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TOX is a critical regulator of tumour-specific T cell differentiation

Nature volume 571, pages 270–274 (2019)Cite this article

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Abstract

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1,2,3,4,5,6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, ‘non-exhausted’ immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.

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Fig. 1: TOX is highly expressed in tumour-infiltrating CD8 T cells of mouse and human tumours.
Fig. 2: Chronic TCR stimulation drives TOX expression in tumour-specific CD8 T cells.
Fig. 3: Ectopic expression of TOX is sufficient to induce a global molecular program characteristic of T cell exhaustion.
Fig. 4: Phenotypic, functional, transcriptional and epigenetic analysis of TOX-deficient T cells.

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Data availability

All data generated and supporting the findings of this study are available within the paper. The RNA-seq and ATAC-seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE126974. Source Data are provided with the online version of the paper. Additional information and materials will be made available upon request.

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Acknowledgements

We thank the members of the Schietinger laboratory, S. Reiner and M. Li for discussions and technical help; R. Nadler for technical assistance; A. Hurwitz and N. Restifo for providing TRP2 transgenic mice; MSKCC Flow Cytometry Core, especially R. Gardner; Integrated Genomics Operation Core, especially A. Viale, N. Mohibullah, A. Farina and R. Patel. We thank the J. Sun laboratory and B. Moltedo (Rudensky laboratory) for providing LCMV strains. D.Z., A. Schietinger, M.D.H. and C.A.K. are members of the Parker Institute for Cancer Immunotherapy, which supports the MSKCC Cancer Immunotherapy Program. This work was supported by NIH-NCI grants DP2 CA225212, R00 CA172371 (to A. Schietinger), U54 CA209975, V Foundation for Cancer Research (to A. Schietinger), the Anna Fuller Foundation (to A. Schietinger), the William and Ella Owens Medical Research Foundation (to A. Schietinger), the Josie Robertson Young Investigator Award (to A. Schietinger), NIH-NCI grant K08 CA158069 (to M.P.), V Foundation Scholar Award (to M.P.), Serodino Family Adventure Allee Fund (to M.P.), the Parker Institute for Cancer Immunotherapy (C.A.K. and A. Schietinger), the Weill Cornell Medicine Core Laboratories Center (P.Z., F.D., D.B.), Francois Wallace Monahan Fellowship (to O.L.), NIH-NIAID U19 AI11143 (to M.S.G.), NIH-NIAID R01 AI054977 (to J.K.), the Damon Runyon Cancer Research Foundation CI-96-18 (to C.A.K.), NCI R33 CA22539 (to C.A.K.), the Manhasset Women’s Coalition Against Breast Cancer (to C.A.K.), the MSK Cancer Center Core Grant P30 CA008748. The Integrated Genomics Operation Core was supported by Cycle for Survival and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology.

Author information

Authors and Affiliations

  1. Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Andrew C. Scott, Mojdeh Shakiba, Prerak Trivedi, Laura Menocal, Heather Appleby, Steven Camara, Olivier Levy, Michael S. Glickman, Mary Philip & Andrea Schietinger

  2. Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, New York, NY, USA

    Andrew C. Scott, Laura Menocal, Niroshana Anandasabapathy, Michael S. Glickman & Andrea Schietinger

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

    Friederike Dündar, Paul Zumbo & Mojdeh Shakiba

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

    Friederike Dündar, Paul Zumbo & Doron Betel

  5. Parker Institute for Cancer Immunotherapy, New York, NY, USA

    Smita S. Chandran, Christopher A. Klebanoff, Dmitriy Zamarin, Matthew R. Femia, Matthew D. Hellmann & Andrea Schietinger

  6. Center for Cell Engineering, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Smita S. Chandran, Christopher A. Klebanoff & Matthew R. Femia

  7. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Christopher A. Klebanoff, Dmitriy Zamarin, Tyler Walther, Alexandra Snyder, Elizabeth A. Comen & Matthew D. Hellmann

