determined by two-way ANOVA with post hoc Šidák multiple comparison test comparing the cumulative number of TCRTAG from tumor-bearing mice and infected mice. c, TNF, IFNγ, and CD107a production following 4h ex vivo TAG peptide stimulation in TCRTAG from LMTAG-infected mice (green) or from tumor-bearing mice (blue) at pooled 48 and 60h time points. GZMB and PRF1 expression was assessed immediately ex vivo. Naive in vivo TCRTAG is shown for comparison (N; grey). Dot plots are concatenated from 3 mice/group. d, IFNγ and TNF production by TCRTAG from spleens and livers of LMTAG-infected mice at 48h time point following 4h ex vivo TAG peptide stimulation (top) and summary plot of percentage TNF+IFNγ+ TCRTAG (bottom) points where bars represent mean and error bars represent standard deviation. ns = not statistically significant, determined by unpaired two-tailed Student’s t-test. e, Dot plots of no peptide stimulation controls from infected and tumor-bearing mice from pooled 48 and 60h time points. Two independent experiments were carried out with 2–4 mice/group.

Source data

Extended Data Fig. 2 Tumor-induced TST effector function impairment begins prior to cell division.

Live CD8+ Thy1.1+ TCRTAG analyzed from spleens of infected mice (green) and livers from tumor-bearing hosts (blue) at 6h, 12h, and 18h. a, Representative histograms of CD44, LAG3, and PD1 expression profiles. Two independent experiments were carried out with 3-4 mice/group. b, Ex vivo GZMB expression. Summary plots are shown to the right with each point representing an individual mouse and n = 3-4 per group. c, Independent experimental replicate of Fig. 2e showing summary plots of percentage TNF+IFNγ+ TCRTAG after peptide stimulation. Each symbol represents an individual mouse with n = 3-4 per group. ***P = 0.0003, ****P <0.0001 determined by two-way ANOVA with post hoc Tukey test.

Source data

Extended Data Fig. 3 Committed effector CD8 T cells rapidly lose function in tumor-bearing hosts.

a, Dot plots of TCRTAG TNF and IFNγ production from spleens and livers of LMTAG-infected animals following 4h TAG peptide stimulation. Gates set based on no peptide stimulation controls. b, Summary plots of percentage TNF+IFNγ+ TCRTAG following 4h TAG peptide stimulation, where open circles represent TCRTAG isolated from livers and closed circles from spleens of infected mice with n = 3 (12h, 7d) or 4 (36h) per group. Each point represents an individual animal, statistics determined by two-way ANOVA with post-hoc Tukey test. c, TCRTAG CFSE dilution and TNF and IFNγ production at 12h and 36h time points from tumor livers following 4h TAG peptide stimulation (left). Data is concatenated from 3-4 biological replicates/timepoint. Summary plots of percentage TNF+IFNγ+ TCRTAG (right) where each symbol represents an individual mouse with n = 3 (12h) or 4 (36h) per group and black bars represent mean. d, Summary plots of percentage TNF+IFNγ+ TCRTAG by cell division 36h post-transfer points where bars represent mean and error bars represent standard deviation. Each symbol represents an individual mouse with n = 3-4 per group.

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Extended Data Fig. 4 Naive and effector TST robustly proliferate in mice with metastatic melanoma but lack effector function.

Live CD8+ Ly5.1+ TCROTI analyzed from spleens of LMOVA infected B6 (green) and B6 with pulmonary B16-OVA metastases (pink) at 16h. a, Representative histograms of CFSE, CD69, and CD44 expression profiles. b, Representative histograms of LAG3 and PD1 expression. c, Ex vivo GZMB expression. Summary plots are shown to the right with each point representing an individual mouse. d, CFSE dilution of E5d TCROTI transferred into time-matched LMOVA infected B6 (green) or B6 with pulmonary B16-OVA metastases (pink) at 24 and 48 hours following transfer. Naive TCROTI (N; grey) shown for comparison. *P = 0.0307 and ***P = 0.0008 determined using unpaired two-tailed Student’s t-test.

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Extended Data Fig. 5 Gating strategy for sorting; dysfunction-associated epigenetic and transcriptional programming begins prior to cell division.

a, Gating strategy to sort TCRTAG from infected spleens or tumor livers for sequencing studies. b, Principal component analysis (PCA) of RNA-SEQ data comparing top 500 most variable genes between naive (N; grey) and TCRTAG differentiating during acute infection (green) and in tumors (blue) at 6, 12, 24h post-transfer. Each symbol represents a single biological replicate. c, Chromatin accessibility profile across the Pdcd1 locus with the exhaustion-associated −23kb peak boxed (left). Summary plot (right) shows the DESeq2-determined log2FC and FDR (two-sided Wald test with Benjamini-Hochberg correction) at the Pdcd1 −23kb peak for early E and T time points as compared to naive (N).

