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.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Custom code used for RNA-seq and ATAC–seq analyses are available at the GitHub links provided above.
Kaech, S. M. & Cui, W. Transcriptional control of effector and memory CD8+ T cell differentiation. Nat. Rev. Immunol. 12, 749–761 (2012).
Wherry, E. J. & Kurachi, M. Molecular and cellular insights into T cell exhaustion. Nat. Rev. Immunol. 15, 486–499 (2015).
Barber, D. L. et al. Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439, 682–687 (2006).
Frebel, H. et al. Programmed death 1 protects from fatal circulatory failure during systemic virus infection of mice. J. Exp. Med. 209, 2485–2499 (2012).
Zajac, A. J. et al. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188, 2205–2213 (1998).
Gallimore, A. et al. Induction and exhaustion of lymphocytic choriomeningitis virus-specific cytotoxic T lymphocytes visualized using soluble tetrameric major histocompatibility complex class I-peptide complexes. J. Exp. Med. 187, 1383–1393 (1998).
Lechner, F. et al. Analysis of successful immune responses in persons infected with hepatitis C virus. J. Exp. Med. 191, 1499–1512 (2000).
Shankar, P. et al. Impaired function of circulating HIV-specific CD8+ T cells in chronic human immunodeficiency virus infection. Blood 96, 3094–3101 (2000).
Pauken, K. E. & Wherry, E. J. Overcoming T cell exhaustion in infection and cancer. Trends Immunol. 36, 265–276 (2015).
Page, D. B., Postow, M. A., Callahan, M. K., Allison, J. P. & Wolchok, J. D. Immune modulation in cancer with antibodies. Annu. Rev. Med. 65, 185–202 (2014).
Hirano, F. et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity. Cancer Res. 65, 1089–1096 (2005).
Doering, T. A. et al. Network analysis reveals centrally connected genes and pathways involved in CD8+ T cell exhaustion versus memory. Immunity 37, 1130–1144 (2012).
Pauken, K. E. et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 354, 1160–1165 (2016).
Sen, D. R. et al. The epigenetic landscape of T cell exhaustion. Science 354, 1165–1169 (2016).
Scott-Browne, J. P. et al. Dynamic changes in chromatin accessibility occur in CD8+ T cells responding to viral infection. Immunity 45, 1327–1340 (2016).
Philip, M. et al. Chromatin states define tumour-specific T cell dysfunction and reprogramming. Nature 545, 452–456 (2017).
Lara-Astiaso, D. et al. Chromatin state dynamics during blood formation. Science 345, 943–949 (2014).
Carty, S. A. et al. The loss of TET2 promotes CD8+ T cell memory differentiation. J. Immunol. 200, 82–91 (2018).
Ghoneim, H. E. et al. De novo epigenetic programs inhibit PD-1 blockade-mediated T cell rejuvenation. Cell 170, 142–157 (2017).
Aliahmad, P., Seksenyan, A. & Kaye, J. The many roles of TOX in the immune system. Curr. Opin. Immunol. 24, 173–177 (2012).
Seehus, C. R. et al. The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor. Nat. Immunol. 16, 599–608 (2015).
Whyte, W. A. et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).
Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).
Wherry, E. J., Blattman, J. N., Murali-Krishna, K., van der Most, R. & Ahmed, R. Viral persistence alters CD8 T-cell immunodominance and tissue distribution and results in distinct stages of functional impairment. J. Virol. 77, 4911–4927 (2003).
Joshi, N. S. et al. Inflammation directs memory precursor and short-lived effector CD8+ T cell fates via the graded expression of T-bet transcription factor. Immunity 27, 281–295 (2007).
Herndler-Brandstetter, D. et al. KLRG1+ effector CD8+ T cells lose KLRG1, differentiate into all memory T cell lineages, and convey enhanced protective immunity. Immunity 48, 716–729 (2018).
Angelosanto, J. M., Blackburn, S. D., Crawford, A. & Wherry, E. J. Progressive loss of memory T cell potential and commitment to exhaustion during chronic viral infection. J. Virol. 86, 8161–8170 (2012).
Paley, M. A. et al. Progenitor and terminal subsets of CD8+ T cells cooperate to contain chronic viral infection. Science 338, 1220–1225 (2012).
Utzschneider, D. T. et al. T cell factor 1-expressing memory-like CD8+ T cells sustain the immune response to chronic viral infections. Immunity 45, 415–427 (2016).
Wu, T. et al. The TCF1–Bcl6 axis counteracts type I interferon to repress exhaustion and maintain T cell stemness. Sci. Immunol. 1, eaai8593 (2016).
Aliahmad, P. et al. TOX provides a link between calcineurin activation and CD8 lineage commitment. J. Exp. Med. 199, 1089–1099 (2004).
Macian, F. NFAT proteins: key regulators of T-cell development and function. Nat. Rev. Immunol. 5, 472–484 (2005).
Martinez, G. J. et al. The transcription factor NFAT promotes exhaustion of activated CD8+ T cells. Immunity 42, 265–278 (2015).
Klein-Hessling, S. et al. NFATc1 controls the cytotoxicity of CD8+ T cells. Nat. Commun. 8, 511 (2017).
Monticelli, S. & Rao, A. NFAT1 and NFAT2 are positive regulators of IL-4 gene transcription. Eur. J. Immunol. 32, 2971–2978 (2002).
