, For the detection of protein aggregates, after overnight PBMC stimulation with or without coated anti-CD3

, NY) was used according to the manufacturer's protocol, before flow cytometry acquisition 25 . Cells treated with 5 ?M of proteasome inhibitor (MG-132) served as positive controls. Aggresome levels were quantified by subtracting ProteoStat median fluorescence intensity (MFI) in the unstimulated from the stimulated samples, Data were collected with a FACSCANTO II multicolor flow cytometer and analyzed with DIVA (BD Biosciences) and FlowJo (Tree Star) softwares

, At the end of the culture period, cytokine determination (IFN-?, TNF-? and IL-2) was performed by intracellular cytokine staining 23 . Specifically, brefeldin-A (10 ?g/ml; Sigma-Aldrich) was added to the cells and left throughout the time of stimulation for ex-vivo assays and for the last 4 h of the 10-days cultures. After washing, cells were stained with anti-CD8, anti-CD4 and anti-CD3, fixed and permeabilized (FIX&PERM Cell Fixation and Permeabilization Kit

, Sigma), the p53 inhibitor Pifithrin-? (tested at concentrations of 10-30 ?M, Sigma), the p38 inhibitor SB203580 (tested at concentrations of 0.01-0.1 ?M, Sigma), the AMPK inhibitor Dorsomorphin, The following compounds were used to restore intracellular signaling, metabolic functions and anti-viral activities: the kinase ATM inhibitor KU-55933

D. G. Bowen and C. M. Walker, Adaptive immune responses in acute and chronic hepatitis C virus infection, Nature, vol.436, pp.946-952, 2005.

B. Rehermann, Pathogenesis of chronic viral hepatitis: differential roles of T cells and NK cells, Nat. Med, vol.19, pp.859-868, 2013.

S. N. Mueller and R. Ahmed, High antigen levels are the cause of T cell exhaustion during chronic viral infection, Proc. Natl Acad. Sci. USA, vol.106, pp.8623-8628, 2009.

S. D. Blackburn, Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection, Nat. Immunol, vol.10, pp.29-37, 2009.

K. E. Pauken and E. J. Wherry, Overcoming T cell exhaustion in infection and cancer, Trends Immunol, vol.36, pp.265-276, 2015.

E. F. Mckinney and K. G. Smith, Metabolic exhaustion in infection, cancer and autoimmunity, Nat. Immunol, vol.19, pp.213-221, 2018.

D. R. Sen, The epigenetic landscape of T cell exhaustion, Science, vol.354, pp.1165-1169, 2016.

C. A. Klebanoff, L. Gattinoni, and N. P. Restifo, CD8+ T-cell memory in tumor immunology and immunotherapy, Immunol. Rev, vol.211, pp.214-224, 2006.

N. Chihara, Induction and transcriptional regulation of the co-inhibitory gene module in T cells, Nature, vol.558, pp.454-459, 2018.

D. L. Barber, Restoring function in exhausted CD8 T cells during chronic viral infection, Nature, vol.439, pp.682-687, 2006.

A. Penna, Dysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection, Hepatology, vol.45, pp.588-601, 2007.

D. Wieland, TCF1+ hepatitis C virus-specific CD8+ T cells are maintained after cessation of chronic antigen stimulation, Nat. Commun, vol.8, p.15050, 2017.

K. E. Pauken, Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade, Science, vol.354, pp.1160-1165, 2016.

N. Patsoukis, PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation, Nat. Commun, vol.6, p.6692, 2015.

C. H. Chang and E. L. Pearce, Emerging concepts of T cell metabolism as a target of immunotherapy, Nat. Immunol, vol.17, pp.364-368, 2016.

B. Bengsch, Epigenomic-guided mass cytometry profiling reveals disease-specific features of exhausted CD8 T cells, Immunity, vol.48, pp.1029-1045, 2018.

R. Wang and D. R. Green, Metabolic checkpoints in activated T cells, Nat. Immunol, vol.13, pp.907-915, 2012.

C. Chang, Posttranscriptional control of T cell effector function by aerobic glycolysis, Cell, vol.153, pp.1239-1251, 2013.

A. T. Phan, Constitutive glycolytic metabolism supports CD8+ T cell effector memory differentiation during viral infection, Immunity, vol.45, pp.1024-1037, 2016.

P. M. Gubser, Rapid effector function of memory CD8+T cells requires an immediate-early glycolytic switch, Nat. Immunol, vol.14, pp.1064-1072, 2013.

E. L. Pearce, Enhancing CD8 T-cell memory by modulating fatty acid metabolism, Nature, vol.460, pp.103-107, 2009.

G. J. Van-der-windt, Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development, Immunity, vol.36, pp.68-78, 2012.

P. Fisicaro, Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B, Nat. Med, vol.23, pp.327-336, 2017.

A. Schurich, Distinct metabolic requirements of exhausted and functional virus-specific CD8 T cells in the same host, Cell Rep, vol.16, pp.1243-1252, 2016.

