TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion

Abstract

Exhausted CD8+ T (Tex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (Teff) or memory (Tmem) CD8+ T cells. Tex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, Tex cells are a distinct immune subset, with a unique chromatin landscape compared with Teff and Tmem cells. However, the mechanisms that govern the transcriptional and epigenetic development of Tex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of Tex cells in mice. TOX is largely dispensable for the formation of Teff and Tmem cells, but it is critical for exhaustion: in the absence of TOX, Tex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in Tex cells. Robust expression of TOX therefore results in commitment to Tex cells by translating persistent stimulation into a distinct Tex cell transcriptional and epigenetic developmental program.

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Fig. 1: Multiple epigenetic modulators, including TOX, are selectively expressed in Tex cells.
Fig. 2: Rapid and sustained TOX expression is associated with key features of exhaustion.
Fig. 3: TOX is required for the development of Tex cells.
Fig. 4: Calcineurin signalling and NFAT2 are necessary and sufficient to induce TOX, but sustained expression becomes calcineurin-independent.
Fig. 5: TOX enforces a Tex cell transcriptional program.
Fig. 6: TOX induces an epigenetic signature of exhaustion in vitro and in vivo.

Data availability

RNA-seq and ATAC–seq data have been deposited in the NCBI Gene Expression Omnibus (GEO) database and are accessible through the GEO SuperSeries accession number: GSE131871. All other relevant data are available from the corresponding author upon reasonable request.

Code availability

Custom code used for RNA-seq and ATAC–seq analyses are available at the GitHub links provided above.

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Acknowledgements

We thank all members of the Wherry laboratory for helpful discussions and critical analysis of the manuscript; J. Kaye for providing the Toxflox/flox and Tox−/− mice; P. M. Porrett for providing FK506; D. Zehn and A. Schietinger for helpful discussions; and H.-Y. Tang and T. Beer of the Wistar Institute Proteomics and Metabolomics Facility for assistance with the analysis of the proteomics data. Support for the Wistar Proteomics and Metabolomics Core Facility was provided by Cancer Center Support Grant CA010815 to the Wistar Institute. Clinical sample acquisition was supported by NIH grant P50CA174523-02, the Wistar Institute, and the Tara Miller Foundation. O.K. was supported by an NIAID F30 fellowship (F30AI129263). This work was funded by the National Institutes of Health (AI105343, AI082630, AI115712, CA210944, AI117950 and AI108545) and the Parker Institute for Cancer Immunotherapy.

Author information

O.K. and E.J.W. conceived the project, designed experiments and wrote the manuscript. O.K. performed the majority of the experiments described herein and performed the in vitro, in vivo and bioinformatics analysis. J.R.G. wrote the script to perform PSEA and, with S. Manne, performed pre-processing of RNA and ATAC–seq data. S. McDonald performed co-immunoprecipitation experiments for western blots. S.F.N. and K.P.P. contributed to in vivo tumour and influenza experiments. M.T.W. performed immunoprecipitation experiments for mass spectrometry. A.C.H., P.Y. and S.M.G. acquired and stained samples of human peripheral blood mononuclear cells and tumour-infiltrating CD8+ T cells. J.E.W., R.P.S., J.R.G. and K.A.A. provided critical edits to the manuscript. W.X., R.K.A., X.X., G.C.K., T.C.M. and L.M.S. provided clinical samples. J.K. provided mice and intellectual input. All authors reviewed the manuscript.

Correspondence to E. John Wherry.

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Competing interests

O.K. is an employee of Arsenal Biosciences. R.K.A. serves as a consultant for Sprint Biosciences, Immunacell and Array Pharmaceuticals and is a founder of Pinpoint Therapeutics. T.C.M. is an advisor to and/or receives honoraria from Aduro, Array, BMS, Incyte, Merck, and Regeneron. S.L.B. receives research funding from Celgene. E.J.W. receives honoraria, consulting fees and/or research support from BMS, Celgene, Dynavax, Eli Lilly, Elstar, Merck, MedImmune, Pieris, Roche, Surface Oncology, and KyMab. E.J.W. is a founder of Arsenal Biosciences. E.J.W. has a patent licensing agreement for the PD-1 pathway.