  8. Weill Cornell Medical College, New York, NY, USA

    Christopher A. Klebanoff, Elizabeth A. Comen & Matthew D. Hellmann

  9. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Hannah Y. Wen

  10. Department of Dermatology, Weill Cornell Medical College, New York, NY, USA

    Niroshana Anandasabapathy & Yong Liu

  11. Department of Cardiothoracic Surgery, Weill Cornell Medicine, New York Presbyterian Hospital, New York, NY, USA

    Nasser K. Altorki

  12. Aduro Biotech, Inc., Berkeley, CA, USA

    Peter Lauer

  13. Research Division of Immunology, Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Jonathan Kaye

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

    Doron Betel

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

    Doron Betel

  16. Department of Medicine, Division of Hematology and Oncology, Vanderbilt University Medical Center, Nashville, TN, USA

    Mary Philip

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  1. Andrew C. Scott
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Contributions

A.C.S., M.P. and A. Schietinger conceived and designed the study. A.C.S., M.P., D.B., F.D., P.Z. and A. Schietinger conceived the computational analyses; D.B., F.D. and P.Z. performed all of the computational analyses. A.C.S., M.P., P.T., L.M., M.S., H.A. and S.S.C. carried out experiments. A.C.S., M.P., F.D., P.Z., D.B., S.S.C., C.A.K. and A. Schietinger interpreted data. S.C. and H.A. assisted with mouse breeding; T.W., A. Snyder, D.Z., M.D.H., M.R.F., E.A.C., H.Y.W. and C.A.K. provided human samples; N.A., Y.L. and N.K.A. contributed to the analysis of human samples. O.L. and M.S.G. provided help in establishing the knockout model. O.L., M.S.G. and J.K. provided mice. P.L. provided Listeria strains. A.C.S., M.P., F.D., P.Z., D.P. and A. Schietinger wrote the manuscript, with all authors contributing to writing and providing feedback.

Corresponding authors

Correspondence to Mary Philip or Andrea Schietinger.

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Competing interests

C.A.K. is a consultant and/or advisor to Aleta Biotherapeutics, Bellicum Pharmaceuticals, Bristol-Meyers Squibb, Cell Design Labs, G1 Therapeutics, Klus Pharma, Obsidian Therapeutics and Rxi Therapeutics. C.A.K. receives research funding unrelated to this work from Kite/Gilead. M.D.H. has received research funding from Bristol-Myers Squibb; is paid as a consultant to Merck, Bristol-Myers Squibb, AstraZeneca, Genentech/Roche, Janssen, Nektar, Syndax, Mirati, and Shattuck Labs; has received travel support/honoraria from AstraZeneca and BMS; a patent has been filed by MSK related to the use of tumour mutation burden to predict response to immunotherapy (PCT/US2015/062208), which has received licensing fees from PGDx. A. Snyder is a current employee and owns stock in Merck. D.Z. reports grants from Merck, and consulting fees from Merck, Synlogic Therapeutics, Trieza Therapeutics, and Tesaro. D.Z. owns a patent concerning the use of Newcastle Disease Virus for cancer therapy. M.S.G. is a paid consultant to Vedanta Biosciences and Takeda for work unrelated to this study. E.A.C. is a consultant to Pfizer, Novartis, Genentech/Roche, Cota, Heron Therapeutics, and Bristol-Myers Squibb; has received travel support/honoraria from Pfizer and Novartis; and receives research funding from Genentech/Roche unrelated to this work.

