, 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
Adaptive immune responses in acute and chronic hepatitis C virus infection, Nature, vol.436, pp.946-952, 2005. ,
Pathogenesis of chronic viral hepatitis: differential roles of T cells and NK cells, Nat. Med, vol.19, pp.859-868, 2013. ,
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. ,
Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection, Nat. Immunol, vol.10, pp.29-37, 2009. ,
Overcoming T cell exhaustion in infection and cancer, Trends Immunol, vol.36, pp.265-276, 2015. ,
Metabolic exhaustion in infection, cancer and autoimmunity, Nat. Immunol, vol.19, pp.213-221, 2018. ,
The epigenetic landscape of T cell exhaustion, Science, vol.354, pp.1165-1169, 2016. ,
CD8+ T-cell memory in tumor immunology and immunotherapy, Immunol. Rev, vol.211, pp.214-224, 2006. ,
Induction and transcriptional regulation of the co-inhibitory gene module in T cells, Nature, vol.558, pp.454-459, 2018. ,
Restoring function in exhausted CD8 T cells during chronic viral infection, Nature, vol.439, pp.682-687, 2006. ,
Dysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection, Hepatology, vol.45, pp.588-601, 2007. ,
TCF1+ hepatitis C virus-specific CD8+ T cells are maintained after cessation of chronic antigen stimulation, Nat. Commun, vol.8, p.15050, 2017. ,
Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade, Science, vol.354, pp.1160-1165, 2016. ,
PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation, Nat. Commun, vol.6, p.6692, 2015. ,
Emerging concepts of T cell metabolism as a target of immunotherapy, Nat. Immunol, vol.17, pp.364-368, 2016. ,
Epigenomic-guided mass cytometry profiling reveals disease-specific features of exhausted CD8 T cells, Immunity, vol.48, pp.1029-1045, 2018. ,
Metabolic checkpoints in activated T cells, Nat. Immunol, vol.13, pp.907-915, 2012. ,
Posttranscriptional control of T cell effector function by aerobic glycolysis, Cell, vol.153, pp.1239-1251, 2013. ,
Constitutive glycolytic metabolism supports CD8+ T cell effector memory differentiation during viral infection, Immunity, vol.45, pp.1024-1037, 2016. ,
Rapid effector function of memory CD8+T cells requires an immediate-early glycolytic switch, Nat. Immunol, vol.14, pp.1064-1072, 2013. ,
Enhancing CD8 T-cell memory by modulating fatty acid metabolism, Nature, vol.460, pp.103-107, 2009. ,
Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development, Immunity, vol.36, pp.68-78, 2012. ,
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. ,
Distinct metabolic requirements of exhausted and functional virus-specific CD8 T cells in the same host, Cell Rep, vol.16, pp.1243-1252, 2016. ,
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. ,
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
Strategies to overcome HBV-specific T cell exhaustion: checkpoint inhibitors and metabolic re-programming, Curr. Opin. Virol, vol.30, pp.1-8, 2018. ,
Mitochondrial phosphoenolpyruvate carboxykinase regulates metabolic adaptation and enables glucose-independent tumor growth, Mol. Cell, vol.60, pp.195-207, 2015. ,
Phosphoenolpyruvate is a metabolic checkpoint of anti-tumor T cell responses, Cell, vol.162, pp.1217-1228, 2015. ,
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. ,
Emerging roles of p53 and other tumour-suppressor genes in immune regulation, Nat. Rev. Immunol, vol.16, pp.741-750, 2016. ,
Activation of the ATM kinase by ionizing radiation and phosphorylation of p53, Science, vol.281, pp.1677-1679, 1998. ,
The DNA-damage response in human biology and disease, Nature, vol.461, pp.1071-1078, 2009. ,
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. ,
Senescence of T lymphocytes: implications for enhancing human immunity, Trends Immunol, vol.37, pp.866-876, 2016. ,
ATM activation by oxidative stress, Science, vol.330, pp.517-521, 2010. ,
The ATM protein kinase and cellular redox signaling: beyond the DNA damage response, Trends Biochem. Sci, vol.37, pp.15-22, 2012. ,
Resveratrol regulates mitochondrial reactive oxygen species homeostasis through Sirt3 signaling pathway in human vascular endothelial cells, Cell Death Dis, vol.5, p.1576, 2014. ,
