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Hepatitis B virus (HBV)-specific CD8 T cells are functionally exhausted in chronic hepatitis B infection, and this condition can be corrected only partially through the modulation of inhibitory pathways, which suggests that a more complex molecular interplay underlies T cell exhaustion. To gain broader insight into this process and identify additional targets for the restoration of T cell function, we compared the transcriptome profiles of HBV-specific CD8 T cells from patients with acute and chronic disease with those of HBV-specific CD8 T cells from patients able to resolve HBV infection spontaneously and influenza (FLU)-specific CD8 T cells from healthy participants. The results indicate that exhausted HBV-specific CD8 T cells are markedly impaired at multiple levels and show substantial downregulation of various cellular processes centered on extensive mitochondrial alterations. A notable improvement of mitochondrial and antiviral CD8 functions was elicited by mitochondrion-targeted antioxidants, which suggests a central role for reactive oxygen species (ROS) in T cell exhaustion. Thus, mitochondria represent promising targets for novel reconstitution therapies to treat chronic hepatitis B infection. © 2017 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.


Alisi A.,University of Rome | Alisi A.,Bambino Ges Childrens Hospital and Research Institute | Ghidinelli M.,University of Rome | Zerbini A.,Laboratory of Viral Immunopathology | And 3 more authors.
Journal of Hepatology | Year: 2011

Background & Aims: Chromosomal aberrations are frequently observed in hepatitis C virus (HCV)- and alcohol-related hepatocellular carcinomas (HCCs). The mechanisms by which chromosomal aberrations occur during hepatocarcinogenesis are still unknown. However, these aberrations are considered to be the result of deregulation of some mitotic proteins, including the alteration of Cyclin B1 and Aurora kinase A expression, and the phosphorylation of gamma-tubulin. Our study aims at investigating changes in expression of the above mentioned proteins and related intracellular pathways, in in vitro and in vivo models of both HCV- and alcohol- dependent HCCs. Methods: In this study, the molecular defects and the mechanisms involved in deregulation of the mitotic machinery were analyzed in human hepatoma cells, expressing HCV proteins treated or not with ethanol, and in liver tissues from control subjects (n = 10) and patients with HCV- (n = 10) or alcohol-related (n = 10) HCCs. Results: Expression of Cyclin B1, Aurora kinase A, and tyrosine-phosphorylated gamma-tubulin was analyzed in models reproducing HCV infection and ethanol treatment in HCC cells. Interestingly, HCV and alcohol increased the expression of Cyclin B, Aurora kinase A, and tyrosine- phosphorylated gamma-tubulin also in tissues from patients with HCV- or alcohol-related HCCs. In vitro models suggest that HCV requires the expression of PKR (RNA-activated protein kinase), as well as JNK (c-Jun N-terminal kinase) and p38MAPK (p38 mitogen-activated protein kinase) proteins; while, ethanol bypasses all these pathways. Conclusions: Our results support the idea that HCV and alcohol may promote oncogenesis by acting through the same mitotic proteins, but via different signaling pathways. © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.


Bertoletti A.,Agency for Science, Technology and Research Singapore | Bertoletti A.,National University of Singapore | Ferrari C.,Laboratory of Viral Immunopathology
Postgraduate Medical Journal | Year: 2013

Knowledge of the immunological events necessary to control hepatitis B virus (HBV) infection has accelerated in recent years, but their translation towards therapeutic strategies able to achieve a durable HBV suppression has been challenging. The scenario of how HBV deals with the host immunity is presented and used to discuss how the immune response can be harnessed to potentially achieve infection control.


Bertoletti A.,Agency for Science, Technology and Research Singapore | Bertoletti A.,National University of Singapore | Ferrari C.,Laboratory of Viral Immunopathology
Gut | Year: 2012

Knowledge of the immunological events necessary to control hepatitis B virus (HBV) infection has accelerated in recent years, but their translation towards therapeutic strategies able to achieve a durable HBV suppression has been challenging. The scenario of how HBV deals with the host immunity is presented and used to discuss how the immune response can be harnessed to potentially achieve infection control.