B. Bengsch, Bioenergetic insufficiencies due to metabolic alterations regulated by the inhibitory receptor PD-1 are an early driver of CD8+ T cell exhaustion, Immunity, vol.45, pp.358-373, 2016.

D. Wolski, Early transcriptional divergence marks virus-specific primary human CD8+ T cells in chronic versus acute infection, Immunity, vol.47, pp.648-663, 2017.
URL : https://hal.archives-ouvertes.fr/inserm-02315536

P. Fisicaro, C. Boni, V. Barili, D. Laccabue, and C. Ferrari, Strategies to overcome HBV-specific T cell exhaustion: checkpoint inhibitors and metabolic re-programming, Curr. Opin. Virol, vol.30, pp.1-8, 2018.

E. E. Vincent, Mitochondrial phosphoenolpyruvate carboxykinase regulates metabolic adaptation and enables glucose-independent tumor growth, Mol. Cell, vol.60, pp.195-207, 2015.

P. Ho, Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses, Cell, vol.162, pp.1217-1228, 2015.

F. Kruiswijk, C. F. Labuschagne, and K. H. Vousden, P53 in survival, death and metabolic health: a lifeguard with a licence to kill, Nat. Rev. Mol. Cell Biol, vol.16, pp.393-405, 2015.

C. Muñoz-fontela, A. Mandinova, S. A. Aaronson, and S. W. Lee, Emerging roles of p53 and other tumour-suppressor genes in immune regulation, Nat. Rev. Immunol, vol.16, pp.741-750, 2016.

C. E. Canman, Activation of the ATM kinase by ionizing radiation and phosphorylation of p53, Science, vol.281, pp.1677-1679, 1998.

S. P. Jackson and J. Bartek, The DNA-damage response in human biology and disease, Nature, vol.461, pp.1071-1078, 2009.

A. Lanna, S. M. Henson, D. Escors, and A. N. Akbar, The kinase p38 activated by the metabolic regulator AMPK and scaffold TAB1 drives the senescence of human T cells, Nat. Immunol, vol.15, pp.965-972, 2014.

A. N. Akbar, S. M. Henson, and A. Lanna, Senescence of T lymphocytes: implications for enhancing human immunity, Trends Immunol, vol.37, pp.866-876, 2016.

Z. Guo, S. Kozlov, M. F. Lavin, M. D. Person, and T. T. Paull, ATM activation by oxidative stress, Science, vol.330, pp.517-521, 2010.

S. Ditch and T. T. Paull, The ATM protein kinase and cellular redox signaling: beyond the DNA damage response, Trends Biochem. Sci, vol.37, pp.15-22, 2012.

X. Zhou, Resveratrol regulates mitochondrial reactive oxygen species homeostasis through Sirt3 signaling pathway in human vascular endothelial cells, Cell Death Dis, vol.5, p.1576, 2014.

V. L. Truong, M. Jun, and W. S. Jeong, Role of resveratrol in regulation of cellular defense systems against oxidative stress, BioFactors, vol.44, pp.36-49, 2018.

K. W. Brudvik and K. Taskén, Modulation of T cell immune functions by the prostaglandin E(2) -cAMP pathway in chronic inflammatory states, Br. J. Pharmacol, vol.166, pp.411-419, 2012.

M. Quigley, Transcriptional analysis of HIV-specific CD8+T cells shows that PD-1 inhibits T cell function by upregulating BATF, Nat. Med, vol.16, pp.1147-1151, 2010.

D. W. Meek and C. W. Anderson, Posttranslational modification of p53: cooperative integrators of function, Cold Spring Harb. Perspect. Biol, vol.1, p.950, 2009.

Y. A. Medvedeva, EpiFactors: a comprehensive database of human epigenetic factors and complexes, Database, p.67, 2015.

J. C. Black, C. Van-rechem, and J. R. Whetstine, Histone lysine methylation dynamics: establishment, regulation, and biological impact, Mol. Cell, vol.48, pp.491-507, 2012.

F. Casciello, K. Windloch, F. Gannon, and J. S. Lee, Functional role of G9a histone methyltransferase in cancer, Front. Immunol, vol.6, p.487, 2015.

K. Lund, P. D. Adams, and M. Copland, EZH2 in normal and malignant hematopoiesis, Leukemia, vol.28, pp.44-49, 2014.

L. M. Snell, T. L. Mcgaha, and D. G. Brooks, Type I interferon in chronic virus infection and cancer, Trends Immunol, vol.38, pp.542-557, 2017.

M. Wong and S. S. Chen, Emerging roles of interferon-stimulated genes in the innate immune response to hepatitis C virus infection, Cell. Mol. Immunol, vol.13, pp.11-35, 2016.

H. Radziewicz, Impaired hepatitis C virus (HCV)-specific effector CD8+ T cells undergo massive apoptosis in the PEripheral Blood during Acute HCV infection and in the liver during the chronic phase of infection, J. Virol, vol.82, pp.9808-9822, 2008.

S. Wesselborg, O. Janssen, and D. Kabelitz, Induction of activation-driven death (Apoptosis) in activated but not resting peripheral blood T cells, J. Immunol, vol.150, pp.4338-4345, 1993.