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Extended data figures and tables

Extended Data Fig. 1 T cell differentiation during tumorigenesis.

a, Scheme of autochthonous liver cancer model to investigate tumour-specific CD8 T cell differentiation and dysfunction. AST×Cre liver cancer model. Cre-mediated deletion of the flox-stop cassette leads to TAG expression and tumour initiation. TAG-specific CD8 T cells isolated from TCRTAG transgenic mice recognize TAG epitope I (shown in red) on major histocompatibility complex (MHC) class I H-2Db. Tamoxifen-inducible Cre-ERT2 (AST×Cre-ERT2) or constitutive Alb-Cre (AST×Alb-Cre) mouse strains are used as indicated. b, Top, scheme of Listeria infection. Bottom, phenotypic characterization of Thy1.1+ effector and memory TCRTAG cells isolated from spleens 7 and more than 35 days after transfer into B6 mice followed by Listeria infection. Gating strategy is shown. KLRG1, CD127, CD44 and CD62L expression levels are shown. c, Naive congenically marked (Thy1.1+) TCRTAG CD8 T cells were adoptively transferred into (Thy1.2+) B6 mice and immunized with TAG-expressing Listeria strain, or were transferred into tumour-bearing (Thy1.2+) AST×Alb-Cre mice. T cells were isolated 7 or more than 20 days after transfer from either spleens (for effector and memory T cells after Listeria infection) or liver tumour lesions of AST×Alb-Cre mice. TOX expression was assessed by flow cytometry. TOX isotype is shown as a control for each sample. Naive TCRTAG cells are shown in grey as a control. d, Flow cytometric analysis of TCRTAG cells isolated from liver lesions of AST×Cre-ERT2 mice more than 20 days after transfer (red). TOX expression with PD-1, LAG-3, 2B4, CD39, TIGIT, TIM-3, CD101, CD38, CTLA4 and TCF-1 expression levels are shown. Naive TCRTAG cells are shown in grey as a control. e–g, Intracellular IFNγ and TNF production of TCRTAG cells isolated at days 7–10 and day 60 after transfer into AST×Cre-ERT2 mice after 4-h ex vivo peptide stimulation with antigen-presenting cells (APCs) (from B6 spleens) (e), or peptide stimulation with in vitro (f, top) or in vivo (f, bottom) LPS-activated splenocytes (f), or stimulation with PMA and ionomycin (g). LPS-mediated activation of APCs was confirmed by flow cytometric analysis assessing the upregulation of MHC-II, CD80, CD86 and CD40 on CD11c+ APCs, CD11b+ APCs and CD19+ B cells (splenocytes). Memory TCRTAG cells are shown as controls. Gates are set based on no-peptide controls. All FACS plots are gated on CD8+Thy1.1+ TCRTAG cells (experiments in f and g are repeated twice). These data are representative of more than ten independent experiments.

Extended Data Fig. 2 Antigen-specific CD8 T cell differentiation during acute and chronic viral LCMV infections, acute Listeria infection, and during tumorigenesis.

a, Top, experimental scheme for acute L. monocytogenes (expressing TAG epitope I) infection (green) and AST×Cre-ERT2 liver tumorigenesis after treatment with tamoxifen (red). Bottom, experimental scheme for acute (Armstrong; blue) and chronic (clone 13; orange) infection with LCMV. b, Expression profiles of TOX, PD-1, LAG-3 and TCF-1 at various time points after infection or tamoxifen treatment. Relative MFI values are shown normalized to naive transgenic TCRP14 T cells (specific for the LCMV epitope GP33) or naive TCRTAG T cells (dashed grey line). c, Top, flow cytometric analysis of TOX, TCF-1, PD-1, LAG-3, 2B4, TIM-3, CD39, TIGIT, CD38 and CTLA4 expression levels of TCRTAG T cells after Listeria infection (green) or tamoxifen treatment (red). Bottom, flow cytometric analysis of TOX, TCF-1, PD-1, LAG-3, 2B4, TIM-3 and CD39 expression levels of GP33-specific T cells at indicated time points after infections with LCMV Armstrong (blue) and LCMV clone 13 (orange). Naive T cells are shown in grey as a control. Data are mean ± s.d. and are representative of two independent experiments with n = 2 (Listeria) and n = 2–3 (AST×Cre-ERT2; LCMV Armstrong; LCMV clone 13) mice per time point.

