Accepted Article
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Article type: Review Article
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Received date: 08/08/2014
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Revised date: 10/13/2014
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Accepted date: 10/14/2014
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For: Hepatology Research - Review
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The Immunobiology of Hepatitis B Virus
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Infection1
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Running Title: The Immunobiology of Hepatitis B Virus Infection
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Masanori Isogawa, and Yasuhito Tanaka
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Department of Virology & Liver Unit
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Nagoya City University Graduate School of Medical Science
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Corresponding Author: This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/hepr.12439 This article is protected by copyright. All rights reserved.
Accepted Article
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Yasuhito Tanaka, M.D. PhD.
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Department of Virology & Liver Unit
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Nagoya City University Graduate School of Medical Science
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Kawasumi 1, Mizuho-ku,
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Nagoya 467-8601, Japan
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Phone: +81-52-853-8191
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Fax:
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email: [email protected]
+81-52-842-0021
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10
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Accepted Article
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Abstract.
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The adaptive immune response, particularly the virus-specific CD8+ T cell response, is
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largely responsible for viral clearance and disease pathogenesis during hepatitis B virus
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(HBV) infection. The HBV-specific CD8+ T cell response is vigorous, polyclonal and
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multispecific in acutely infected patients who successfully clear the virus and relatively
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weak and narrowly focused in chronically infected patients. The immunological basis for
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this dichotomy is unclear. A recent study using HBV transgenic mice and HBV-specific
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T cell receptor (TCR) transgenic mice suggests that intrahepatic antigen presentation by
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HBV positive hepatocytes suppresses HBV-specific CD8+ T cell responses through a
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coinhibitory molecule, programmed cell death 1 (PD-1). In contrast, antigen presentation
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by activated professional antigen presenting cells (pAPCs) induces functional
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differentiation of HBV-specific CD8+ T cells. These findings suggest that the outcome of
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T cell priming is largely dependent on the nature of antigen presenting cells. Another
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study suggests that the timing of HBV-specific CD4+ T cell priming regulates the
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magnitude of the HBV-specific CD8+ T cell response. Other factors that could regulate
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HBV-specific cellular immune responses are high viral loads, mutational epitope
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inactivation, T cell receptor antagonism and infection of immunologically privileged
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tissues. However, these pathways become apparent only in the setting of an ineffective
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cellular immune response, which is therefore the fundamental underlying cause.
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Understanding the cellular and molecular mechanisms by which HBV evades host
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immune responses will eventually help develop new immunotherapeutic strategies
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designed to terminate chronic HBV infection.
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Accepted Article
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Key words: Hepatitis B virus, Immune response, Immunological priming,
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Immunotherapy, T cells
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Introduction
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Hepatitis B virus (HBV) is a partially double stranded DNA virus that causes
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necroinflammatory liver disease of variable severity.1 More than 240 million people
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worldwide are persistently infected with HBV. Persistent infection by HBV is often
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associated with chronic liver disease that can lead to the development of cirrhosis and
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hepatocellular carcinoma (HCC). HBV is thought to be largely non-cytopathic for the
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infected hepatocyte under normal circumstances,2,3 but could be cytopathic in
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immunodeficient chimeric mice carrying human hepatocytes,4 suggesting that
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unmitigated HBV replication and gene expression trigger cellular stresses and cell death.
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The immune response mediates the clearance of HBV and disease pathogenesis.2
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Interestingly, HBV appears to be a poor inducer of innate immune responses, acting as a
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stealth virus in this regard by evading recognition by innate immune sensor molecules.5
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Despite the weak innate immune responses, acute HBV infection of adults is usually self-
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limited and cleared by functional HBV-specific CD8+ T cell responses. The induction of
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functional HBV-specific CD8+ T cell responses is dependent on early CD4+ T cell
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priming prior to HBV spread,6 which might explain why patients infected with human
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immunodeficiency virus (HIV) become persistently infected following horizontal
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transmission of HBV.7,8 Once chronic HBV infection is established, HBV-specific CD8+
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T cells are subjected to powerful immunoregulatory mechanisms in the liver. Ample
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evidence suggests that HBV actively suppresses adoptive immune responses, particularly
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Accepted Article
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HBV-specific CD8+ T cell responses. Reduction of viral antigen expression by antiviral
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treatment can overcome CD8+ T cell hyporesponsiveness in patients with chronic HBV
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infection, suggesting that T cells are present in these subjects but suppressed by high viral
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loads.9 In addition, recent evidence suggests that antigen recognition in the liver
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suppresses HBV-specific CD8+ T cell effector functions by signaling through a
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coinhibitory molecule, programmed cell death 1 (PD-1).10,11 Furthermore, several viral
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proteins have been shown to regulate the adaptive immune response to HBV,12-14 which
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might explain the varying chronicity rate between genotypes that express different levels
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of viral antigens.15,16 We herein review our current knowledge of the host immune
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responses to HBV as well as the cellular and molecular mechanisms by which HBV
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evades these immune responses. We also discuss how the accumulating knowledge of
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HBV immunobiology will help us to develop more inventive therapeutic strategies that
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are designed to reinvigorate HBV-specific immune responses, thereby terminating
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chronic infection with this deadly virus.
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1. The Innate Immune Response
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1-1. Absence of an Innate Response by Infected Cells during Acute HBV Infection.
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Virus infection usually triggers a robust innate immune response that is heralded by rapid
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induction of IFNα/β by the infected cell.17 Production of IFNα/β induces the
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transcriptional expression of a large number of interferon inducible genes (ISGs), which
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in turn exert a variety of intracellular antiviral mechanisms.17,18 Surprisingly, intrahepatic
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gene expression profiling in acutely HBV infected chimpanzees revealed that HBV acts
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like a stealth virus early after infection because it does not induce any cellular gene
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expression, including ISGs, as it spreads through the liver.5 This contrasts strikingly to
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the induction of many ISGs during the spread of hepatitis C virus (HCV) that is highly
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visible to the innate immune system.19 The relative invisibility of HBV to the innate
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sensing machinery of the cells probably reflects its replication strategy. HBV retains its
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transcriptional template, namely covalently closed circular DNA (cccDNA), in the
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nucleus20 and the HBV mRNA is capped and polyadenylated, resembling normal cellular
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transcripts.21,22 In addition, HBV replicates within viral capsid particles in the
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cytoplasm20 and is therefore shielded from innate sensing machinery. Thus, the typical
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widespread expansion of HBV in the liver may reflect the absence of IFNα/β production
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to which the virus is exquisitely sensitive, as shown in HBV transgenic mice.23,24
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1-2. The Natural Killer and Natural Killer T Cell Responses during HBV Infection
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The role of natural killer (NK) cells in acute HBV infection remains largely unknown.
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Studies that analyzed NK cell functions during acute HBV infection have provided
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conflicting evidence. A recent study demonstrated that the activation and cytokine
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producing ability of NK cells were impaired in acute HBV patients,25 while others
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showed enhanced IFN-γ production by, and cytotoxicity of, NK cells.26,27 The cause of
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this discrepancy is unclear. On the other hand, NK cells in chronic HBV patients seem
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generally cytolytic but their cytokine producing ability is selectively impaired.28,29 A
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recent report demonstrated a strong correlation between liver disease, IFN-α production
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and NK cell expression of tumor necrosis factor related apoptosis-inducing ligand
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(TRAIL) during chronic HBV infection,30 suggesting a pathogenic role of NK cells
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(Figure 1, grey area). Interestingly, NK cells seem highly immunoregulatory as well,
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because they can apparently delete HBV-specific, but not cytomegalovirus (CMV)-
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specific, CD8+ T cells through TRAIL-TRAIL receptor 2 (TRAIL-R2) interaction.31
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Taken together, NK cells appear to exert a detrimental effect on the host during chronic
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HBV infection. The precise role of NK cells during chronic HBV infection must be fully
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elucidated to develop new NK cell based therapeutic approaches against chronic HBV
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infections. The role of natural killer T (NKT) cells during natural HBV infection is even
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less clear. Therapeutic potential of NKT cell activation against HBV infection was 32
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initially indicated in HBV transgenic mice model
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and woodchuck hepatitis virus (WHV) suggested that NKT cells were activated soon
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after infection.33,34 However, the degree of NKT cell activation was relatively modest in
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both cases, and the physiological importance of such activation has not been addressed in
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these studies. Studies with several mouse models provided more convincing evidence that
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CD1d-restricted NKT cells are important for the control of HBV replication and gene
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expression.35,36 It is well known, however, that hepatic NKT cell repertoires are
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phenotypically and functionally very different between humans and mice.37 Further
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studies are required to confirm the role of NKT cell during HBV infections.