Fisicaro P.,Laboratory of Viral Immunopathology | Valdatta C.,Laboratory of Viral Immunopathology | Massari M.,Unit of Infectious Diseases | Loggi E.,University of Bologna | And 6 more authors.
Gastroenterology | Year: 2010

Background & Aims: The antiviral function of peripheral hepatitis B virus (HBV)-specific T cells can be increased in patients with chronic hepatitis B by blocking the interaction of programmed death (PD)-1 with its ligand PD-L1. However, no information is available about the effects of this blockade on intrahepatic lymphocytes. We studied T-cell exhaustion and the effects of PD-1/PD-L1 blockade on intrahepatic and circulating HBV-specific T cells in patients with chronic hepatitis B. Methods: A total of 42 patients with chronic HBV infection who underwent liver biopsy were studied. The ex vivo phenotype of peripheral and intrahepatic HBV-specific CD8+ T cells was assessed by flow cytometry with class I tetramers and antibodies to T-cell differentiation molecules. Functional recovery was evaluated by analyzing expansion and production of interferon (IFN)-γ and interleukin (IL)-2 after short-term incubation of T cells with HBV peptides in the presence of anti-PD-L1 or control antibodies. Results: Intrahepatic HBV-specific CD8+ cells expressed higher levels of PD-1 and lower levels of CD127 than their peripheral counterparts. Blockade of PD-1/PD-L1 interaction increased CD8+ cell proliferation and IFN-γ and IL-2 production by circulating intrahepatic lymphocytes, even though anti-PD-L1 had a stronger effect on intrahepatic compared with peripheral T cells. Conclusions: T-cell exhaustion by high antigen concentrations promotes HBV-specific T-cell dysfunction by affecting phenotype and function of peripheral and intrahepatic T cells. By restoring antiviral T-cell functions, not only in peripheral but also in intrahepatic lymphocytes, anti-PD-L1 might be a good therapeutic candidate for chronic HBV infection. © 2010 AGA Institute.


Fisicaro P.,Laboratory of Viral Immunopathology | Valdatta C.,Laboratory of Viral Immunopathology | Massari M.,Unit of Infectious Diseases | Loggi E.,University of Bologna | And 8 more authors.
Gastroenterology | Year: 2012

Background & Aims: In patients with chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, antiviral functions of T cells are impaired; these might be increased by blocking T-cell co-inhibitory pathways, such as preventing interaction between the receptor programmed death (PD)-1 and its ligand, PD-L1. We attempted to optimize the restoration of T-cell functions in patients with chronic HBV or HCV infection with a combination of reagents that block PD-1 interaction with PD-L1 and stimulate T-cell signaling via CD137, a member of the tumor necrosis factor-receptor family. Methods: We assessed the effects of CD137 stimulation (via CD137L), alone or in combination with antibodies that block PD-1 interaction with PD-L1 (anti-PD-L1), on proliferation and production of interferon-γ and interleukin-2 by intrahepatic and peripheral T cells from patients with chronic HBV or HCV infection. We also analyzed expression of different co-stimulatory molecules on virus-specific CD8+ and forkhead box P3+CD4+ cells by flow cytometry. Results: Incubation of intrahepatic T cells with CD137L and anti-PD-L1 increased their responses to HBV, but not HCV. However, HCV-specific T cells isolated from peripheral blood were sensitive to these reagents. Virus-specific T cells from some, but not all patients, had increased responses to anti-PD-L1 when CD137L was added because in some cases the combination of anti-PD-L1 and CD137L overstimulated T cells, leading to their inhibition. Intrahepatic HBV- and HCV-specific CD8+ T cells had different costimulatory profiles; liver cells from patients with chronic HBV infection had a higher proportion of forkhead box P3+ regulatory T cells, with higher levels of PD-1, compared with liver cells from patients with chronic HCV infection. Conclusions: A combination of reagents that prevent interaction between PD-1 and its ligand and activate CD137 signaling increase responses of intrahepatic HBV-specific T cells and circulating HCV-specific T cells. This strategy might be developed to increase T-cell responses to these viruses in patients with chronic hepatitis B or C, and tailoring the dose of CD137L administered will help optimize results. © 2012 AGA Institute.