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Extended data figures and tables

Extended Data Fig. 1 Expression of epigenetic modifiers in acute and chronic LCMV infection.

a, Data points indicate the z-score of each gene in clusters 1–5 plotted against time post-infection with Armstrong or clone 13. Grey and blue lines represent the moving average of z-score (with shading indicating the 95% confidence interval) in P14 T cells from Armstrong and clone-13 infection, respectively. b, Expression of selected genes within cluster 1 plotted as normalized array intensity against time post-infection. Grey and blue shading represent P14 T cells from infection with Armstrong and clone 13, respectively. c, Distribution of the ATAC–seq signal across loci in naive T, Teff, Tmem and Tex P14 T cells. Loci above the horizontal dashed lines denote putative super enhancers. The rank of the Tox locus among all identified potential super enhancers is shown.

Extended Data Fig. 2 Dynamics of TOX expression in mouse and human disease.

a, TOX expression in P14 T cells from peripheral blood at day 208 post-infection with Armstrong or clone 13. b, Top, Teff and Tmem cell markers relative to TOX expression in P14 T cells or endogenous CD8+ T cells on day 6 post-infection with clone 13. Bottom left, frequency of Tmem-cell and Teff-cell subsets within TOX+ and TOX P14 T cell populations. Bottom right, TOX median fluorescence intensity in KLRG1+ and KRLG1 P14 T cells. c, TOX versus transcription-factor expression after 8 (top) or 30 (bottom) days of clone-13 infection. d, e, TOX versus inhibitory-receptor expression in P14 T cells after 8 days (d) or 30 days (e) of clone-13 infection. f, TOX expression in antigen-specific CD8+ T cells after influenza, VSV or Listeria monocytogenes infection compared with LCMV Armstrong or clone-13 infection. g, TOX versus PD-1 and quantification of TOX expression in activated CD8+CD44+ T cells from control tissues or tumours. Control T cells for mouse tumour models were acquired from the spleen, whereas in humans, T cells from the peripheral blood of normal donors served as controls. h, Radar plots of median gene expression in single-cell RNA-sequencing data from tumour biopsies and peripheral blood of patients with non-small-cell lung cancer (NSCLC) or hepatocellular carcinoma (HCC)61,62. Median expression was calculated on cell clusters that were defined by key driver genes and represent canonical T cell populations61,62. i, Top, P14 T cell infiltration in GP33-expressing B16 tumours. Bottom, cytokine production in TOX+ or TOX tumour-infiltrating P14 T cells. Contour and histogram plots are from one representative experiment of at least 2 independent experiments consisting of at least 4 mice per group. Unless otherwise noted, P14 T cells were analysed from the spleens of infected mice. In the summarized experiments, each data point represents one mouse and the error is reported as s.d. For e, five human melanoma biopsy samples were analysed. Statistical significance (*P < 0.01) was determined using the Student’s t-test.

Extended Data Fig. 3 Response of TOX-deficient T cells in acute and chronic LCMV.