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2. The Adoptive Immune Response
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2-1. The Antibody Response
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Antibodies to the HBV envelope antigen (HBsAg) neutralize HBV infection presumably
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by preventing virus attachment to hepatocytes. They also facilitate viral clearance by This article is protected by copyright. All rights reserved.
, and subsequent studies with HBV
Accepted Article
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complexing with free viral particles and removing them from the circulation. The
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induction of anti-HBs by immunization is a T cell-dependent process and requires CD4+
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T cell help.38 While anti-HBs neutralizing antibodies are essential for providing
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protection after vaccination, their importance in resolving natural HBV infection is
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somewhat underappreciated, mostly because the appearance of anti-HBs occurs relatively
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late after HBV exposure39 (Figure 1, blue lines). It is generally believed that anti-HBs
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neutralizing antibodies prevent viral spread from the rare cells that remain infected, even
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after resolution of HBV infection. Supporting this notion, HBV is frequently re-activated
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long after resolution of infection in patients who receive conventional chemotherapy or a
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newly developed monoclonal antibody (Rituximab) therapy that targets the CD20 antigen
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expressed on the surface of normal and malignant B lymphocytes.40 More detailed
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analysis of HBV-specific B cell responses might reveal a heretofore-unappreciated role
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of humoral responses in the control of HBV infection.
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2-2. The CD4+ T Cell Response
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Ample evidence suggests a clear relationship between the CD4+ T cell response to HBV
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and the outcome of HBV infection. For example, the peripheral blood CD4+ T cell
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response to HBV is vigorous and multispecific in patients with acute hepatitis who
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ultimately clear the virus, while it is relatively weak in chronic HBV patients39,41,42
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(Figure 1, red areas). Despite the association between a strong CD4+ T cell response and
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viral clearance, CD4+ T cell depletion at the peak of HBV infection had no effect on viral
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replication and liver disease in infected chimpanzees.43 In contrast, CD4+ T cell depletion
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before HBV infection resulted in quantitatively and qualitatively reduced HBV-specific
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CD8+ T cell responses, leading to persistent HBV infection.6 These results suggest that
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CD4+ T cells probably contribute indirectly to the control of HBV infection by
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facilitating the induction and maintenance of the virus-specific CD8+ T cell responses. It
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is likely that CD4+ T cells help induce functional HBV-specific CD8+ T cell responses by
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producing IL-2 that is essential for T cell proliferation and by activating professional
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antigen presenting cells (pAPCs) that are capable of providing co-stimulation to T cells.44
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In contrast, in the absence of early CD4+ T cell responses, CD8+ T cell priming could
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occur in the liver, resulting in T cell inactivation, tolerance or apoptosis,11,45,46 as
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summarized later in this review. This requirement of CD4+ T cells for the induction of
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HBV-specific CD8+ T cells is one of the reasons patients infected with HIV (low CD4
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count) become persistently infected after horizontal transmission of HBV7,8 and why
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genetic variants in the HLA-DP locus are associated with the risk of developing chronic
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HBV infection.47 Interestingly, early CD4+ T cell priming does not occur when the size of
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inoculum is small (i.e. 1 to 10 copies) and the appearance of HBV DNA is delayed after
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infection,6 suggesting that slow spread of HBV might facilitate chronicity, as often
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observed with genotype A infections.15,16 Interestingly, recent studies indicated that the
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frequency of IL-17 producing CD4 T cells (Th-17 cells) is increased in chronic HBV
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patients,
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liver disease.49 These results suggest that CD4 T cells not only help induce antiviral
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cellular immune responses during acute HBV infections, but also mediate inflammatory
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responses during chronic HBV infection.
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2-3. The CD8+ T Cell Response
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and IL-17 and another Th-17 related cytokine IL-22 are shown to exacerbate
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Accepted Article
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The HBV-specific CD8+ T cell response plays a fundamental role in viral clearance and
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liver disease. A vigorous polyclonal CD8+ T cell response is readily detectable in the
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peripheral blood of patients with acute hepatitis B who ultimately clear the virus. In
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contrast, the peripheral blood T cell response in chronically infected patients is weak and
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narrowly focused2,50 (Figure 1, green areas). The livers of these patients contain virus-
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specific T cells that likely contribute to disease pathogenesis but, for functional and/or
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quantitative reasons, are unable to clear the infection. Interestingly, a recent study that
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examined the relationship between the number of intrahepatic HBV-specific CD8+ T
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cells, extent of liver disease, and levels of HBV replication in chronically infected
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patients indicated that inhibition of virus replication could be independent of liver
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damage and that the functionality of HBV-specific CD8+ T cells was more important than
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the number of T cells in controlling HBV replication.51 Experiments in chimpanzees have
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shown that the viral clearance and the onset of liver disease coincide with the
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accumulation of virus-specific CD8+ T cells and the induction of IFN-γ and IFN-
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inducible genes in the liver.52 Importantly, depletion of CD8+ T cells at the peak of
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viremia delays viral clearance and the onset of viral hepatitis until the T cells return,
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providing the most definitive evidence that viral clearance and liver disease are mediated
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by virus specific CD8+ T cells.43
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3. Non-cytolytic and cytolytic T cell effector functions emerge alternately during
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HBV clearance
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It is widely believed that virus specific CD8+ T cells clear the viral infection by killing
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infected cells. However, CD8+ T cell killing is an inefficient process, requiring direct
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Accepted Article
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physical contact between the CD8+ T cells and the infected cells. Therefore, it may not be
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possible for HBV-specific CD8+ T cells to kill all the HBV infected cells if the CD8+ T
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cells are greatly outnumbered, as occurs during HBV infection in which as many as 1011
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hepatocytes can be infected.53 Hence, although the liver disease in HBV infection is
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clearly due to the cytopathic activity of the CD8+ T cells, viral clearance may require
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more efficient CD8+ T cell functions than killing. Important insights into the pathogenic
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and noncytopathic antiviral functions of the CD8+ T cell response have come from
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studies of HBV transgenic mice that develop an acute necroinflammatory liver disease
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after adoptive transfer of HBV-specific effector or effector memory CD8+ T cells that
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were generated by immunizing non-transgenic mice.10,54 In that model, the HBV-specific
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CD8+ T cells rapidly enter the liver and recognize viral antigen and secrete interferon
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gamma (IFN-γ), which non-cytopathically inhibits HBV replication in the remaining
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hepatocytes54 by preventing the assembly of HBV RNA-containing capsids in the
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cytoplasm23,55 in a proteasome-56 and kinase-dependent57 process. During this remarkable
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process, the viral nucleocapsids disappear from the cytoplasm of the hepatocytes, yet the
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hepatocytes remain perfectly healthy.54 Antibody blocking and knockout experiments in
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the HBV transgenic mouse model further demonstrated that the cytopathic and antiviral
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functions of CD8+ T cells are completely independent of each other.58,59 Following the
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secretion of IFN-γ and the suppression of HBV replication, HBV-specific CD8+ T cells
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expand vigorously in situ. Surprisingly, however, the ability of HBV-specific CD8+ T
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cells to produce IFN-γ is quickly down-regulated as they expand. Despite the suppression
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of IFN-γ secretion, the cytotoxic capabilities of the HBV-specific CD8+ T cells increase
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over time, causing necroinflammatory liver disease, and complete inhibition of HBV
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Accepted Article
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mRNA expression.10 Taken together, these results suggest that, in the context of a
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vigorous immune response during acute HBV infection, the sequential and altering
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patterns of non-cytolytic and cytolytic effector function, coupled with T cell expansion
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and contraction, cooperate to minimize the extent of tissue injury required for viral
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clearance. Conversely, we suggest that diminution of the CD8+ T cell response or poor
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coordination of these responses could lead to viral persistence and varying degrees of
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chronic liver disease.
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4. Where, how, and what kind of HBV-specific CD8+ T cells are generated?