Ferrari C.,Laboratory of Viral Immunopathology
Liver International | Year: 2015

Hepatitis B virus (HBV) infection acquired in adult life is generally self-limited while chronic persistence of the virus is the prevalent outcome when infection is acquired perinatally. Both control of infection and liver cell injury are strictly dependent upon protective immune responses, because hepatocyte damage is the price that the host must pay to get rid of intracellular virus. Resolution of acute hepatitis B is associated with functionally efficient, multispecific antiviral T-cell responses which are preceded by a poor induction of intracellular innate responses at the early stages of infection. Persistent control of infection is provided by long-lasting protective memory, which is probably sustained by continuous stimulation of the immune system by trace amounts of virus which are never totally eliminated, persisting in an occult episomic form in the nucleus of liver cells even after recovery from acute infection. Chronic virus persistence is instead characterized by a lack of protective T-cell memory maturation and by an exhaustion of HBV-specific T-cell responses. Persistent exposure of T cells to high antigen loads is a key determinant of functional T-cell impairment but also other mechanisms can contribute to T-cell inhibition, including the tolerogenic effect of the liver environment. The degree of T-cell impairment is variable and its severity is related to the level of virus replication and antigen load. The antiviral T-cell function is more efficient in patients who can control infection either partially, such as inactive HBsAg carriers with low levels of virus replication, or completely, such as patients who achieve HBsAg loss either spontaneously or after antiviral therapy. Thus, understanding the features of the immune responses associated with control of infection is needed for the successful design of novel immune modulatory therapies based on the reconstitution of efficient antiviral responses in chronic HBV patients. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.


Boni C.,Laboratory of Viral Immunopathology | Laccabue D.,Laboratory of Viral Immunopathology | Lampertico P.,University of Milan | Giuberti T.,Laboratory of Viral Immunopathology | And 9 more authors.
Gastroenterology | Year: 2012

BACKGROUND & AIMS: In patients with chronic hepatitis B virus (HBV) infection, persistent exposure to high concentrations of antigen can disrupt T-cell functions. It is not clear to what extent long-term suppression of HBV by nucleos(t)ide analogues can restore antiviral T-cell functions. We compared HBV-specific T-cell responses of patients treated with nucleos(t)ide analogues with those detected in other conditions of HBV control. METHODS: We analyzed intracellular levels of interferon gamma, interleukin-2, and tumor necrosis factor α in HBV-specific T cells after 10 days of stimulation with peptides covering the overall HBV genotype D sequence and ex vivo with selected CD8 epitopes and the corresponding HLA-A2 dextramers. Findings from patients treated with nucleos(t)ide analogues who had complete (HBV DNA negative/antibody to hepatitis B surface antigen positive) or partial (HBV DNA negative/hepatitis B surface antigen positive) control of their infections were compared with those of patients with spontaneous or interferon alfa-induced resolution of acute or chronic infections, inactive HBV carriers, or untreated hepatitis B e antigen-negative patients with chronic infections. RESULTS: Although HBV-specific T cells from nucleos(t)ide analogue-treated patients with complete control of infection were dysfunctional ex vivo, they had efficient responses after in vitro expansion. These responses were comparable to those of patients who spontaneously resolved acute HBV infection. Nucleos(t)ide analogue-treated patients who were HBV DNA negative but hepatitis B surface antigen positive had lower levels of T-cell responses but responses greater than those of untreated patients with chronic infection. CONCLUSIONS: In vitro reactivity can be restored to T cells from patients with suppressed HBV infection following long-term treatment with nucleos(t)ide analogues, despite prolonged exposure to large loads of antigen. Immune therapies that increase the antiviral T-cell response might increase the likelihood of complete HBV control in patients undergoing long-term nucleos(t)ide analogue treatment. © 2012 AGA Institute.