L. Van-parijs, A. Ibraghimov, and A. K. Abbas, The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance, Immunity, vol.4, pp.321-328, 1996.

K. W. Yoon, Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53, Science, vol.349, pp.1261669-1261669, 2015.

A. Banerjee, Lack of p53 augments antitumor functions in cytolytic T cells, Cancer Res, vol.76, pp.5229-5240, 2016.

G. J. Martinez, The transcription factor NFAT promotes exhaustion of activated CD8+ T cells, Immunity, vol.42, pp.265-278, 2015.

A. J. Sadler and B. R. Williams, Interferon-inducible antiviral effectors, Nat. Rev. Immunol, vol.8, pp.559-568, 2008.

M. Narita, A novel role for high-mobility group A proteins in cellular senescence and heterochromatin formation, Cell, vol.126, pp.503-514, 2006.

B. Kakaradov, Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA sequencing, Nat. Immunol, vol.18, pp.422-432, 2017.

A. N. Henning, R. Roychoudhuri, and N. P. Restifo, Epigenetic control of CD8+ T cell differentiation, Nat. Rev. Immunol, vol.18, pp.340-356, 2018.

P. S. De-araújo-souza, S. C. Hanschke, and J. P. Viola, Epigenetic control of interferon-gamma expression in CD8 T cells, J. Immunol. Res, pp.1-7, 2015.

S. M. Gray, S. M. Kaech, and M. M. Staron, The interface between transcriptional and epigenetic control of effector and memory CD8(+) T-cell differentiation, Immunol. Rev, vol.261, pp.157-168, 2014.

S. M. Gray, R. A. Amezquita, T. Guan, S. H. Kleinstein, and S. M. Kaech, Polycomb repressive complex 2-mediated chromatin repression guides effector CD8+t cell terminal differentiation and loss of multipotency, Immunity, vol.46, pp.596-608, 2017.

S. Scheer and C. Zaph, The lysine methyltransferase G9a in immune cell differentiation and function, Front. Immunol, vol.8, p.429, 2017.

S. Chang and T. M. Aune, Dynamic changes in histone-methylation 'marks' across the locus encoding interferon-gamma during the differentiation of T helper type 2 cells, Nat. Immunol, vol.8, pp.723-731, 2007.

H. Müller, E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis, Genes Dev, vol.15, pp.267-285, 2001.

K. H. Kim and C. W. Roberts, Targeting EZH2 in cancer, Nat. Med, vol.22, pp.128-134, 2016.

B. Martin, Restoration of HCV-specific CD8+ T cell function by interferon-free therapy, J. Hepatol, vol.61, pp.538-543, 2014.

B. Callendret, Persistent hepatitis C viral replication despite priming of functional CD8+ T cells by combined therapy with a vaccine and a directacting antiviral, Hepatology, vol.63, pp.1442-1454, 2016.

G. Missale, Lack of full CD8 functional restoration after antiviral treatment for acute and chronic hepatitis C virus infection, Gut, vol.61, pp.1076-1084, 2012.

E. J. Wherry, Molecular signature of CD8+ T cell exhaustion during chronic viral infection, Immunity, vol.27, pp.670-684, 2007.

T. A. Doering, Network analysis reveals centrally connected genes and pathways involved in CD8+ T cell exhaustion versus memory, Immunity, vol.37, pp.1130-1144, 2012.

A. I. Saeed, TM4: A free, open-source system for microarray data management and analysis, Biotechniques, vol.34, pp.374-378, 2003.

P. Martini, G. Sales, M. S. Massa, M. Chiogna, and C. Romualdi, Along signal paths: an empirical gene set approach exploiting pathway topology, Nucleic Acids Res, vol.41, pp.19-19, 2013.

G. Sales, E. Calura, P. Martini, and C. Romualdi, Graphite Web: web tool for gene set analysis exploiting pathway topology, Nucleic Acids Res, vol.41, pp.89-97, 2013.

A. Subramanian, H. Kuehn, J. Gould, P. Tamayo, and J. P. Mesirov, GSEA-P: a desktop application for gene set enrichment analysis, Bioinformatics, vol.23, pp.3251-3253, 2007.

D. Merico, R. Isserlin, O. Stueker, A. Emili, and G. D. Bader, Enrichment map: a network-based method for gene-set enrichment visualization and interpretation, PLoS ONE, vol.5, p.13984, 2010.

R. Saito, A travel guide to Cytoscape plugins, Nat. Methods, vol.9, pp.1069-1076, 2012.

D. Szklarczyk, STRING v10: protein-protein interaction networks, integrated over the tree of life, Nucleic Acids Res, vol.43, pp.447-452, 2015.

A. Cossarizza, Guidelines for the use of flow cytometry and cell sorting in immunological studies, Eur. J. Immunol, vol.47, pp.1584-1797, 2017.
URL : https://hal.archives-ouvertes.fr/pasteur-01619848


, Correspondence and requests for materials should be addressed to C

, Peer review information Nature Communications thanks T. Jake Liang, Paul Pharoah, and other, anonymous, reviewer(s) for their contribution to the peer review of this work