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The results described above were generated by adoptively transferring splenocytes from
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HBV-immunized mice to HBV transgenic mice. While these results suggest that
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intrahepatic antigen recognition has a profound impact on the distribution, expansion, and
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effector functions of primed effector-memory CD8+ T cells, they may not reflect the
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events that occur when a naïve individual becomes infected with HBV, because
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immunologically naïve T cells are known to behave very differently than memory T
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cells.60-62 Accordingly, we generated transgenic mice whose CD8+ T cells express T cell
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receptors (TCRs) specific for HBV, to study where and how immunologically naïve T
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cells are primed in response to intrahepatic HBV.
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When HBV-specific naïve CD8+ T cells were adoptively transferred into HBV transgenic
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mice, they were predominantly activated rapidly in the liver, independent of T cell
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homing to lymphoid organs, suggesting that HBV-specific native T cells are primed in
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the liver.11 While this intrahepatic priming of HBV-specific CD8+ T cells challenges the
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Accepted Article
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current T cell priming model postulating that naïve T cells are primed by activated
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professional antigen presenting cells (pAPCs) in lymphoid organs,63,64 several studies
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previously indicated that the liver is an exception to this rule.45,46,65 This rather unusual
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ability of the liver to prime naïve T cells presumably reflects the unique architecture of
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the hepatic sinusoid, which is characterized by a discontinuous endothelium, the absence
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of a basement membrane, and a very slow flow rate,66 allowing circulating T cells to
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make prolonged direct contact with resident liver cells, including hepatocytes.
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Furthermore, the liver is replete with diverse and unique antigen presenting cell
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populations, including liver sinusoidal endothelial cells (LSECs), hepatic stellate cells
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(HSCs), Kupffer cells, and conventional and plasmacytoid dendritic cells, all of which
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are capable of priming and/or tolerizing naïve T cells.67 In our model, HBV-specific
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CD8+ T cells are presumably primed by HBV-expressing hepatocytes, because HBV-
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specific CD8+ T cells were activated when co-cultured with hepatocytes isolated from
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HBV transgenic mice but not with LSECs, liver residential dendritic cells (DCs) or
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Kupffer cells. Importantly, the intrahepatically primed HBV-specific CD8+ T cells
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expand vigorously in the liver but are functionally impaired because they do not express
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IFN-γ or the cytolytic granule Granzyme B, suggesting that intrahepatic priming by
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HBV-expressing hepatocytes expands functionally defective CD8+ T cells. Consequently,
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such intrahepatically primed T cells are incapable of suppressing HBV gene expression
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or inducing liver disease.11 Interestingly, functionally defective HBV-specific CD8+ T
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cells expressed a co-inhibitory molecule, PD-1, after antigen recognition in the liver.11
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Furthermore, a significant fraction of PD-1 deficient HBV-specific naïve T cells
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expanded more vigorously than PD-1 positive controls, and differentiated into effector T
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Accepted Article
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cells capable of producing IFN-γ, as well as Granzyme B, which in turn suppressed HBV
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gene expression and caused liver disease.11 Collectively, these results suggest that PD-1
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signaling impairs the effector functions of HBV-specific CD8+ T cells. Importantly, PD-1
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expression was also observed on HBV-specific CD8+ T cells in chronic HBV patients,68
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indicating that PD-1 mediated functional suppression is active during natural HBV
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infection. Taken together, these results suggest that intrahepatic priming induces T cell
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tolerance and such tolerance is at least partially mediated by PD-1 signaling. This PD-1
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meditated intrahepatic suppression might be the underlying cause of the weak T cell
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responsiveness during chronic HBV infection.
10 11
5. Other Mechanisms of HBV Persistence
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The potential mechanisms by which HBV evades the immune responses are listed on
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Table 1.
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following mother-infant transmission.69,70 The underlying cause of adult onset chronic
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HBV infection is probably multifaceted. Potential contributing factors include mutational
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escape leading to inactivation of B-cell and T-cell epitopes and specific inhibition of the
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adaptive immune response by viral proteins.71 For example, HBeAg has been shown to
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suppress the antibody and T cell response to HBcAg in T cell receptor transgenic
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mice.12,13 The immunosuppressive potential of HBeAg might explain the clinical
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observation that viral mutations precluding the production of HBeAg are often associated
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with exacerbations of liver disease and, sometimes, even with viral clearance from
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chronically infected patients.72,73 HBsAg might also prevent immune elimination of
Neonatal tolerance to HBV is probably responsible for viral persistence
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Accepted Article
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infected cells by functioning as a high dose tolerogen, because extremely high serum
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HBsAg titers in the mg/ml range are often seen in chronically infected patients74,75 and
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chronically infected patients have an absence or subnormal levels of HBsAg-specific
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CD8+ T cells.75 In addition, HBV X protein, a transcriptional transactivator that is
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required for the initiation of infection,76,77 can inhibit cellular proteasome activity when it
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is overexpressed78 and might interfere with antigen processing and presentation. Finally,
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genotypic variation clearly influences the outcome of HBV infection. Up to 10% of adult
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onset infections with genotype A become persistent, while less than 5% of infections with
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other genotypes do so.15 The precise mechanism for the difference is unclear, but the high
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levels of HBsAg expression and low replication activity of genotype A infections might
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facilitate immune evasion.16
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In both neonate and adult onset of chronic HBV infections, weak CD4 and CD8 T cell
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responses are clearly responsible for the viral persistence. The mechanism that suppresses
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HBV-specific immune responses during chronic HBV infection is likely multifactorial. In
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addition to PD-1, other negative signaling molecules, such as cytotoxic T lymphocyte
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antigen-4 (CTLA-4) and T-cell immunoglobulin and mucin 3 (Tim-3), are expressed on
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functionally compromised HBV-specific CD8 T cells in chronic HBV patients.
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example, a fraction of CD8 T cells in patients with CHB express the coinhibitory
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molecule CTLA-4, and its expression level is correlated with serum HBV DNA titer and
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proapoptotic protein Bim expression. Blockade of CTLA-4 increases the expansion of
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interferon gamma (IFN-γ) producing HBV-specific CD8 T cells in vitro. Similarly, Tim-
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3 is also highly expressed on functionally impaired HBV-specific CD8 T cells in chronic
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HBV patients, and the blockade of Tim-3 in vitro restored effector functions of the CD8 This article is protected by copyright. All rights reserved.
79
For
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T cells,
suggesting that multiple layers of negative co-regulation contribute to T cell
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exhaustion in chronic HBV infection. Interestingly, Tim-3 expressing CD8 T cells appear
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to have a immunosuppressive function similar to regulatory T cells (Tregs), limiting
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proliferation of antigen-specific effector T cells in vitro and in vivo.80 Extrinsic factors
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could also contribute to the reduced T cell responses during chronic HBV infection. The
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livers of chronic HBV patients have been shown to express elevated levels of IL-10,81
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TGF-β,82 and arginase,83 all of which are known to suppress cellular immune responses.
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These immunosuppressive soluble factors might be produced by regulatory cells, as the
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frequency of regulatory T cells (Tregs) is increased in chronic HBV patients.84
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Interestingly, recently identified myeloid-derived suppressor cells (MDSCs) are shown to
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induce T cell tolerances in mouse models of HBV infection.85,86 In addition, several
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studies suggest that the number and functionality of DCs are reduced in chronic HBV
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patients compared with healthy donors, and the reduction of HBV DNA titer by a
14
nucleotide analogue adefovir dipivoxil significantly restored the number and
15
functionality of DCs. Caution should be exercised, however, to interpret the physiological
16
relevance of the antigen nonspecific immunosuppression during chronic HBV infections,
17
because chronic HBV patients are no more susceptible than healthy people to other
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infectious agents such as influenza virus, etc.
19 20
6. New immunotherapeutic strategies to cure chronic HBV infections
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The management of chronic HBV has improved significantly in the last decade, mainly
22
because of the development of very effective and safe nucleoside analogues (NAs) that This article is protected by copyright. All rights reserved.
Accepted Article
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primarily inhibit reverse transcription and DNA replication.87 Although NAs strongly
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inhibit HBV replication and have much fewer side effects than IFN-based therapies, they
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usually do not achieve sustained viral suppression. Failure to sustain viral suppression by
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NAs presumably reflects the persistence of cccDNA, which is untouched by reverse
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transcriptase inhibitors. Because the elimination of cccDNA usually requires the turnover
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of infected hepatocytes, the induction of functional HBV-specific CD8+ T cells that can
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specifically kill virus infected hepatocytes has been deemed the most promising approach
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to cure chronic HBV infections. Immunization of chronically infected patients is the most
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straightforward approach to boost HBV-specific CD8+ T cell responses. Several
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strategies have been tested in clinical trials with disappointing results,88 indicating that
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more inventive approaches are required to induce functional HBV-specific CD8+ T cell
12
responses. There are three approaches that could effectively reinvigorate HBV-specific
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CD8+ T cell responses (Table 2).
