Thanks to additional intravital microscopy studies it was also recently shown that after the initial platelet-dependent arrest, effector CD8+ T cells actively crawl along liver sinusoids (at an average speed of about 10 μm/min) and extend cellular protrusions through sinusoidal endothelial fenestrate to probe underlying hepatocytes for the presence of antigen [34]. Unexpectedly, hepatocellular recognition of HBV antigens leading to cytokine production and hepatocyte killing occurs in a diapedesis-independent manner (Figs. 4.2 and 4.3), i.e., when effector CD8+ T cells are still intravascular and before they extravasate into the parenchyma [34]. Notably, CD8+ T cell antigen recognition and effector functions are inhibited by sinusoidal defenestration and capillarization—two pathological conditions that typify liver fibrosis (see below)—suggesting that the process of liver fibrosis might reduce CD8+ T cell immune surveillance towards infected or transformed hepatocytes [34]. Altogether, the abovementioned studies highlight the notion that rather peculiar mechanisms regulate the ways by which HBV-specific CD8+ T cells recognize hepatocellular antigens and perform effector functions aimed at viral clearance.

There is little doubt that target cell killing by effector CD8+ T cells represents a highly relevant means by which effector CD8+ T cells contribute to HBV clearance. Target cell killing, however, is an intrinsically inefficient process, requiring physical contact between the infected hepatocyte and the T cell. As such, it may not be possible for the effector CD8+ T cells to reach and kill all infected hepatocytes, particularly if one considers that (a) all of the hepatocytes (~10¹¹ cells) are routinely infected during HBV infection in chimpanzees and (b) relatively few virus-specific effector CD8+ T cells circulate in the bloodstream of these animals [24]. Thus, viral clearance may require more efficient T cell functions than killing. Important insights into such functions have spawned from studies in HBV replication-competent transgenic mice. There, it was demonstrated that rapid inhibition of HBV replication by effector CD8+ T cells is mostly mediated by noncytolytic mechanisms involving the local production of IFN-γ by these cells [37]. Indeed, IFN-γ, largely via its ability to induce nitric oxide in the liver [38], was shown to prevent the hepatocellular assembly of replication-competent HBV RNA-containing capsids in a proteasome- and kinase-dependent manner [39, 40]. During this process, the levels of viral nucleocapsids in the cytoplasm of hepatocytes rapidly decline, and viral RNAs are destabilized in the hepatocellular nucleus by an SSB/La-dependent mechanism [41–43]. Antibody blocking and knockout experiments in the HBV transgenic mouse model further demonstrated that the cytolytic and antiviral functions of effector CD8+ T cells are completely independent of each other [37]. Thus, effector CD8+ T cells have the potential to inhibit viral gene expression and replication noncytopathically. Similar antiviral activities were recently shown to extend, via a gradient of IFN-γ, more than 80 μm beyond the site of antigen presentation, promoting pathogen clearance in the absence of immunological synapse formation [44].

Of note, additional work in the HBV transgenic mouse model also indicates that, upon entry into the liver, effector CD8+ T cells rapidly lose the capacity of secreting IFN-γ (i.e., the IFN-γ-producing phenotype is maintained only for the few days during which HBV antigens are cleared from the liver), and this is followed by the intrahepatic expansion of IFN-γ- non-producing virus-specific effector CD8+ T cells with unaltered cytotoxic capabilities [45]. These results suggest that sustained antigen stimulation, as occurs during chronic infection, may create an environment in which antiviral (i.e., production of IFN-γ) but not pathogenic (i.e., killing of hepatocytes) functions of intrahepatic virus-specific effector CD8+ T cells are relatively impaired (see below).

Even when the adaptive immune response effectively clears a virus, immune-mediated mechanisms can cause significant injury to host tissues. Besides, viruses like HBV can persist in the presence of an active adaptive immune response, predisposing the host to chronic tissue damage (see below). Thus, the balance between the protective and the harmful effects of immunity in some cases clearly shifts to immunity being the primary cause of tissue pathology. In these cases, virus-induced tissue damage is referred to as immunopathology.