Role of resveratrol in regulation of cellular defense systems against oxidative stress, BioFactors, vol.44, pp.36-49, 2018. ,
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. ,
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. ,
Posttranslational modification of p53: cooperative integrators of function, Cold Spring Harb. Perspect. Biol, vol.1, p.950, 2009. ,
EpiFactors: a comprehensive database of human epigenetic factors and complexes, Database, p.67, 2015. ,
Histone lysine methylation dynamics: establishment, regulation, and biological impact, Mol. Cell, vol.48, pp.491-507, 2012. ,
Functional role of G9a histone methyltransferase in cancer, Front. Immunol, vol.6, p.487, 2015. ,
EZH2 in normal and malignant hematopoiesis, Leukemia, vol.28, pp.44-49, 2014. ,
Type I interferon in chronic virus infection and cancer, Trends Immunol, vol.38, pp.542-557, 2017. ,
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. ,
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. ,
Induction of activation-driven death (Apoptosis) in activated but not resting peripheral blood T cells, J. Immunol, vol.150, pp.4338-4345, 1993. ,
The roles of costimulation and Fas in T cell apoptosis and peripheral tolerance, Immunity, vol.4, pp.321-328, 1996. ,
Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53, Science, vol.349, pp.1261669-1261669, 2015. ,
Lack of p53 augments antitumor functions in cytolytic T cells, Cancer Res, vol.76, pp.5229-5240, 2016. ,
The transcription factor NFAT promotes exhaustion of activated CD8+ T cells, Immunity, vol.42, pp.265-278, 2015. ,
Interferon-inducible antiviral effectors, Nat. Rev. Immunol, vol.8, pp.559-568, 2008. ,
A novel role for high-mobility group A proteins in cellular senescence and heterochromatin formation, Cell, vol.126, pp.503-514, 2006. ,
Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA sequencing, Nat. Immunol, vol.18, pp.422-432, 2017. ,
Epigenetic control of CD8+ T cell differentiation, Nat. Rev. Immunol, vol.18, pp.340-356, 2018. ,
Epigenetic control of interferon-gamma expression in CD8 T cells, J. Immunol. Res, pp.1-7, 2015. ,
The interface between transcriptional and epigenetic control of effector and memory CD8(+) T-cell differentiation, Immunol. Rev, vol.261, pp.157-168, 2014. ,
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. ,
The lysine methyltransferase G9a in immune cell differentiation and function, Front. Immunol, vol.8, p.429, 2017. ,
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. ,
E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis, Genes Dev, vol.15, pp.267-285, 2001. ,
Targeting EZH2 in cancer, Nat. Med, vol.22, pp.128-134, 2016. ,
Restoration of HCV-specific CD8+ T cell function by interferon-free therapy, J. Hepatol, vol.61, pp.538-543, 2014. ,
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. ,
Lack of full CD8 functional restoration after antiviral treatment for acute and chronic hepatitis C virus infection, Gut, vol.61, pp.1076-1084, 2012. ,
Molecular signature of CD8+ T cell exhaustion during chronic viral infection, Immunity, vol.27, pp.670-684, 2007. ,
Network analysis reveals centrally connected genes and pathways involved in CD8+ T cell exhaustion versus memory, Immunity, vol.37, pp.1130-1144, 2012. ,
TM4: A free, open-source system for microarray data management and analysis, Biotechniques, vol.34, pp.374-378, 2003. ,
Along signal paths: an empirical gene set approach exploiting pathway topology, Nucleic Acids Res, vol.41, pp.19-19, 2013. ,
Graphite Web: web tool for gene set analysis exploiting pathway topology, Nucleic Acids Res, vol.41, pp.89-97, 2013. ,
GSEA-P: a desktop application for gene set enrichment analysis, Bioinformatics, vol.23, pp.3251-3253, 2007. ,
Enrichment map: a network-based method for gene-set enrichment visualization and interpretation, PLoS ONE, vol.5, p.13984, 2010. ,
A travel guide to Cytoscape plugins, Nat. Methods, vol.9, pp.1069-1076, 2012. ,
STRING v10: protein-protein interaction networks, integrated over the tree of life, Nucleic Acids Res, vol.43, pp.447-452, 2015. ,
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
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, 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