14
The first approach is to block signaling pathways that negatively regulate HBV-specific
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CD8+ T cell responses. As described in section 4, we showed that blockade of PD-1
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signaling induced functional differentiation of intrahepatically primed, HBV-specific
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CD8 T cells in an HBV transgenic mouse model, indicating that PD-1 could be a new
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therapeutic target to treat chronic HBV infection. HBV-specific CD8 T cell responses
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might be restored by blocking other negative signaling molecules, such as CTLA-4, Tim-
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3, and arginase, as well as by depleting Tregs. More evidence is needed to support this
21
approach.
22
The second approach is to provide extra stimulus to override negative signaling that
23
suppresses effector functions of HBV-specific CD8 T cells. One of the promising
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Accepted Article
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approaches is to activate professional antigen presenting cells (pAPCs), particularly
2
dendritic cells (DCs). Activated DCs are capable of providing the second and third
3
signals necessary for the functional differentiation of HBV-specific CD8+ T cells. Indeed,
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we showed that activation of myeloid DCs (mDC) with an agonistic anti-CD40 antibody
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(αCD40) restored HBV-specific CD8+ T cell responses that are otherwise suppressed by
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PD-1 signaling.11 Interestingly, PD-1 expression on the functional HBV-specific CD8+ T
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cells is strongly down-regulated in αCD40 treated HBV transgenic mice. These results
8
suggest that activation of myeloid dendritic cells directly or indirectly suppresses PD-1
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expression, thereby rescuing HBV-specific CD8+ T cells from PD-1 mediated functional
10
suppression. The data also illustrate the therapeutic potential of mDC activation by
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αCD40 for the treatment of chronic HBV infection. Other signaling pathways that
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stimulate pAPCs, such as toll-like receptor (TLR) signaling pathways, should be tested
13
for their ability to restore HBV-specific CD8+ T cell responses. In addition, cytokines
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required for T cell expansion and differentiation, particularly common gamma-chain
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cytokines such as IL-2, IL-4, IL-7, IL-15, and IL-21, could serve as effective adjuvants
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for therapeutic vaccine against chronic HBV infections.
17
The third approach is to genetically engineer peripheral blood T cells from chronic HBV
18
patients to express T cell receptors (TCRs) specific for HBV-derived epitopes. The first
19
and second strategies described above require the presence of HBV-specific CD8+ T cells
20
that can efficiently recognize HBV-derived T cell epitopes presented on infected
21
hepatocytes. However, such T cells are often deleted in chronic HBV patients after years
22
of exposure to HBV antigens. To circumvent this problem, genes encoding TCRs that
23
efficiently recognize HBV derived epitopes can be introduced into the patient’s
This article is protected by copyright. All rights reserved.
Accepted Article
19
1
peripheral blood T cells using retroviral or lentiviral vectors. These TCR redirected
2
autologous T cells could be subsequently transferred into the chronic HBV patient. Such
3
approaches are employed in treating cancer patients89,90 and the results so far have been
4
very encouraging. In addition, T cells with redirected specificity toward the HBV
5
envelope protein have been shown to recognize and lyse HCC cell lines with natural
6
HBV DNA integration in in vitro and animal models.91 Safety concerns, cost
7
effectiveness, and ethical issues should be fully addressed before this promising approach
8
is adapted to chronic HBV patients.
9
10
Summary and Conclusions
11 12
In summary, HBV acts like a stealth virus early in infection, remaining undetected and
13
spreading until the onset of the adaptive immune response several weeks later. The
14
relative invisibility of HBV to the innate sensing machinery of the cells probably reflects
15
its replication strategy, with the replicating viral genome being sheltered within viral
16
capsid particles in the cytoplasm. On the other hand, HBV can be controlled when
17
properly activated HBV-specific CD8+ T cells enter the liver, recognize antigen, kill
18
infected cells, and secrete IFN-γ, which triggers a broad-based cascade that amplifies the
19
inflammatory process and has noncytopathic antiviral activity against HBV. However,
20
HBV-specific CD8+ T cell responses are subjected to a powerful immunoregulatory
21
mechanism in the liver and the induction of an effective HBV-specific CD8+ T cell
22
response is dependent on activation of pAPCs. Failure to activate pAPCs induces
23
functionally impaired CD8+ T cell responses due to intrahepatic priming by HBVThis article is protected by copyright. All rights reserved.
Accepted Article
20
1
expressing hepatocytes, leading to persistent infection (Figure 2). The precise mechanism
2
by which pAPCs are activated during HBV infection remains to be elucidated but HBV-
3
specific CD4+ T cells probably play a critical role. The functional impairment is
4
mediated, at least partially, by PD-1 signaling but other co-inhibitory molecules are likely
5
to be involved as well. Thus, activating pAPCs and suppressing PD-1 and other negative
6
signaling pathways might have therapeutic potential to treat chronic HBV patients.
7
Adoptive transfer of genetically engineered T cells with redirected specificity towards
8
HBV might represent a powerful immune strategy to cure chronic HBV infection, but
9
safety, cost, and ethical issues should be fully addressed before clinical trials.
10 11
Acknowledgements
12
This work was supported in part by a grant-in-aid from the Ministry of Health, Labour,
13
and Welfare of Japan and the Ministry of Education, Culture, Sports, Science and
14
Technology, Japan.
15
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Accepted Article
21
1 2 3
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32
1
Figure legends
2 3
Figure 1. Schematic representation of immune responses during acute and chronic
4
HBV infections.
5
Figures (a) and (b) schematically represent the time course of serum HBV titer, and
6
immune responses during acute and chronic HBV infections. The upper panel in each
7
figure represents the timing of HBV infection as an arrow, the serum HBV DNA as a
8
dashed black line, and the NK cell response as gray area. The lower panel in each figure
9
represents the timing if CD4 T cell priming to HBV as an arrow, the CD4 T cell response
10
as red area, the CD8 T cell response as green area, the blue line as the antibody response.
11 12
Figure 2. The outcome of T cell priming is determined by the antigen presenting cell
13
population.
14
(a) HBV-specific naive CD8 T cells that are primed by HBV positive hepatocytes receive
15
coinhibitory signals rather than costimulatory signals, resulting in the expansion of
16
functionally impaired CD8 T cells. (b) T cell priming by professional antigen presenting
17
cells, such as DCs, that can deliver costimualtiory signals is presumably required to
18
induce functional HBV-specific CD8 T cell responses.
19 20 21
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1 2
Potential mechanisms of HBV persistence
Neonatal tolerance Peripheral deletion/exhaustion (due to high viral load and negative signaling) Anergy Ignorance Weak CD4 T cell response (associated with low viral titer and HIV infection) Reduced dendritic cell numbers and functions Negative signaling (PD-1/CTLA-4/Tim-3) Immunosuppressive microenvironment (IL-10, TGF-β) Regulatory T cells/Myeloid derived suppressor cells Escape mutations in T cell and B cell epitopes Inhibit antigen processing or presentation Inhibit cytokine signaling
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3 4 5 6
Tabl e1
Accepted Article
34
1
2
Strategies to reinvigorate T cell responses
3 1. Block negative signaling pathways (PD-1, CTLA-4, Tim-3, etc.) 4 2. Stimulate pAPCs (anti-CD40, TLR, etc.) and cytokine signaling 5 3. Redirect peripheral T cells with high avidity TCRs
This article is protected by copyright. All rights reserved.
Table 2
Accepted Article
35
1 2
hepr_12439_f1
3
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Accepted Article
36
1 2
hepr_12439_f2
This article is protected by copyright. All rights reserved.
1
1
Article type: Review Article
2
Received date: 08/08/2014
3
Revised date: 10/13/2014
4
Accepted date: 10/14/2014
5
For: Hepatology Research - Review
6
7
The Immunobiology of Hepatitis B Virus
8
Infection1
9
Running Title: The Immunobiology of Hepatitis B Virus Infection
10 11
Masanori Isogawa, and Yasuhito Tanaka
12 13
Department of Virology & Liver Unit
14
Nagoya City University Graduate School of Medical Science
15 16 17
Corresponding Author: This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/hepr.12439 This article is protected by copyright. All rights reserved.