Source Data

Extended Data Fig. 3 Phenotypic and functional characterization of TILs from mouse and human tumours.

a–c, TCRTRP2 (TRP2) and TCRPMEL (PMEL) TILs in mouse B16 melanoma tumours. a, TOX expression and TCF-1, PD-1, LAG-3, CD39, 2B4 and TIM-3 expression levels of TRP2 (Thy1.1+) TILs (red; top) and PMEL (Thy1.1+) TILs (red; bottom) isolated more than 15 days after adoptive transfer from established B16 melanoma tumours growing subcutaneously in B6 (Thy1.2+) mice. Naive CD8 T cells are shown in grey as a control. T cells are gated on CD8+Thy1.1+ cells. b, Intracellular IFNγ and TNF production of TRP2 and PMEL TILs after 4-h peptide stimulation ex vivo. c, Relative MFI values of TOX, TCF-1 and PD-1 of the indicated tumour models and TIL specificities shown on a log10 scale. Each symbol represents an individual mouse. Data are mean ± s.e.m of n = 2 (PMEL); n = 4 (TRP2); and n = 5 (TAG) mice, and are representative of two independent experiments. d–g, Phenotypic characterization and TOX expression profiles of human TILs and PBMCs isolated from patients with melanoma, lung, breast and ovarian cancer. d, Flow cytometric analysis of PBMCs and TILs of patients with breast cancer. TOX expression of TILs and matched PBMC CD8+ T cells. Gating strategy is shown. CD45RO+PD-1hiCD39hi (TILs; red), CD45RO+PD-1hi (PBMCs; blue), CD45RO+PD-1lo (PBMCs; green), and CD45RA+CD45RO− (naive PBMCs; grey). TOX isotypes are shown as controls for each sample. e, Top, TOX expression in human CD45RO+PD-1loCD39lo (dark blue) and CD45RO+PD-1hiCD39hi (red) TILs isolated from human primary melanoma. Isotypes are shown and data correspond to Fig. 1f. Bottom, TOX expression of TILs and matched PBMC CD8+ T cells from patients with melanoma. CD45RO+PD-1hi (TIL; red; n = 4), CD45RO+PD-1hi (PBMCs; blue, n = 4). TOX isotypes are shown as controls for each sample/patient. Bar plot shows MFI values for TOX. Each symbol represents an individual TIL and PBMC matched pair. f, TOX expression in human PD-1hi TILs isolated from human primary ovarian tumours. Flow plots are gated on CD8+CD45RO+PD-1hi T cells (red). CD8+CD45RO+ T cells from healthy donors are shown in grey. Gating strategy is shown. Each symbol represents a patient or healthy donor sample. g, TOX, CD39, TIM-3 and LAG-3 expression of CD8+CD45RO+PD-1hi (red) and CD8+CD45RO+PD-1lo (blue) TILs from human melanoma (n = 5), breast (n = 5) and lung (n = 6) tumours. Each symbol represents an individual matched PD-1hi/PD-1lo patient sample. Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-sided Student’s t-test. ns, not significant.

Source Data

Extended Data Fig. 4 Phenotypic, functional, transcriptional and epigenetic characterization of TCRTAG and TCROT1 cells in liver tumours.