Bengsch, B. et al. Epigenomic-guided mass cytometry profiling reveals disease-specific features of exhausted CD8 T cells. Immunity 48, 1029–1045 (2018).
Johnson, J. L. et al. Lineage-determining transcription factor TCF-1 initiates the epigenetic identity of T cells. Immunity 48, 243–257 (2018).
Lalonde, M. E. et al. Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity. Genes Dev. 27, 2009–2024 (2013).
Miotto, B. & Struhl, K. HBO1 histone acetylase activity is essential for DNA replication licensing and inhibited by Geminin. Mol. Cell 37, 57–66 (2010).
Yu, B. et al. Epigenetic landscapes reveal transcription factors that regulate CD8+ T cell differentiation. Nat. Immunol. 18, 573–582 (2017).
Boiani, M. & Schöler, H. R. Regulatory networks in embryo-derived pluripotent stem cells. Nat. Rev. Mol. Cell Biol. 6, 872–881 (2005).
Iwai, Y., Terawaki, S. & Honjo, T. PD-1 blockade inhibits hematogenous spread of poorly immunogenic tumor cells by enhanced recruitment of effector T cells. Int. Immunol. 17, 133–144 (2005).
Strome, S. E. et al. B7-H1 blockade augments adoptive T-cell immunotherapy for squamous cell carcinoma. Cancer Res. 63, 6501–6505 (2003).
Blank, C. et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T cell receptor (TCR) transgenic CD8+ T cells. Cancer Res. 64, 1140–1145 (2004).
Anderson, K. L. et al. Transcription factor PU.1 is necessary for development of thymic and myeloid progenitor-derived dendritic cells. J. Immunol. 164, 1855–1861 (2000).
Fontenot, J. D., Gavin, M. A. & Rudensky, A. Y. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat. Immunol. 4, 330–336 (2003).
Hori, S., Nomura, T. & Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science 299, 1057–1061 (2003).
Weber, B. N. et al. A critical role for TCF-1 in T-lineage specification and differentiation. Nature 476, 63–68 (2011).
Aliahmad, P. & Kaye, J. Development of all CD4 T lineages requires nuclear factor TOX. J. Exp. Med. 205, 245–256 (2008).
Blattman, J. N., Wherry, E. J., Ha, S. J., van der Most, R. G. & Ahmed, R. Impact of epitope escape on PD-1 expression and CD8 T-cell exhaustion during chronic infection. J. Virol. 83, 4386–4394 (2009).
Odorizzi, P. M., Pauken, K. E., Paley, M. A., Sharpe, A. & Wherry, E. J. Genetic absence of PD-1 promotes accumulation of terminally differentiated exhausted CD8+ T cells. J. Exp. Med. 212, 1125–1137 (2015).
Araki, K. et al. Pathogenic virus-specific T cells cause disease during treatment with the calcineurin inhibitor FK506: implications for transplantation. J. Exp. Med. 207, 2355–2367 (2010).
Kurachi, M. et al. Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function. Nat. Protoc. 12, 1980–1998 (2017).
Huang, A. C. et al. T-cell invigoration to tumour burden ratio associated with anti-PD-1 response. Nature 545, 60–65 (2017).
Medvedeva, Y. A. et al. EpiFactors: a comprehensive database of human epigenetic factors and complexes. Database (Oxford) 2015, bav067 (2015).
Shi, J. et al. Discovery of cancer drug targets by CRISPR–Cas9 screening of protein domains. Nat. Biotechnol. 33, 661–667 (2015).
Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).
Dou, Z. et al. Autophagy mediates degradation of nuclear lamina. Nature 527, 105–109 (2015).
Dawson, M. A. et al. Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature 478, 529–533 (2011).
Jäger, S. et al. Global landscape of HIV–human protein complexes. Nature 481, 365–370 (2012).
Guo, X. et al. Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing. Nat. Med. 24, 978–985 (2018).
Zheng, C. et al. Landscape of infiltrating T cells in liver cancer revealed by single-cell sequencing. Cell 169, 1342–1356 (2017).
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.
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.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
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.
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.
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, f–k) or clone 13 (c–e). 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. i–k, 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 (e–l), error is reported as s.d.
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.
a–d, 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.
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.
RNA-Seq of Tox-/- vs. WT P14 following 8 days of Cl-13 infection.
RNA-Seq of Tox vs. control GFP transduced in vitro activated CD8+ T cells.
RNA-Seq of Tox vs. control GFP RV transduced NIH3T3 fibroblasts.
ATAC-Seq of Tox-/- vs. WT P14 following 8 days of Cl-13 infection.
ATAC-Seq of Tox vs. control GFP RV transduced in vitro activated CD8+ T cells.
MiST analysis following Tox immunoprecipitation and mass spectrometry in EL4 cells.
Epigenetic-modulating genes identified from GO, EpiFactors, and Shi et al. Nature Biotechnology 2015 (see methods for more detail).
Sequencing and alignment statistics for RNA-Seq and ATAC-Seq experiments.
About this article
Striking a Balance—Cellular and Molecular Drivers of Memory T Cell Development and Responses to Chronic Stimulation
Frontiers in Immunology (2019)
Nature Reviews Immunology (2019)
Nature Immunology (2019)