Accepted Article
2
1
Yasuhito Tanaka, M.D. PhD.
2
Department of Virology & Liver Unit
3
Nagoya City University Graduate School of Medical Science
4
Kawasumi 1, Mizuho-ku,
5
Nagoya 467-8601, Japan
6
Phone: +81-52-853-8191
7
Fax:
8
email: [email protected]
+81-52-842-0021
9
10
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Accepted Article
3
1
Abstract.
2
The adaptive immune response, particularly the virus-specific CD8+ T cell response, is
3
largely responsible for viral clearance and disease pathogenesis during hepatitis B virus
4
(HBV) infection. The HBV-specific CD8+ T cell response is vigorous, polyclonal and
5
multispecific in acutely infected patients who successfully clear the virus and relatively
6
weak and narrowly focused in chronically infected patients. The immunological basis for
7
this dichotomy is unclear. A recent study using HBV transgenic mice and HBV-specific
8
T cell receptor (TCR) transgenic mice suggests that intrahepatic antigen presentation by
9
HBV positive hepatocytes suppresses HBV-specific CD8+ T cell responses through a
10
coinhibitory molecule, programmed cell death 1 (PD-1). In contrast, antigen presentation
11
by activated professional antigen presenting cells (pAPCs) induces functional
12
differentiation of HBV-specific CD8+ T cells. These findings suggest that the outcome of
13
T cell priming is largely dependent on the nature of antigen presenting cells. Another
14
study suggests that the timing of HBV-specific CD4+ T cell priming regulates the
15
magnitude of the HBV-specific CD8+ T cell response. Other factors that could regulate
16
HBV-specific cellular immune responses are high viral loads, mutational epitope
17
inactivation, T cell receptor antagonism and infection of immunologically privileged
18
tissues. However, these pathways become apparent only in the setting of an ineffective
19
cellular immune response, which is therefore the fundamental underlying cause.
20
Understanding the cellular and molecular mechanisms by which HBV evades host
21
immune responses will eventually help develop new immunotherapeutic strategies
22
designed to terminate chronic HBV infection.
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Accepted Article
4
1
Key words: Hepatitis B virus, Immune response, Immunological priming,
2
Immunotherapy, T cells
3
Introduction
4
Hepatitis B virus (HBV) is a partially double stranded DNA virus that causes
5
necroinflammatory liver disease of variable severity.1 More than 240 million people
6
worldwide are persistently infected with HBV. Persistent infection by HBV is often
7
associated with chronic liver disease that can lead to the development of cirrhosis and
8
hepatocellular carcinoma (HCC). HBV is thought to be largely non-cytopathic for the
9
infected hepatocyte under normal circumstances,2,3 but could be cytopathic in
10
immunodeficient chimeric mice carrying human hepatocytes,4 suggesting that
11
unmitigated HBV replication and gene expression trigger cellular stresses and cell death.
12
The immune response mediates the clearance of HBV and disease pathogenesis.2
13
Interestingly, HBV appears to be a poor inducer of innate immune responses, acting as a
14
stealth virus in this regard by evading recognition by innate immune sensor molecules.5
15
Despite the weak innate immune responses, acute HBV infection of adults is usually self-
16
limited and cleared by functional HBV-specific CD8+ T cell responses. The induction of
17
functional HBV-specific CD8+ T cell responses is dependent on early CD4+ T cell
18
priming prior to HBV spread,6 which might explain why patients infected with human
19
immunodeficiency virus (HIV) become persistently infected following horizontal
20
transmission of HBV.7,8 Once chronic HBV infection is established, HBV-specific CD8+
21
T cells are subjected to powerful immunoregulatory mechanisms in the liver. Ample
22
evidence suggests that HBV actively suppresses adoptive immune responses, particularly
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Accepted Article
5
1
HBV-specific CD8+ T cell responses. Reduction of viral antigen expression by antiviral
2
treatment can overcome CD8+ T cell hyporesponsiveness in patients with chronic HBV
3
infection, suggesting that T cells are present in these subjects but suppressed by high viral
4
loads.9 In addition, recent evidence suggests that antigen recognition in the liver
5
suppresses HBV-specific CD8+ T cell effector functions by signaling through a
6
coinhibitory molecule, programmed cell death 1 (PD-1).10,11 Furthermore, several viral
7
proteins have been shown to regulate the adaptive immune response to HBV,12-14 which
8
might explain the varying chronicity rate between genotypes that express different levels
9
of viral antigens.15,16 We herein review our current knowledge of the host immune
10
responses to HBV as well as the cellular and molecular mechanisms by which HBV
11
evades these immune responses. We also discuss how the accumulating knowledge of
12
HBV immunobiology will help us to develop more inventive therapeutic strategies that
13
are designed to reinvigorate HBV-specific immune responses, thereby terminating
14
chronic infection with this deadly virus.
15 16
1. The Innate Immune Response
17
1-1. Absence of an Innate Response by Infected Cells during Acute HBV Infection.
18
Virus infection usually triggers a robust innate immune response that is heralded by rapid
19
induction of IFNα/β by the infected cell.17 Production of IFNα/β induces the
20
transcriptional expression of a large number of interferon inducible genes (ISGs), which
21
in turn exert a variety of intracellular antiviral mechanisms.17,18 Surprisingly, intrahepatic
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Accepted Article
6
1
gene expression profiling in acutely HBV infected chimpanzees revealed that HBV acts
2
like a stealth virus early after infection because it does not induce any cellular gene
3
expression, including ISGs, as it spreads through the liver.5 This contrasts strikingly to
4
the induction of many ISGs during the spread of hepatitis C virus (HCV) that is highly
5
visible to the innate immune system.19 The relative invisibility of HBV to the innate
6
sensing machinery of the cells probably reflects its replication strategy. HBV retains its
7
transcriptional template, namely covalently closed circular DNA (cccDNA), in the
8
nucleus20 and the HBV mRNA is capped and polyadenylated, resembling normal cellular
9
transcripts.21,22 In addition, HBV replicates within viral capsid particles in the
10
cytoplasm20 and is therefore shielded from innate sensing machinery. Thus, the typical
11
widespread expansion of HBV in the liver may reflect the absence of IFNα/β production
12
to which the virus is exquisitely sensitive, as shown in HBV transgenic mice.23,24
13
1-2. The Natural Killer and Natural Killer T Cell Responses during HBV Infection
14
The role of natural killer (NK) cells in acute HBV infection remains largely unknown.
15
Studies that analyzed NK cell functions during acute HBV infection have provided
16
conflicting evidence. A recent study demonstrated that the activation and cytokine
17
producing ability of NK cells were impaired in acute HBV patients,25 while others
18
showed enhanced IFN-γ production by, and cytotoxicity of, NK cells.26,27 The cause of
19
this discrepancy is unclear. On the other hand, NK cells in chronic HBV patients seem
20
generally cytolytic but their cytokine producing ability is selectively impaired.28,29 A
21
recent report demonstrated a strong correlation between liver disease, IFN-α production
22
and NK cell expression of tumor necrosis factor related apoptosis-inducing ligand
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Accepted Article
7
1
(TRAIL) during chronic HBV infection,30 suggesting a pathogenic role of NK cells
2
(Figure 1, grey area). Interestingly, NK cells seem highly immunoregulatory as well,
3
because they can apparently delete HBV-specific, but not cytomegalovirus (CMV)-
4
specific, CD8+ T cells through TRAIL-TRAIL receptor 2 (TRAIL-R2) interaction.31
5
Taken together, NK cells appear to exert a detrimental effect on the host during chronic
6
HBV infection. The precise role of NK cells during chronic HBV infection must be fully
7
elucidated to develop new NK cell based therapeutic approaches against chronic HBV
8
infections. The role of natural killer T (NKT) cells during natural HBV infection is even
9
less clear. Therapeutic potential of NKT cell activation against HBV infection was 32
10
initially indicated in HBV transgenic mice model
11
and woodchuck hepatitis virus (WHV) suggested that NKT cells were activated soon
12
after infection.33,34 However, the degree of NKT cell activation was relatively modest in
13
both cases, and the physiological importance of such activation has not been addressed in
14
these studies. Studies with several mouse models provided more convincing evidence that
15
CD1d-restricted NKT cells are important for the control of HBV replication and gene
16
expression.35,36 It is well known, however, that hepatic NKT cell repertoires are
17
phenotypically and functionally very different between humans and mice.37 Further
18
studies are required to confirm the role of NKT cell during HBV infections.