a, Approximately 3 × 104 TCRTAG (TAG, red; Thy1.1+) and TCROT1 (OT1, black; Ly5.1+) T cells were transferred into wild-type B6 mice or liver tumour-bearing AST×Alb-Cre mice and immunized with 5 × 106 CFU of Listeria LmTAG-I-OVA. Three to four weeks after immunization, livers from AST×Alb-Cre mice and spleens from B6 mice were analysed for the presence of donor TAG and OT1 T cells by FACS; the percentages of CD8 T cells are shown. Expression of CD62L, CD44, CD69 and Ki67 of TAG and OT1 T cells. Naive T cells are shown in grey as a control. CD107 expression after 4-h TAG or OVA peptide stimulation of TAG and OT1 TILs isolated 3–4 weeks after transfer. Flow plots are gated on CD8+Thy1.1+ and CD8+Ly5.1+ cells. Data are representative of three independent experiments. b, Heat map of RNA-seq-normalized expression values (log2(counts per million)) across all samples (colour corresponds to z-scores) for genes differentially expressed between TAG and OT1 T cells (FDR < 0.05). c, GSEA of RNA-seq data generated from TAG and OT1 T cells isolated from AST×Cre liver lesions 3 weeks after adoptive transfer and Listeria infection. Gene sets used: T cell exhaustion during chronic viral infection20 (GEO accession GSE30962) and mutant/constitutively-active form of NFAT1-overexpressing CD8 T cells21. NES, normalized enrichment score. d, Venn diagrams showing the numbers and percentage of significantly opening (left) and closing (right) peaks between TAG and OT1 T cells (FDR < 0.05, log2-transformed fold change > 2). e, Genome browser view of ATAC-seq signal intensities of TAG and OT1 T cells at Pdcd1, Entpd1, Cd38 and Cd244 loci. Red or blue boxes indicate peaks that become significantly more accessible or inaccessible in TAG versus OT1 T cells, respectively. ATAC-seq peaks from naive TAG T cells are shown in grey as a control. f, Chromatin accessibility heat map for TAG and OT1 T cells. Each row represents one peak (differentially accessible between TAG and OT1 T cells; FDR < 0.05) displayed over a 2-kb window centred on the peak summit; regions were clustered using k-means clustering. Genes associated with individual clusters are highlighted.

Extended Data Fig. 5 Chromatin accessibility of the mouse and human Tox locus.

a, Accessibility of TOX and TCF7 loci in human tumour-infiltrating PD-1hiCD8+ T cells. ATAC-seq signal profiles of TOX (left) and TCF7 (right) in naive CD8+CD45RA+ (grey), CD8+CD45RO+CD62L+ central memory T cells (green) and CD8+CD45RO+PD-1hi TILs isolated from patients with melanoma and lung cancer (red). Red or blue boxes, respectively, indicate peaks that become accessible or inaccessible in PD-1hi TILs as compared to naive or memory T cells. Naive and memory T cells were isolated from PBMCs of healthy donors. b, c, NFAT1 binds to differentially accessible regions in the Tox locus in mice and pharmacological targeting of NFAT1 reduces TOX expression. b, Genome browser view of the Tox locus and numerous ATAC-seq and ChIP–seq tracks. On top, ATAC-seq signals of naive (N; grey), effector (E5, E7; green), memory (M; green), dysfunctional liver tumour-infiltrating TCRTAG cells (blue series, with D indicating the days after transfer when T cells were isolated from liver lesions) are shown. These data are from ref. 5. These are followed by newly generated ATAC-seq data from TCRTAG (TAG; orange) and TCROT1 (OT1; green) cells from AST×Cre liver lesions (as described in Fig. 2) as well as NFAT1 ChIP–seq tracks generated previously22 representing wild-type NFAT1 (blue) and mutant/constitutive active NFAT1-overexpressing T cells (red) (with and without stimulation). The vertical bars at the bottom of the plot represent statistically significantly enriched NFAT1-binding sites (peaks) as well as regions with statistically significantly changing accessibility between ATAC-seq of OT1 and TAG T cells. Red stars and pink boxes highlight NFAT1-binding sites that overlap with regions of increased chromatin accessibility in dysfunctional TCRTAG compared to TCROT1 cells. c, Pharmacological targeting of NFAT signalling decreases TOX expression in vivo. Naive TCRTAG (Thy1.1+) cells were transferred into AST×Cre-ERT2 (Thy1.2+) mice, which were treated with tamoxifen (Tam) 1 day later. At days 2–9, mice were treated with the calcineurin inhibitor FK506 (2.5 mg per kg per mouse; blue, n= 3) or PBS (control group; black, n = 3). At day 10, TCRTAG cells were isolated from livers and assessed for expression of CD44, TOX, PD-1 and TCF-1. Linear regression analysis of MFI values are shown. Naive TCRTAG cells are shown in grey as a control (n = 1). Each symbol represents an individual mouse. R2 = 0.6886 (TOX/TCF-1); R2 = 0.947 (TOX/PD-1); data are representative of two independent experiments. Dotted lines represent 95% confidence interval.