a, The gating strategy used in co-adoptive transfer and infection experiments. b, Expression of activation markers and transcription factors in naive wild-type and Toxflox/floxCd4cre P14 T cells before adoptive transfer. Wild-type and TOX cKO T cells were mixed 1:1 and adoptively transferred into congenic wild-type mice followed by infection with Armstrong (c, d, fk) or clone 13 (ce). c, Frequency of wild-type or TOX cKO P14 T cells during infection with Armstrong or clone 13. d, TOX expression in wild-type and TOX cKO P14 T cells after infection with Armstrong or clone 13. e, Ki-67 expression on day 8 of clone-13 infection. f, g, Frequency of memory populations on day 8 (f) or day 30 (g) of Armstrong infection. h, Transcription-factor expression in wild-type and TOX cKO P14 T cells on day 30 post-infection with Armstrong. ik, Cytokine and effector molecule (i), inhibitory-receptor (j), and transcription-factor (k) expression on day 8 post-infection with Armstrong. Inhibitory-receptor expression is reported as the ratio of the median fluorescent intensity between TOX cKO and wild-type P14 T cells (j, right). l, GSEA of transcriptional signatures associated with naive T or Tmem cells compared to the differentially expressed genes in Tox−/− versus wild-type P14 T cells. m, Expression of genes associated with the terminal short-lived subset of Teff cells26. n, Comparison of the transcriptional signature of TOX cKO and TCF-1 cKO30 T cells after 8 days of clone-13 infection. Genes differentially expressed relative to wild-type (FDR < 0.05 and log-fold change > 0.6) were compared between datasets. Contour and histogram plots are representative of at least 4 independent experiments with at least 4 mice. Statistical significance (*P < 0.01) was determined by a pairwise t-test with Holm–Sidak correction (c) or the Student’s t-test (el), error is reported as s.d.

Extended Data Fig. 4 Effect of calcium and NFAT2 perturbation on TOX expression.

a, Normalized microarray expression of Nfatc1 (which encodes NFAT2) and Nfatc2 (which encodes NFAT1) in P14 T cells after infection with Armstrong or clone 13. b, CD8+ T cells were enriched, activated and transduced with control (CT), wild-type NFAT2 or CA-NFAT2 encoding retroviruses. T cells were expanded and differentiated in vitro in the presence of IL-2 for 6 days before analysis. c, Expression of activation markers and transcription factors in naive wild-type and Nfatc1flox/floxCd4cre (NFAT2 cKO) P14 T cells from the blood before adoptive transfer. d, P14 T cells were adoptively transferred into wild-type hosts followed by infection with clone 13. Top, on day 3–7 of infection, mice were treated with PBS or FK506 and splenocytes were collected on day 8 post-infection. Bottom, CD44 expression in P14 T cells on day 8 post-infection with clone 13 and treatment with PBS or FK506 on day 3–7. e, NFAT2 cKO CD8+ T cells were enriched from naive mice, activated with antibodies against CD3 and CD28 and transduced with retroviruses encoding TOX or GFP-only control. Twenty-four hours later, cells were sorted and transferred into clone-13-infected mice. Protein expression was analysed on day 8 post-infection. f, P14 T cells were transferred into wild-type mice followed by infection with clone 13. On day 25–29 post-infection, recipient mice were treated with PBS, FK506 or cyclosporin A (CsA) and splenocytes were collected on day 30 post-infection for analysis. g, Protein expression in P14 T cells after treatment with cyclosporin A or PBS on day 25–29 of clone-13 infection. All contour and histogram plots are representative of at least 3 independent experiments consisting of at least 3 mice per group. Error is reported as s.d.

Extended Data Fig. 5 Enforced expression of TOX in T cells and fibroblasts.