19
2. The Adoptive Immune Response
20
2-1. The Antibody Response
21
Antibodies to the HBV envelope antigen (HBsAg) neutralize HBV infection presumably
22
by preventing virus attachment to hepatocytes. They also facilitate viral clearance by This article is protected by copyright. All rights reserved.
, and subsequent studies with HBV
Accepted Article
8
1
complexing with free viral particles and removing them from the circulation. The
2
induction of anti-HBs by immunization is a T cell-dependent process and requires CD4+
3
T cell help.38 While anti-HBs neutralizing antibodies are essential for providing
4
protection after vaccination, their importance in resolving natural HBV infection is
5
somewhat underappreciated, mostly because the appearance of anti-HBs occurs relatively
6
late after HBV exposure39 (Figure 1, blue lines). It is generally believed that anti-HBs
7
neutralizing antibodies prevent viral spread from the rare cells that remain infected, even
8
after resolution of HBV infection. Supporting this notion, HBV is frequently re-activated
9
long after resolution of infection in patients who receive conventional chemotherapy or a
10
newly developed monoclonal antibody (Rituximab) therapy that targets the CD20 antigen
11
expressed on the surface of normal and malignant B lymphocytes.40 More detailed
12
analysis of HBV-specific B cell responses might reveal a heretofore-unappreciated role
13
of humoral responses in the control of HBV infection.
14
2-2. The CD4+ T Cell Response
15
Ample evidence suggests a clear relationship between the CD4+ T cell response to HBV
16
and the outcome of HBV infection. For example, the peripheral blood CD4+ T cell
17
response to HBV is vigorous and multispecific in patients with acute hepatitis who
18
ultimately clear the virus, while it is relatively weak in chronic HBV patients39,41,42
19
(Figure 1, red areas). Despite the association between a strong CD4+ T cell response and
20
viral clearance, CD4+ T cell depletion at the peak of HBV infection had no effect on viral
21
replication and liver disease in infected chimpanzees.43 In contrast, CD4+ T cell depletion
22
before HBV infection resulted in quantitatively and qualitatively reduced HBV-specific
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1
CD8+ T cell responses, leading to persistent HBV infection.6 These results suggest that
2
CD4+ T cells probably contribute indirectly to the control of HBV infection by
3
facilitating the induction and maintenance of the virus-specific CD8+ T cell responses. It
4
is likely that CD4+ T cells help induce functional HBV-specific CD8+ T cell responses by
5
producing IL-2 that is essential for T cell proliferation and by activating professional
6
antigen presenting cells (pAPCs) that are capable of providing co-stimulation to T cells.44
7
In contrast, in the absence of early CD4+ T cell responses, CD8+ T cell priming could
8
occur in the liver, resulting in T cell inactivation, tolerance or apoptosis,11,45,46 as
9
summarized later in this review. This requirement of CD4+ T cells for the induction of
10
HBV-specific CD8+ T cells is one of the reasons patients infected with HIV (low CD4
11
count) become persistently infected after horizontal transmission of HBV7,8 and why
12
genetic variants in the HLA-DP locus are associated with the risk of developing chronic
13
HBV infection.47 Interestingly, early CD4+ T cell priming does not occur when the size of
14
inoculum is small (i.e. 1 to 10 copies) and the appearance of HBV DNA is delayed after
15
infection,6 suggesting that slow spread of HBV might facilitate chronicity, as often
16
observed with genotype A infections.15,16 Interestingly, recent studies indicated that the
17
frequency of IL-17 producing CD4 T cells (Th-17 cells) is increased in chronic HBV
18
patients,
19
liver disease.49 These results suggest that CD4 T cells not only help induce antiviral
20
cellular immune responses during acute HBV infections, but also mediate inflammatory
21
responses during chronic HBV infection.
22
2-3. The CD8+ T Cell Response
48
and IL-17 and another Th-17 related cytokine IL-22 are shown to exacerbate
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Accepted Article
10
1
The HBV-specific CD8+ T cell response plays a fundamental role in viral clearance and
2
liver disease. A vigorous polyclonal CD8+ T cell response is readily detectable in the
3
peripheral blood of patients with acute hepatitis B who ultimately clear the virus. In
4
contrast, the peripheral blood T cell response in chronically infected patients is weak and
5
narrowly focused2,50 (Figure 1, green areas). The livers of these patients contain virus-
6
specific T cells that likely contribute to disease pathogenesis but, for functional and/or
7
quantitative reasons, are unable to clear the infection. Interestingly, a recent study that
8
examined the relationship between the number of intrahepatic HBV-specific CD8+ T
9
cells, extent of liver disease, and levels of HBV replication in chronically infected
10
patients indicated that inhibition of virus replication could be independent of liver
11
damage and that the functionality of HBV-specific CD8+ T cells was more important than
12
the number of T cells in controlling HBV replication.51 Experiments in chimpanzees have
13
shown that the viral clearance and the onset of liver disease coincide with the
14
accumulation of virus-specific CD8+ T cells and the induction of IFN-γ and IFN-
15
inducible genes in the liver.52 Importantly, depletion of CD8+ T cells at the peak of
16
viremia delays viral clearance and the onset of viral hepatitis until the T cells return,
17
providing the most definitive evidence that viral clearance and liver disease are mediated
18
by virus specific CD8+ T cells.43
19
3. Non-cytolytic and cytolytic T cell effector functions emerge alternately during
20
HBV clearance
21
It is widely believed that virus specific CD8+ T cells clear the viral infection by killing
22
infected cells. However, CD8+ T cell killing is an inefficient process, requiring direct
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Accepted Article
11
1
physical contact between the CD8+ T cells and the infected cells. Therefore, it may not be
2
possible for HBV-specific CD8+ T cells to kill all the HBV infected cells if the CD8+ T
3
cells are greatly outnumbered, as occurs during HBV infection in which as many as 1011
4
hepatocytes can be infected.53 Hence, although the liver disease in HBV infection is
5
clearly due to the cytopathic activity of the CD8+ T cells, viral clearance may require
6
more efficient CD8+ T cell functions than killing. Important insights into the pathogenic
7
and noncytopathic antiviral functions of the CD8+ T cell response have come from
8
studies of HBV transgenic mice that develop an acute necroinflammatory liver disease
9
after adoptive transfer of HBV-specific effector or effector memory CD8+ T cells that
10
were generated by immunizing non-transgenic mice.10,54 In that model, the HBV-specific
11
CD8+ T cells rapidly enter the liver and recognize viral antigen and secrete interferon
12
gamma (IFN-γ), which non-cytopathically inhibits HBV replication in the remaining
13
hepatocytes54 by preventing the assembly of HBV RNA-containing capsids in the
14
cytoplasm23,55 in a proteasome-56 and kinase-dependent57 process. During this remarkable
15
process, the viral nucleocapsids disappear from the cytoplasm of the hepatocytes, yet the
16
hepatocytes remain perfectly healthy.54 Antibody blocking and knockout experiments in
17
the HBV transgenic mouse model further demonstrated that the cytopathic and antiviral
18
functions of CD8+ T cells are completely independent of each other.58,59 Following the
19
secretion of IFN-γ and the suppression of HBV replication, HBV-specific CD8+ T cells
20
expand vigorously in situ. Surprisingly, however, the ability of HBV-specific CD8+ T
21
cells to produce IFN-γ is quickly down-regulated as they expand. Despite the suppression
22
of IFN-γ secretion, the cytotoxic capabilities of the HBV-specific CD8+ T cells increase
23
over time, causing necroinflammatory liver disease, and complete inhibition of HBV
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Accepted Article
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1
mRNA expression.10 Taken together, these results suggest that, in the context of a
2
vigorous immune response during acute HBV infection, the sequential and altering
3
patterns of non-cytolytic and cytolytic effector function, coupled with T cell expansion
4
and contraction, cooperate to minimize the extent of tissue injury required for viral
5
clearance. Conversely, we suggest that diminution of the CD8+ T cell response or poor
6
coordination of these responses could lead to viral persistence and varying degrees of
7
chronic liver disease.