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Extended Data Fig. 6 Ectopic expression of TOX in T cells in vitro induces a molecular signature of T cell exhaustion.

a, Gating strategy for TOX–GFP-expressing (blue) and GFP-expressing (green) TCRTAG cells, and their corresponding TOX expression levels. TOX isotypes are shown for each sample. Naive TCRTAG cells adoptively transferred into AST×Cre mice and isolated from liver tumours after transfer (red), and naive TCRTAG cells (grey) are shown as controls. Inset numbers show MFI values. b, Heat map of RNA-seq expression values (row normalized log2(counts per million)) for genes differentially expressed between TOX–GFP and GFP TCRTAG cells (FDR < 0.10). c, Relative expression of selected genes as determined by digital droplet PCR. Data show raw droplet counts normalized to the housekeeping gene, Gapdh; n = 2 (TOX–GFP, GFP). d, Flow cytometric analysis of PD-1, 2B4, CD160, CD39 and TIM-3 expression levels of TOX–GFP (n = 3) or GFP (n = 3)-expressing TCRTAG cells. e, FACS analysis of TOX expression (left) on day 6 after spinfection of TCRTAG cells transduced with TOX–GFP (n = 2) or GFP (n = 2), and cytokine production (right) after 4-h peptide stimulation. f, Percentage of Ki67+ cells (top), and GZMB+ cells (with or without 4-h peptide stimulation) (bottom) in TCRTAG cells transduced with TOX–GFP (blue, n = 3) or GFP (green, n = 3). Naive TCRTAG cells are shown in grey as a control (n = 1). Data are mean ± s.e.m and representative of two independent experiments (n = 3 per experiment, with n representing a biological replicate/individual transduced spleen). *P ≤ 0.05, **P ≤ 0.01, two-sided Student’s t-test. g, GSEA of TCRTAG cells transduced with TOX–GFP or GFP. T cell exhaustion gene sets used: tumour-specific T cell dysfunction5 (left), and T cell exhaustion during chronic viral infection20 (GEO accession GSE30962) (right). Corresponding heat maps with selected genes with significant enrichment scores are shown below.

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Extended Data Fig. 7 Phenotypic and functional characterization of TOX wild-type and knockout TCRTAG mice.