Missale G.,Laboratory of Viral Immunopathology | Pilli M.,Laboratory of Viral Immunopathology | Zerbini A.,Instituto Of Ricovero E Cura A Carattere Scientifico | Penna A.,Laboratory of Viral Immunopathology | And 7 more authors.
Gut | Year: 2012

Background: Hepatitis C virus (HCV) persistence is associated with impaired CD8 functions. Whether functional restoration of CD8 T cells chronically exposed to antigen can be obtained once the antigen is removed remains to be clarified. Objective: To determine whether clearance of HCV by antiviral treatment can fully restore the antiviral function of HCV-specific CD8 cells. Design: Peripheral blood HCV-, Flu- and cytomegalovirus (CMV)-specific CD8 cells were quantified by tetramer staining in 28 patients whose HCV infection resolved after peginterferon or peginterferon/ribavirin treatment for either acute or chronic hepatitis and in eight subjects with acute HCV infection which resolved spontaneously for comparison. HCV-specific CD8 cells were evaluated for their phenotypic and functional characteristics by comparing different patient groups and CD8 cells with different viral specificities in the same patients. Results: Sustained viral response (SVR) did not lead to full maturation of a functional memory CD8 cell response. In particular, SVR in chronic infection was associated with a greater level of T cell dysfunction than responders after acute infection, who showed HCV-specific CD8 responses comparable to those of spontaneous resolvers but weaker than those of Flu-specific CD8 cells. Higher programmed death (PD)-1 expression was detected on HCV than on Flu- and CMV-specific CD8 cells and the effect of PD-1/PD-L1 blockade was better in SVRs after chronic than after acute HCV infection. Conclusion: A better restoration of HCV-specific CD8 function was detectable after SVR in patients with acute hepatitis than in those with chronic disease. Thus, the difficulty in achieving a complete restoration of the antiviral T cell function should be considered in the design of immunomodulatory therapies.


Amadei B.,Laboratory of Viral Immunopathology | Urbani S.,Laboratory of Viral Immunopathology | Cazaly A.,University of Southampton | Fisicaro P.,Laboratory of Viral Immunopathology | And 5 more authors.
Gastroenterology | Year: 2010

Background & Aims: Natural killer (NK) cells are essential early after infection, not only for viral containment but also for timely and efficient induction of adaptive responses. An inhibitory effect of hepatitis C virus (HCV)-E2 proteins on NK cells has been reported, but the features of NK cell responses in the acute phase of hepatitis C are still largely undefined. Therefore, the aim of this study was to characterize the function and phenotype of NK cells in the acute phase of infection and compare individuals with chronic and self-limited outcomes. Methods: Twenty-two individuals with acute HCV infection, 14 with chronic evolution, and 8 with self-limited infection, were studied using NK phenotypic and functional assays. Results: An increased expression of NKG2D on both CD56bright and CD56dim NK cells was detected in patients with acute HCV, irrespective of the outcome, as compared with healthy controls. Also, interferon gamma production and cytotoxicity by NK cells were higher in individuals with acute HCV infection than in healthy controls. Subset analysis showed increased interferon gamma production in both NK cell subsets carrying group 1 and group 2 HLA-C-specific killer cell immunoglobulin-like receptors. However, increased CD107a was noted only on NK cells expressing the group 1 HLA-C-specific killer cell immunoglobulin-like receptor and was maximal in self-limited infection. Conclusions: Our data show that in the acute phase of HCV infection, NK cells are activated regardless of outcome, with no evidence of a suppressive effect of HCV on NK cell function. © 2010 AGA Institute.

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