8 9
4. Where, how, and what kind of HBV-specific CD8+ T cells are generated?
10
The results described above were generated by adoptively transferring splenocytes from
11
HBV-immunized mice to HBV transgenic mice. While these results suggest that
12
intrahepatic antigen recognition has a profound impact on the distribution, expansion, and
13
effector functions of primed effector-memory CD8+ T cells, they may not reflect the
14
events that occur when a naïve individual becomes infected with HBV, because
15
immunologically naïve T cells are known to behave very differently than memory T
16
cells.60-62 Accordingly, we generated transgenic mice whose CD8+ T cells express T cell
17
receptors (TCRs) specific for HBV, to study where and how immunologically naïve T
18
cells are primed in response to intrahepatic HBV.
19
When HBV-specific naïve CD8+ T cells were adoptively transferred into HBV transgenic
20
mice, they were predominantly activated rapidly in the liver, independent of T cell
21
homing to lymphoid organs, suggesting that HBV-specific native T cells are primed in
22
the liver.11 While this intrahepatic priming of HBV-specific CD8+ T cells challenges the
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Accepted Article
13
1
current T cell priming model postulating that naïve T cells are primed by activated
2
professional antigen presenting cells (pAPCs) in lymphoid organs,63,64 several studies
3
previously indicated that the liver is an exception to this rule.45,46,65 This rather unusual
4
ability of the liver to prime naïve T cells presumably reflects the unique architecture of
5
the hepatic sinusoid, which is characterized by a discontinuous endothelium, the absence
6
of a basement membrane, and a very slow flow rate,66 allowing circulating T cells to
7
make prolonged direct contact with resident liver cells, including hepatocytes.
8
Furthermore, the liver is replete with diverse and unique antigen presenting cell
9
populations, including liver sinusoidal endothelial cells (LSECs), hepatic stellate cells
10
(HSCs), Kupffer cells, and conventional and plasmacytoid dendritic cells, all of which
11
are capable of priming and/or tolerizing naïve T cells.67 In our model, HBV-specific
12
CD8+ T cells are presumably primed by HBV-expressing hepatocytes, because HBV-
13
specific CD8+ T cells were activated when co-cultured with hepatocytes isolated from
14
HBV transgenic mice but not with LSECs, liver residential dendritic cells (DCs) or
15
Kupffer cells. Importantly, the intrahepatically primed HBV-specific CD8+ T cells
16
expand vigorously in the liver but are functionally impaired because they do not express
17
IFN-γ or the cytolytic granule Granzyme B, suggesting that intrahepatic priming by
18
HBV-expressing hepatocytes expands functionally defective CD8+ T cells. Consequently,
19
such intrahepatically primed T cells are incapable of suppressing HBV gene expression
20
or inducing liver disease.11 Interestingly, functionally defective HBV-specific CD8+ T
21
cells expressed a co-inhibitory molecule, PD-1, after antigen recognition in the liver.11
22
Furthermore, a significant fraction of PD-1 deficient HBV-specific naïve T cells
23
expanded more vigorously than PD-1 positive controls, and differentiated into effector T
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Accepted Article
14
1
cells capable of producing IFN-γ, as well as Granzyme B, which in turn suppressed HBV
2
gene expression and caused liver disease.11 Collectively, these results suggest that PD-1
3
signaling impairs the effector functions of HBV-specific CD8+ T cells. Importantly, PD-1
4
expression was also observed on HBV-specific CD8+ T cells in chronic HBV patients,68
5
indicating that PD-1 mediated functional suppression is active during natural HBV
6
infection. Taken together, these results suggest that intrahepatic priming induces T cell
7
tolerance and such tolerance is at least partially mediated by PD-1 signaling. This PD-1
8
meditated intrahepatic suppression might be the underlying cause of the weak T cell
9
responsiveness during chronic HBV infection.
10 11
5. Other Mechanisms of HBV Persistence
12
The potential mechanisms by which HBV evades the immune responses are listed on
13
Table 1.
14
following mother-infant transmission.69,70 The underlying cause of adult onset chronic
15
HBV infection is probably multifaceted. Potential contributing factors include mutational
16
escape leading to inactivation of B-cell and T-cell epitopes and specific inhibition of the
17
adaptive immune response by viral proteins.71 For example, HBeAg has been shown to
18
suppress the antibody and T cell response to HBcAg in T cell receptor transgenic
19
mice.12,13 The immunosuppressive potential of HBeAg might explain the clinical
20
observation that viral mutations precluding the production of HBeAg are often associated
21
with exacerbations of liver disease and, sometimes, even with viral clearance from
22
chronically infected patients.72,73 HBsAg might also prevent immune elimination of
Neonatal tolerance to HBV is probably responsible for viral persistence
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Accepted Article
15
1
infected cells by functioning as a high dose tolerogen, because extremely high serum
2
HBsAg titers in the mg/ml range are often seen in chronically infected patients74,75 and
3
chronically infected patients have an absence or subnormal levels of HBsAg-specific
4
CD8+ T cells.75 In addition, HBV X protein, a transcriptional transactivator that is
5
required for the initiation of infection,76,77 can inhibit cellular proteasome activity when it
6
is overexpressed78 and might interfere with antigen processing and presentation. Finally,
7
genotypic variation clearly influences the outcome of HBV infection. Up to 10% of adult
8
onset infections with genotype A become persistent, while less than 5% of infections with
9
other genotypes do so.15 The precise mechanism for the difference is unclear, but the high
10
levels of HBsAg expression and low replication activity of genotype A infections might
11
facilitate immune evasion.16
12
In both neonate and adult onset of chronic HBV infections, weak CD4 and CD8 T cell
13
responses are clearly responsible for the viral persistence. The mechanism that suppresses
14
HBV-specific immune responses during chronic HBV infection is likely multifactorial. In
15
addition to PD-1, other negative signaling molecules, such as cytotoxic T lymphocyte
16
antigen-4 (CTLA-4) and T-cell immunoglobulin and mucin 3 (Tim-3), are expressed on
17
functionally compromised HBV-specific CD8 T cells in chronic HBV patients.
18
example, a fraction of CD8 T cells in patients with CHB express the coinhibitory
19
molecule CTLA-4, and its expression level is correlated with serum HBV DNA titer and
20
proapoptotic protein Bim expression. Blockade of CTLA-4 increases the expansion of
21
interferon gamma (IFN-γ) producing HBV-specific CD8 T cells in vitro. Similarly, Tim-
22
3 is also highly expressed on functionally impaired HBV-specific CD8 T cells in chronic
23
HBV patients, and the blockade of Tim-3 in vitro restored effector functions of the CD8 This article is protected by copyright. All rights reserved.
79
For
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16
79
1
T cells,
suggesting that multiple layers of negative co-regulation contribute to T cell
2
exhaustion in chronic HBV infection. Interestingly, Tim-3 expressing CD8 T cells appear
3
to have a immunosuppressive function similar to regulatory T cells (Tregs), limiting
4
proliferation of antigen-specific effector T cells in vitro and in vivo.80 Extrinsic factors
5
could also contribute to the reduced T cell responses during chronic HBV infection. The
6
livers of chronic HBV patients have been shown to express elevated levels of IL-10,81
7
TGF-β,82 and arginase,83 all of which are known to suppress cellular immune responses.
8
These immunosuppressive soluble factors might be produced by regulatory cells, as the
9
frequency of regulatory T cells (Tregs) is increased in chronic HBV patients.84
10
Interestingly, recently identified myeloid-derived suppressor cells (MDSCs) are shown to
11
induce T cell tolerances in mouse models of HBV infection.85,86 In addition, several
12
studies suggest that the number and functionality of DCs are reduced in chronic HBV
13
patients compared with healthy donors, and the reduction of HBV DNA titer by a
14
nucleotide analogue adefovir dipivoxil significantly restored the number and
15
functionality of DCs. Caution should be exercised, however, to interpret the physiological
16
relevance of the antigen nonspecific immunosuppression during chronic HBV infections,
17
because chronic HBV patients are no more susceptible than healthy people to other
18
infectious agents such as influenza virus, etc.
19 20
6. New immunotherapeutic strategies to cure chronic HBV infections
21
The management of chronic HBV has improved significantly in the last decade, mainly
22
because of the development of very effective and safe nucleoside analogues (NAs) that This article is protected by copyright. All rights reserved.