a, Mouse strains generated and used in this study. We define wild type as littermate controls TCRTAG;dLck-Cre;Tox+/+ or TCRTAG;Toxfl/fl. We define knockout as TOX-deficient T cells from TCRTAG;dLck-Cre;Toxfl/fl mice. b, Thymocytes and peripheral CD8 T cells from knockout mice develop normally. CD4 and CD8 flow staining of thymocytes isolated from knockout (red, n = 5) or littermate controls (grey, n= 3). TCR Vβ7 and CD44 expression, and enumeration of single-positive CD8+ thymocytes from knockout and wild-type mice. c, Enumeration of total splenocytes (n = 5) and CD8+ splenocytes (n = 4) of knockout and wild-type mice. d, e, TOX is not required for effector and memory CD8 T cell differentiation during acute Listeria infection. d, Approximately 1 × 105 congenically marked naive wild-type and knockout TCRTAG T cells were adoptively transferred into B6 mice, and infected with Listeria 1 day later. Flow cytometric analysis of CD44, CD62L, CD127 and KLRG1 expression directly ex vivo (inset numbers show percentage in respective quadrants) of wild-type and knockout effector TCRTAG cells isolated from spleens of LmTAG-immunized B6 mice 7 days after immunization. e, Flow cytometric analysis of CD44, CD62L, CD127 and KLRG1 expression of wild-type and knockout memory TCRTAG cells isolated from spleens of LmTAG-immunized B6 mice 3 weeks after immunization. Right, intracellular IFNγ and TNF production after 4-h ex vivo TAG peptide stimulation of wild-type (n = 4) and knockout (n = 4) memory TCRTAG T cells. Flow plots are gated on CD8+Thy1.1+ cells. Data are representative of at least three independent experiments. f–i, Phenotypic and functional characterization of TOX wild-type and knockout TCRTAG cells differentiating in developing liver tumours of AST×Cre mice. f, Top, CD44, CD69, CD25 and PD-1 expression and CellTrace Violet (CTV) dilution of adoptively transferred, CTV-labelled naive wild-type (black) or knockout (red) TCRTAG cells isolated from livers of AST×Cre mice 3 days after transfer. Data are representative of three independent experiments. Middle, expression of CD44 and proliferation (CTV dilution) of wild-type (black) or knockout (red) TCRTAG cells isolated from AST×Cre liver lesions 5 days after transfer. CTV-labelled TCRTAG cells transferred into B6 control mice are shown in grey as controls transferred and isolated at the same time points. Bottom, PD-1 and LAG-3 expression together with TOX expression of wild-type and knockout TCRTAG cells isolated from the livers of AST×Cre mice 8 days after transfer. All FACS plots are gated on CD8+ and Thy1.1+. g, Flow cytometric analysis of intracellular IFNγ and TNF production (top), CD107 degranulation (middle), and GZMB expression (bottom) of day 7–10 wild-type (black) or knockout (red) TCRTAG cells after 4-h peptide stimulation. h, i, PMA and ionomycin stimulation (h) or 4-h peptide stimulation using in vivo LPS-activated APCs (i). Each sample is gated on its respective no-peptide control. All flow plots are gated on CD8+Thy1.1+ T cells. Data are representative of three independent experiment and shown as mean ± s.e.m. P values determined by two sided Student’s t-test.

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Extended Data Fig. 8 TOX wild-type and knockout TCRTAG cells reveal differences in genes and proteins associated with apoptosis.

a, Flow cytometric analysis of PD-1 (n = 3 (KO); n = 5 (WT)), LAG-3 (n = 4 (KO); n = 5 (WT)), CD38 (n = 4 (KO); n = 5 (WT)), 2B4 (n = 2 (KO); n = 3 (WT)), and TCF-1 (n = 4 (KO); n = 5 (WT)), expression levels in wild-type (black) or knockout (red) TCRTAG cells isolated from liver lesions approximately 3 weeks after adoptive transfer. Data are representative of at least three independent experiments. b, Flow cytometric analysis of TOX wild-type (black) and knockout (red) TCRTAG cells isolated 7–10 days after transfer from AST×Cre liver lesions. BIM, BCL-2 and BCL-xL expression levels were assessed directly ex vivo. Each pair of symbols represents an individual mouse (n = 4). Data are representative of two independent experiments. c, Flow cytometric analysis of active caspases 3 and 7 in TOX wild-type (black) and knockout (red) TCRTAG cells. These data are combined results of two experiments (n = 11). Each pair of symbols represents an individual mouse. d, Representative histograms and quantification of annexin V+ wild-type (black, n = 3) and knockout (red, n = 3) TCRTAG cells isolated 7–10 days after transfer from AST×Cre liver lesions. e, GSEA of DEGs between TOX wild-type and knockout T cells. ‘Hallmark_apoptosis’ and ‘wikipathways_MM_apoptosis_WP254’ gene sets show normalized enrichment score (NES) of −1.52 and −1.1, respectively, and the corresponding heat maps of genes with significant enrichment scores are shown. Data are mean ± s.e.m. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-sided Student’s t-test.