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Extended Data Fig. 6 STAT1 motif enrichment is accompanied by increased gene expression in CD8 T cells activated during acute infection; tumor- and infection-activated CD8 T cells show similar enrichment of T cell activation-associated genes.

a, Heatmap showing expression of differentially-expressed genes with peaks containing STAT1 motifs (by ChromVar; Fig. 5e) in effector (E, left) or tumor (T, right) across time points. Heatmaps are z-score normalized across rows. b, Gene set enrichment analysis (GSEA) of E versus N or T versus N at 6h, 12h, and 24h post transfer for activation associated gene sets and KEGG, HALLMARK, and REACTOME gene sets. Color represents normalized enrichment score (NES) and circle size represents the negative log10 (FDR).

Extended Data Fig. 7 Pre-division tumor-induced TST chromatin remodeling is reinforced with time and tumor antigen exposure.

a, Chromatin accessibility heatmap showing naive (N) and TCRTAG from 6h to 60+d in liver tumors of ASTxCre (left) and in LMTAG-infected B6 (right). Each row represents one of 29,884 (left) or 31,756 (right) peaks (differentially accessible between at least one sequential time point comparison; FDR <0.05, |log2FC| >1.5) displayed over 2kb window centered on the peak summit. Scale units are RPGC normalized to 1x sequencing depth for 20 bp bins with blue indicating closed chromatin and red open chromatin. Peaks are clustered by k-means (k = 6). b, PCA comparing chromatin accessibility in TCRTAG from 6h to M during infection. Each symbol represents a single biological replicate. c, Upper panel shows peak changes for early (E24h) → intermediate (E5d) transition (log2FC E5d/E24h) versus int (E7d) → late (M) transition (log2FC M/E7d) (upper). Lower panel shows peak changes for early (T24h) → int (T5d) transition (log2FC T5d/T24h) versus int (T7d) → late (T14d) transition (log2FC T14d/T7d). Each point represents an individual peak colored according to the scheme in Fig. 5c. To the right are shown corresponding bar plots showing the number of peaks in e.

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Extended Data Fig. 8 Early TST uniformly express TCF1.

a, Experimental scheme: CFSE-labeled naive TCRTAG (Thy1.1) were adoptively transferred into B6 (Thy1.2) or ASTxAlb-Cre mice (Thy1.2). TCRTAG were re-isolated at 36h (T36h), 5d (T5d), and 60d (T60d) from spleens and livers for flow cytometric analysis (Naive in vivo (N; grey); tumor (T; blue)). b, Dot plots of TST TCF1 and PD1 expression by CFSE dilution across time points. c, Dot plots comparing CD38 and CD101 (top) or TOX and TCF1 (bottom) expression in TST isolated from tumor-bearing livers across time points. Gates set based on N with inset numbers indicating percentage of cells in each gate. Plots are concatenated from 3-4 samples/time point.

Extended Data Fig. 9 Duration of tumor antigen exposure determines dysfunction stability and imprinting.

Chromatin accessibility profiles across selected gene loci for TCRTAG activated in tumor for 24h (T24h), 5d (T5d), 10d (T10d) pre-parking (blue) and post-parking (P24h, P5d, P10d) (purple) with naive (grey) and memory (green). a, Pdcd1 gene locus with −23 kb enhancer peak boxed in red. Summary plot (right) shows the DESeq2-determined log2FC and FDR (two-sided Wald test with Benjamini-Hochberg correction) at the Pdcd1 −23kb peak for pre- (blue) and post-park (purple) relative to naive. b, Tox locus with blue box representing region without epigenetic scarring and red box showing region with epigenetic scarring. Star denotes representative +100 kb peak from scar region used for quantification on right. Late dysfunctional TCRTAG activated in tumor (D35) added for comparison. Summary plot (right) shows the DESeq2-determined log2FC and FDR (two-sided Wald test with Benjamini-Hochberg correction) at the Tox +220 kb and +100 kb peaks for pre- (blue) and post-park (purple) relative to naive. c, Ifng locus with black box denoting +14kb peak open in memory but not in pre- or post-parking samples, and red box denoting +19kb peak open in both pre- and post-parking samples. Summary plot (right) shows the DESeq2-determined log2FC and FDR (two-sided Wald test with Benjamini-Hochberg correction) for the Ifng +14kb and +19 kb peak area pre- (blue) and post-park (purple) relative to memory.

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Rudloff, M.W., Zumbo, P., Favret, N.R. et al. Hallmarks of CD8+ T cell dysfunction are established within hours of tumor antigen encounter before cell division. Nat Immunol 24, 1527–1539 (2023). https://doi.org/10.1038/s41590-023-01578-y

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