Virus-specific T cells can promote immunopathology by directly killing infected cells, by releasing cytokines or other soluble mediators with intrinsic cytotoxic properties and, also, by recruiting antigen-nonspecific inflammatory cells that have the potential to amplify tissue damage. Studies in HBV-infected patients have indeed shown that hepatic infiltrates contain a large antigen-nonspecific component, whose extent correlates with the degree of liver damage [46]. This observation goes along with data obtained in mouse models of HBV immunopathogenesis, where it was shown that the initial apoptotic process triggered by passively transferred virus-specific effector CD8+ T cells involves only a relatively small number of hepatocytes. Using these same models, it was also shown that Kupffer cells (the resident macrophages of the liver) rapidly remove apoptotic hepatocytes in a manner largely dependent on scavenger receptors [47]. As time progresses, though, apoptotic hepatocytes not readily removed by Kupffer cells become secondarily necrotic and release damage-associated molecular pattern molecules (DAMPs) such as the high-mobility group box 1 (HMGB1 ) protein [48]. HMGB1 is an abundant nuclear protein acting as an architectural chromatin-binding factor that can be passively released by necrotic, but not apoptotic, cells [49]. Once discharged by necrotic hepatocytes, HMGB1 chemo-attracts mainly polymorphonuclear cells (e.g., neutrophils), the first antigen-nonspecific inflammatory cells arriving at the site of disease [48]. Neutrophil activation leading to production of matrix metalloproteinases (MMPs) rapidly degrades matrix components (e.g., collagen, laminin, fibronectin, and proteoglycans) that are deposited de novo by stellate cells, myofibroblasts and fibroblasts during the process of liver repair [50]. In turn, these matrix-degrading events favor the intrahepatic arrival of numerous antigen-nonspecific mononuclear cells (e.g., antigen-nonspecific CD8+ and CD4 T cells, B cells, monocytes), which respond to their own chemoattractants (mostly chemokines such as CXCL9 and CXCL10 produced locally by parenchymal and non-parenchymal liver cells [51]) and exacerbate disease severity [50]. The pathogenic mechanisms whereby antigen-nonspecific mononuclear cells thus recruited induce organ damage are not well understood and may involve the local production of pro-inflammatory and cytotoxic mediators (including TNF-α, perforin, hydrogen peroxide, superoxide anion, and nitric oxide) by these cells. Moreover, antigen-nonspecific mononuclear cells (in particular NK cells, NKT cells, and T-helper cells) and platelets express Fas-L, a glycoprotein that triggers hepatocellular apoptosis by ligating Fas on the hepatocyte membrane [1].

Observations in acutely infected chimpanzees depleted of CD4+ T cells at the peak of acute HBV infection indicate that the liver disease in this animal is comparable to that detected in immunologically unmanipulated controls [24]. Thus, CD4+ T helper cells may contribute to HBV pathogenesis mainly by facilitating the induction and maintenance of virus-specific effector CD8+ T cells, as has been suggested for other viruses such HCV [52]. In keeping with this, relatively vigorous HBV-specific T helper responses are always associated with quantitatively and qualitatively significant effector CD8+ T cell responses in humans and chimpanzees that resolve HBV infection [1].

Based on the studies abovementioned, it is apparent that the adaptive immune response to HBV, particularly the CD8+ T cell response, plays key roles in viral clearance and liver disease. Thus, it is reasonable to assume that HBV persistence demands that such response must be either not induced or deficient, or if present it must be overwhelmed, counteracted or evaded.

Notably, both viral and host factors can be involved in the establishment of chronicity. Among the former, it has been suggested that circulating hepatitis B e-antigen (HBeAg) functions as a tolerogenic protein that induces anergy of HBcAg/HBeAg cross-reactive T cells [53, 54]. The capacity of circulating HBeAg to functionally suppress HBcAg/HBeAg-specific T cell responses may explain clinical observations whereby HBeAg-negative variants are frequently cleared following neonatal exposure and they are usually associated with more severe courses of liver disease in adults [55]. Through its capacity to function as high dose tolerogen, circulating hepatitis B surface antigen (HBsAg) is also considered a viral factor that retains immune suppressive potential [55]. Indeed, the extremely high-serum HBsAg titers observed in certain chronically infected patients are often associated with absence of peripheral HBsAg-specific T cell responses [55]. Mutational inactivation of HBV-derived B cell or T cell epitopes is also thought to facilitate viral persistence [55], albeit this process is likely to play a more prominent role during infection with viruses (such as HCV) that intrinsically possess a much higher mutation rate. Nonetheless, mutations involving epitope residues that anergize or antagonize recognition by the T cell receptor have been reported to arise during HBV infections evolving towards a chronic phase [56].

Among the latter, the notion that immune tolerance is likely responsible for viral persistence in most neonatal HBV infections coupled with the fact that a vigorous, multispecific, and polyclonal cellular immune response is associated with viral clearance in immunocompetent adults strongly suggest that host factors significantly determine infection outcome [1]. Why T cell responses are quantitatively weak and qualitatively inadequate to terminate infection in some adult onset infections remains to be fully determined. An increasing body of studies in HBV infected patients or surrogate animal models suggests that several nonexclusive mechanisms favor viral persistence; they include the inhibition of functional T cell priming as a results of antigen presentation by the hepatocyte [57] or the induction of anergy and exhaustion of initially vigorous T cell responses as a result of (a) antigen overload and excessive T cell stimulation [1], (b) action of regulatory T cells [1], and (c) activation of negative regulatory pathways in T cells (such as those promoted by programmed cell death protein 1 [PD1], cytotoxic T-lymphocyte antigen-4 [CTLA-4], or T-cell immunoglobulin and mucin 3 (Tim-3) [1, 55, 58, 59]. Additional factors contributing to suppression of pre-existing T-cell responses during chronic HBV infection may relate to the relative intrahepatic abundance of selected cytokines (e.g., IL-10 or TGF-β) or enzymes (e.g., arginase) possessing immunosuppressive potential [60–62]. All together, these results indicate that both primary and secondary immunological unresponsiveness to HBV presumably occurs, and this likely contributes to the establishment of persistent infection.