ad, Naive P14 T cells were activated with antibodies against CD3 and CD28 for 24 h before transduction with retroviruses encoding TOX (TOXOE) or control GFP. Twenty-four hours after transduction, GFP+ cells were sorted and transferred into day-2 Armstrong-infected recipients. Eight days after transfer, transduced P14 T cells were isolated from spleens and assayed for KLRG1+ Teff cell frequency (a), inhibitory-receptor expression (b), cytokine production after 5 h of restimulation with GP33 peptide (c) and transcription-factor expression (d). e, f, Distribution of memory T cell subsets and PD-1 expression in TOX- versus control-transduced P14 T cells after 30 days of Armstrong infection. g, Genes upregulated (blue) or downregulated (grey) in TOXOE compared with control cells were analysed for enrichment in the transcripts that were differentially expressed in P14 T cells on days 8, 15 and 30 of infection with clone 13 or Armstrong12. Normalized GSEA enrichment scores are plotted against time post-infection. h, The experimental procedure used to generate the datasets analysed in i, j and Fig. 5e, f. NIH3T3 cells were transduced with retroviruses encoding TOX + GFP (TOXOE) or control GFP-only. Cells were cultured for 48 h, then collected and processed for RNA-seq analysis. i, Gene ontology analysis of biological processes differentially regulated in TOXOE versus control fibroblasts. j, As in g, genes upregulated (blue) or downregulated (grey) in fibroblasts were assayed for enrichment in the genes differentially expressed in P14 T cells on days 6, 8, 15 and 30 of infection with clone 13 or Armstrong12. All contour and histogram plots are representative of at least two independent experiments consisting of at least five mice per group. Unless otherwise noted, P14 T cells were analysed from the spleens of infected mice. Statistical significance (*P < 0.01) was determined using the Student’s t-test, error is reported as s.d.

Extended Data Fig. 6 Epigenetic program regulated by TOX.

a, Left, location of differentially accessible ATAC–seq peaks from Fig. 6a (top) or Fig. 6f (bottom). Right, distribution of all peaks in CD8+ T cells that are above background levels. b, c, ATAC–seq and RNA-seq tracks of Teff-cell-associated (b) or Tmem-cell-associated (c) loci. Peaks uniquely opened (b) or closed (c) in Tox−/− relative to wild-type T cells are highlighted with grey bars. d, Enumeration of significantly differentially accessible sites (FDR < 0.05) in wild-type and Tox−/− T cells at Tex-cell-specific and Teff-cell-specific loci13. e, PSEA of chromatin regions specifically accessible in naive T, Teff, Tmem cells13 in Tox−/− compared with wild-type P14 T cells. f, Fold change in ATAC accessibility versus RNA expression. Key genes for Tex and Teff cells are highlighted and genes associated with multiple peaks are connected with a red line. Inset, a table enumerating the number of gene–ATAC peak pairs in each quadrant. g, PSEA of chromatin regions specifically accessible in naive T, Teff, Tmem cells in TOXOE compared with control P14 T cells. h, ATAC–seq tracks of naive T, Teff, Tmem and Tex cells13 compared with control and TOXOE T cells at the Pdcd1 locus. The grey bar highlights the Tex-cell-specific −23.8-kb enhancer. i, Abundance, specificity and reproducibility plot of proteins identified by mass spectrometry after TOX immunoprecipitation compared with IgG control in EL4 cells. Hits are coloured using the MiST score (blue signifies >0.75). j, Gene ontology biological process enrichment of TOX-bound proteins identified in i with MiST score >0.75.

Supplementary information

Reporting Summary

Supplementary Table 1

Gene-cluster associations.

Supplementary Table 2

RNA-Seq of Tox-/- vs. WT P14 following 8 days of Cl-13 infection.

Supplementary Table 3

RNA-Seq of Tox vs. control GFP transduced in vitro activated CD8+ T cells.

Supplementary Table 4

RNA-Seq of Tox vs. control GFP RV transduced NIH3T3 fibroblasts.

Supplementary Table 5

ATAC-Seq of Tox-/- vs. WT P14 following 8 days of Cl-13 infection.

Supplementary Table 6

ATAC-Seq of Tox vs. control GFP RV transduced in vitro activated CD8+ T cells.

Supplementary Table 7

MiST analysis following Tox immunoprecipitation and mass spectrometry in EL4 cells.

Supplementary Table 8

Epigenetic-modulating genes identified from GO, EpiFactors, and Shi et al. Nature Biotechnology 2015 (see methods for more detail).

Supplementary Table 9

Sequencing and alignment statistics for RNA-Seq and ATAC-Seq experiments.

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