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Extended Data Fig. 9 TOX wild-type and knockout TCRTAG cells reveal transcriptional and chromatin accessibility changes.

a, Heat map of RNA-seq expression (row normalized log2(counts per million)) for genes differentially expressed between TOX wild-type and knockout TCRTAG cells (FDR < 0.05). b, GSEA between wild-type and knockout TCRTAG. T cell exhaustion gene sets used: tumour-specific T cell dysfunction5 (left) and T cell exhaustion during chronic viral infection20 (GEO accession GSE30962) (right). Selected genes with significant enrichment score are listed. c, Pie chart showing the proportions of reproducible ATAC-seq peaks in indicated regions for all peaks within the atlas. d, Venn diagrams showing the numbers and percentages of significantly opening (top) and closing (bottom) peaks between TOX wild-type and knockout TCRTAG cells (FDR < 0.05, log2-transformed fold change > 2). e, Gains and losses of regulatory elements for the top 100 most DEGs between TOX wild-type and knockout TCRTAG cells that were part of the gene set of tumour-specific T cell dysfunction5. The plot is divided into top and bottom 50 genes with the highest and lowest respective log2-transformed fold change of gene expression. Each gene is illustrated by a stack of diamonds, in which each diamond represents a region of high chromatin accessibility (peak) overlapping with the locus of the respective gene. Red diamonds denote peaks that are more accessible in wild-type (and less accessible in TOX KO) T cells; blue diamonds denote peaks that are more accessible in TOX knockout T cells. f, Molecular function (GO terms) enriched in genes associated with peaks that are more accessible in TOX knockout versus wild-type T cells. g, ATAC-seq signal profiles across the Pdcd1 and Entpd1 loci. Peaks less accessible in knockout TCRTAG cells are highlighted in red.

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Extended Data Fig. 10 Comparison of functional TOXlow OT1 and dysfunctional TOX knockout T cells in tumours with proposed model on the role of TOX in tumour-specific CD8 T cell exhaustion and dysfunction.

a, DEGs of the TAG versus OT1 comparison (see Fig. 2) were compared with DEGs of the wild-type versus TOX-knockout comparison (see Fig. 4). There were 389 genes identified to be significantly differentially expressed in both (WT vs KO and TAG vs OT1). b, Heat map of normalized expression values (log2(counts per million)) across all samples (colour corresponds to z-scores) for these 389 genes. Selected genes of interest are highlighted. c, Proposed model on the role of TOX in tumour-specific CD8 T cell exhaustion and dysfunction. Top, antigen-specific T cells in solid tumours are continuously triggered with tumour antigen. Chronic TCR stimulation leads to NFAT-mediated expression of TOX. TOX induces a transcriptional and epigenetic program and phenotype associated with exhaustion, including the expression of numerous inhibitory receptors (for example, PD-1, LAG-3, 2B4, CD39 and CD38) and downregulation of transcription factors (such as TCF-1). The TOX-mediated exhaustion program prevents T cells from overactivation or overstimulation and activation-induced cell death. Bottom, TOX-deficient T cells do not upregulate inhibitory receptors, become overstimulated or overactivated, and eventually undergo activation-induced cell death. Despite their non-exhausted phenotype, TOX-deficient T cells are dysfunctional.

Supplementary information

Reporting Summary

Supplementary Table 1

Summary of differentially expressed genes by RNA-seq.

Supplementary Table 2

Summary of differentially accessible peaks by ATAC-seq.

Supplementary Table 3

Summary of differentially expressed genes by RNA-seq.

Supplementary Table 4

Summary of NFAT1 ChIP-seq samples from Martinez et al. (GSE64407) used for analysis.

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Scott, A.C., Dündar, F., Zumbo, P. et al. TOX is a critical regulator of tumour-specific T cell differentiation. Nature 571, 270–274 (2019). https://doi.org/10.1038/s41586-019-1324-y

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