As mentioned above, HBV has the capacity to persist in face of an active, albeit functionally inefficient, adaptive immune response. Indeed, chronic HBV infection could be characterized by a dysfunctional virus-specific CD8+ T cell response that fails to eliminate HBV from the liver but maintains continuous cycles of low-level hepatocellular injury, promoting the development of liver fibrosis/cirrhosis and, ultimately, HCC.

Of note, liver fibrosis and cirrhosis are pathological conditions characterized by an imbalance between fibrogenesis and fibrolysis, resulting in the excessive intrahepatic deposition by stellate cells, myofibroblasts, and fibroblasts of extracellular matrix (ECM) that is qualitatively different in its composition and organization from that of normal liver repair [63]. As a result of this process, a dense, reticulated ECM is initially deposited around the portal areas of the liver and, as a function of time, the fibrosis progressively expands into the lobules with the formation of septa that can eventually connect portal and central veins [63]. Liver cirrhosis represents the final stage of fibrosis in which fibrous septa surround nodules of regenerating hepatocytes, causing profound architectural distortion of the liver, functional insufficiency and diversion of venous blood containing intestinal toxins into the systemic circulation [63]. As mentioned above, liver fibrosis and cirrhosis are also associated with a reduction in number and size of sinusoidal fenestrae (a process often described as “ defenestration” of the hepatic sinusoids) and the formation of a basal membrane separating hepatocytes from sinusoidal blood (a process often described as “capillarization” of the hepatic sinusoids) [64]. These events alter the normal exchange of soluble factors between blood and hepatocytes [64] and worsen HBV morbidity (possibly, as stated earlier, because of reduced immune surveillance). The severity and duration of chronic liver disease positively influence liver fibrosis/cirrhosis, and the same is true for HCC where almost all cases take place after many years (usually several decades) of a chronic hepatitis characterized by a sustained liver disease with associated hepatocellular regeneration (i.e., cellular DNA synthesis) and inflammation (i.e., the production of mutagens) [1]. Chronic liver cell injury, therefore, also appears to be a premalignant state promoting cellular processes, like enhanced cellular DNA synthesis and production of inflammatory mutagens, which are oncogenic. Persistence of these events for a sufficiently long period of time results in the random/multiple genetic and chromosomal alterations that contribute to HCC development [1]. Consistent with this, it has been shown in mouse models of immune-mediated chronic HBV infection that the maintenance of low-level liver cell destruction caused by a dysfunctional and detrimental virus-specific CD8+ T cell response is sufficient to cause the development of liver fibrosis/cirrhosis and HCC, and this occurs in the absence of cofactors (e.g., viral integration, HBV X gene expression, or genotoxic agents) that have been proposed to contribute to the development of hepatocellular carcinoma in humans [65, 66].

The notion that a virus-specific CD8+ T cell response, although inefficient and essentially harmful, remains detectable in the liver of patients chronically infected with HBV can be exploited therapeutically. One reasonable approach is to restore the functionality of such response to the levels that are observed in patients undergoing self-limited acute infection. There, a number of different hurdles must be overcome and, in particular, the severe exhaustion that typifies T cells chronically exposed to large amounts of antigens (with the hope that these cells do not carry dysfunctional signatures that are permanent) [67]. Another approach, conceptually different, is to further reduce the capacity of T cells to induce chronic liver damage with the idea that—in so doing—the onset of liver fibrosis/cirrhosis might be prevented or delayed. In keeping with this and building on the observation that platelets are instrumental to intrahepatic effector CD8+ T cell homing, a recent mouse study demonstrated that clinically achievable doses of the antiplatelet drugs aspirin and clopidogrel, when administered continuously after the onset of liver disease, can prevent the development of advanced fibrosis and HCC, greatly improving overall survival [66]. These outcomes were preceded by and associated with reduced hepatic accumulation of pathogenic virus-specific effector CD8+ T cells and pathogenic virus-nonspecific inflammatory cells, and reduced hepatocellular injury and hepatocellular proliferation [66]. Irrespective of antiplatelet treatment , intrahepatic virus-specific effector CD8+ T cells analyzed at multiple times during chronic liver injury were found to express virtually no IFN-γ [66]; this is consistent with the abovementioned observation that HBV-specific effector CD8+ T cells rapidly abandon the ability to produce this antiviral cytokine after intrahepatic antigen recognition in mice [45] and that IFN-γ-nonproducing HBV-specific CD8+ T cells are commonly present in the liver of chronically infected patients [68]. Altogether, the abovementioned results indicate that the antiplatelet drugs aspirin and clopidogrel effectively prevent or delay the onset of severe liver fibrosis HCC and improve survival, supporting the concept that platelets promote CD8+ T cell-induced liver immunopathology. The results also reinforce the notion that a detrimental CD8+ T cell response is both necessary and sufficient to induce the complications of chronic viral hepatitis and they suggest that future drugs targeting platelet function or other functions linked to disease severity may be a therapeutic option in patients with chronic HBV infection.