Accepted Article
17
1
primarily inhibit reverse transcription and DNA replication.87 Although NAs strongly
2
inhibit HBV replication and have much fewer side effects than IFN-based therapies, they
3
usually do not achieve sustained viral suppression. Failure to sustain viral suppression by
4
NAs presumably reflects the persistence of cccDNA, which is untouched by reverse
5
transcriptase inhibitors. Because the elimination of cccDNA usually requires the turnover
6
of infected hepatocytes, the induction of functional HBV-specific CD8+ T cells that can
7
specifically kill virus infected hepatocytes has been deemed the most promising approach
8
to cure chronic HBV infections. Immunization of chronically infected patients is the most
9
straightforward approach to boost HBV-specific CD8+ T cell responses. Several
10
strategies have been tested in clinical trials with disappointing results,88 indicating that
11
more inventive approaches are required to induce functional HBV-specific CD8+ T cell
12
responses. There are three approaches that could effectively reinvigorate HBV-specific
13
CD8+ T cell responses (Table 2).
14
The first approach is to block signaling pathways that negatively regulate HBV-specific
15
CD8+ T cell responses. As described in section 4, we showed that blockade of PD-1
16
signaling induced functional differentiation of intrahepatically primed, HBV-specific
17
CD8 T cells in an HBV transgenic mouse model, indicating that PD-1 could be a new
18
therapeutic target to treat chronic HBV infection. HBV-specific CD8 T cell responses
19
might be restored by blocking other negative signaling molecules, such as CTLA-4, Tim-
20
3, and arginase, as well as by depleting Tregs. More evidence is needed to support this
21
approach.
22
The second approach is to provide extra stimulus to override negative signaling that
23
suppresses effector functions of HBV-specific CD8 T cells. One of the promising
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Accepted Article
18
1
approaches is to activate professional antigen presenting cells (pAPCs), particularly
2
dendritic cells (DCs). Activated DCs are capable of providing the second and third
3
signals necessary for the functional differentiation of HBV-specific CD8+ T cells. Indeed,
4
we showed that activation of myeloid DCs (mDC) with an agonistic anti-CD40 antibody
5
(αCD40) restored HBV-specific CD8+ T cell responses that are otherwise suppressed by
6
PD-1 signaling.11 Interestingly, PD-1 expression on the functional HBV-specific CD8+ T
7
cells is strongly down-regulated in αCD40 treated HBV transgenic mice. These results
8
suggest that activation of myeloid dendritic cells directly or indirectly suppresses PD-1
9
expression, thereby rescuing HBV-specific CD8+ T cells from PD-1 mediated functional
10
suppression. The data also illustrate the therapeutic potential of mDC activation by
11
αCD40 for the treatment of chronic HBV infection. Other signaling pathways that
12
stimulate pAPCs, such as toll-like receptor (TLR) signaling pathways, should be tested
13
for their ability to restore HBV-specific CD8+ T cell responses. In addition, cytokines
14
required for T cell expansion and differentiation, particularly common gamma-chain
15
cytokines such as IL-2, IL-4, IL-7, IL-15, and IL-21, could serve as effective adjuvants
16
for therapeutic vaccine against chronic HBV infections.
17
The third approach is to genetically engineer peripheral blood T cells from chronic HBV
18
patients to express T cell receptors (TCRs) specific for HBV-derived epitopes. The first
19
and second strategies described above require the presence of HBV-specific CD8+ T cells
20
that can efficiently recognize HBV-derived T cell epitopes presented on infected
21
hepatocytes. However, such T cells are often deleted in chronic HBV patients after years
22
of exposure to HBV antigens. To circumvent this problem, genes encoding TCRs that
23
efficiently recognize HBV derived epitopes can be introduced into the patient’s
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Accepted Article
19
1
peripheral blood T cells using retroviral or lentiviral vectors. These TCR redirected
2
autologous T cells could be subsequently transferred into the chronic HBV patient. Such
3
approaches are employed in treating cancer patients89,90 and the results so far have been
4
very encouraging. In addition, T cells with redirected specificity toward the HBV
5
envelope protein have been shown to recognize and lyse HCC cell lines with natural
6
HBV DNA integration in in vitro and animal models.91 Safety concerns, cost
7
effectiveness, and ethical issues should be fully addressed before this promising approach
8
is adapted to chronic HBV patients.
9
10
Summary and Conclusions
11 12
In summary, HBV acts like a stealth virus early in infection, remaining undetected and
13
spreading until the onset of the adaptive immune response several weeks later. The
14
relative invisibility of HBV to the innate sensing machinery of the cells probably reflects
15
its replication strategy, with the replicating viral genome being sheltered within viral
16
capsid particles in the cytoplasm. On the other hand, HBV can be controlled when
17
properly activated HBV-specific CD8+ T cells enter the liver, recognize antigen, kill
18
infected cells, and secrete IFN-γ, which triggers a broad-based cascade that amplifies the
19
inflammatory process and has noncytopathic antiviral activity against HBV. However,
20
HBV-specific CD8+ T cell responses are subjected to a powerful immunoregulatory
21
mechanism in the liver and the induction of an effective HBV-specific CD8+ T cell
22
response is dependent on activation of pAPCs. Failure to activate pAPCs induces
23
functionally impaired CD8+ T cell responses due to intrahepatic priming by HBVThis article is protected by copyright. All rights reserved.
Accepted Article
20
1
expressing hepatocytes, leading to persistent infection (Figure 2). The precise mechanism
2
by which pAPCs are activated during HBV infection remains to be elucidated but HBV-
3
specific CD4+ T cells probably play a critical role. The functional impairment is
4
mediated, at least partially, by PD-1 signaling but other co-inhibitory molecules are likely
5
to be involved as well. Thus, activating pAPCs and suppressing PD-1 and other negative
6
signaling pathways might have therapeutic potential to treat chronic HBV patients.
7
Adoptive transfer of genetically engineered T cells with redirected specificity towards
8
HBV might represent a powerful immune strategy to cure chronic HBV infection, but
9
safety, cost, and ethical issues should be fully addressed before clinical trials.
10 11
Acknowledgements
12
This work was supported in part by a grant-in-aid from the Ministry of Health, Labour,
13
and Welfare of Japan and the Ministry of Education, Culture, Sports, Science and
14
Technology, Japan.
15
This article is protected by copyright. All rights reserved.
Accepted Article
21
1 2 3
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32
1
Figure legends
2 3
Figure 1. Schematic representation of immune responses during acute and chronic
4
HBV infections.
5
Figures (a) and (b) schematically represent the time course of serum HBV titer, and
6
immune responses during acute and chronic HBV infections. The upper panel in each
7
figure represents the timing of HBV infection as an arrow, the serum HBV DNA as a
8
dashed black line, and the NK cell response as gray area. The lower panel in each figure
9
represents the timing if CD4 T cell priming to HBV as an arrow, the CD4 T cell response
10
as red area, the CD8 T cell response as green area, the blue line as the antibody response.
11 12
Figure 2. The outcome of T cell priming is determined by the antigen presenting cell
13
population.
14
(a) HBV-specific naive CD8 T cells that are primed by HBV positive hepatocytes receive
15
coinhibitory signals rather than costimulatory signals, resulting in the expansion of
16
functionally impaired CD8 T cells. (b) T cell priming by professional antigen presenting
17
cells, such as DCs, that can deliver costimualtiory signals is presumably required to
18
induce functional HBV-specific CD8 T cell responses.
19 20 21
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Accepted Article
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1 2
Potential mechanisms of HBV persistence
Neonatal tolerance Peripheral deletion/exhaustion (due to high viral load and negative signaling) Anergy Ignorance Weak CD4 T cell response (associated with low viral titer and HIV infection) Reduced dendritic cell numbers and functions Negative signaling (PD-1/CTLA-4/Tim-3) Immunosuppressive microenvironment (IL-10, TGF-β) Regulatory T cells/Myeloid derived suppressor cells Escape mutations in T cell and B cell epitopes Inhibit antigen processing or presentation Inhibit cytokine signaling
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3 4 5 6
Tabl e1
Accepted Article
34
1
2
Strategies to reinvigorate T cell responses
3 1. Block negative signaling pathways (PD-1, CTLA-4, Tim-3, etc.) 4 2. Stimulate pAPCs (anti-CD40, TLR, etc.) and cytokine signaling 5 3. Redirect peripheral T cells with high avidity TCRs
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Table 2
Accepted Article
35
1 2
hepr_12439_f1
3
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Accepted Article
36
1 2
hepr_12439_f2
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