Role of T cell death in maintaining immune tolerance during persistent viral hepatitis

Abstract

Virus-specific T cells play an important role in the resolution of hepatic infection. However, during chronic hepatitis infection these cells lack their effector functions and fail to control the virus. Hepatitis B virus and hepatitis C virus have developed several mechanisms to generate immune tolerance. One of these strategies is the depletion of virus-specific T cells by apoptosis. The immunotolerogenic liver has unique property to retain and activate naïve T cell to avoid the over reactivation of immune response against antigens which is exploited by hepatotropic viruses to persist. The deletion of the virus-specific T cells occurs by intrinsic (passive) apoptotic mechanism. The pro-apoptotic molecule Bcl-2 interacting mediator (Bim) has attracted increasing attention as a pivotal involvement in apoptosis, as a regulator of tissue homeostasis and an enhancer for the viral persistence. Here, we reviewed our current knowledge on the evidence showing critical role of Bim in viral-specific T cell death by apoptotic pathways and helps in the immune tolerance.

Key Words: T cell death, Specific cytotoxic T lymphocytes, Hepatitis C virus immune tolerance, Apoptosis, Bcl-2 interacting mediator, Liver tolerance, Apoptotic pathways, Viral hepatitis

INTRODUCTION

Hepatotropic, non-cytopathic viruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV) behave as intracellular parasites. The activation of cellular immune response by priming of naïve specific CD4+ and CD8+ T cells in the lymph nodes is very important to control viral infection. However, the unique ability of the liver to retain and activate naïve CD8+ T cells leads to liver tolerance, by-passing normal activation in the lymph nodes. The continuous triggering of antigen presenting cells (APCs) in the sinusoids by the antigen-rich blood leads to peripheral tolerance to protect the liver tissue. This physiological feature can be used by hepatotropic viruses as a persistence mechanism. The depletion of liver activated CD8+ T cells is the critical part of the peripheral tolerance in HBV/HCV infection. The anticipated mechanisms for immune tolerance in liver specific pathogens are linked to virus-specific T cells death. The vital role of pro-apoptotic molecule, Bcl-2 interacting mediator (Bim) in the death of the virus-specific T cells has been shown after intrahepatic T cell activation by hepatocytes[1], in chronic HBV and HCV infection[2,3]. Therefore, this review provides glimpse of the recent advances to understand the cellular and molecular mechanism involved on “T cell death” during viral hepatitis as a viral escape mechanism through the induction of a specific-immunotolerant status on the host.

VIRAL HEPATITIS

HBV and HCV viruses are two hepatotropic non-cytopathic, human blood-born viruses. HBV is a small, enveloped DNA virus that undergoes a pro-viral state to persist in the host. HCV is an enveloped virus with a plus-strand RNA genome. It has been estimated that more than 350 million for HBV and 170 million people for HCV are infected. Approximately 80% of infections in HCV and > 90% of infected neonates, 5%-10% of infected adults in HBV succeed in establishing a chronic infection, with the potential for developing severe liver diseases such as cirrhosis and hepatocellular carcinoma[4,5].

Highly productive and replicative viruses such as HBV and HCV are associated with ineffective antiviral immunity during persistent viral infections. The complex ineffective immunity involves the functional deterioration of antiviral T cell responses and contraction of the size of this response. In persistent HBV/HCV infections, T cells are continuously challenged by high levels of viral antigens that eventually result in limiting the antiviral T cell response and ultimately leading to T cell exhaustion. This is a progressive process, starting with the deficiency in cytokine production, proliferation and survival[6], to end with physical deletion of specific antiviral T-cell populations[7].

Meticulously, cytotoxic T lymphocytes (CTLs) play a vital role in viral eradication[8] and in the pathogenesis of hepatitis[9-11]. A strong, multi-specific and long-lasting T-cell immune response emerges to be important for control of viral infection[12-14]. Appropriate, polyclonal, vigorous and multi-specific CTL responses can facilitate complete viral clearance, in which long-lasting protective T cell response is observed. However, specific CTL responses are usually not strong enough to eradicate the virus, hence contributing to persistent infection[15,16].

HBV and HCV are hepatotropic viruses that replicate in the liver. This organ features a unique immune tissue, where the deletion of antiviral T cell populations has been shown, being involved in local and systemic immune tolerance.

LIVER AS A FOUNDATION OF IMMUNE TOLERANCE

Liver situates at a hemodynamic convergence, receiving the splanchnic stream, which means an intense contact with exogenous antigens. This fact leads to the development of tolerance mechanisms to avoid inappropriate immune system activation, but it also allows antigen presentation by resident cells. Therefore, the liver is progressively more being recognized as an immune organ[17]. Liver sinusoids, hepatic arteries and portal venous carry blood containing digested nutrients and micro antigens from intestine, and as a primary metabolic organ, the liver produces multiple neo-antigens. All these molecules pass through sinusoids and finally are taken up and metabolized by different hepatic resident cells. The liver has acquired specialized mechanisms of immune tolerance to avoid the over reactivation of immune response against antigens that are metabolized in the liver. In fact, this process may be beneficial for inducing tolerance to liver grafts but also to the liver specific pathogens. Therefore, hepatotropic viruses exploit these immunotolerogenic liver features to persist. It is important to remind that the liver has the ability to retain and activate naïve CD8+ T cells ineffectively, in contrast to other lymphoid tissues. This fact may allow pathogens to escape from T cell mediated immunity and establish a persistent hepatic infection due to immune tolerance induction. This immunotolerant state can be reached by the development of T cell anergy but also by specific T cell deletion.

Uniqueness of the liver

The unique character of the hepatic tissue to tolerate liver allograft across major histocompatibility complex (MHC) mismatch in the pig without immunosupression was described by first time in 1969[17]. Later studies confirmed that this occurred because of the induction of immunological tolerance in the liver[18]. Initially, “graveyard theory” suggested that the exclusive ability of the liver to get rid of activated T cells, programmed to undergo apoptosis, was the root of the hepatic tolerance effect[19]. This theory proposed two functions of the liver as a T cell graveyard: (1) passive killer of the liver cells after their life cycle; and (2) efficient killer of the activated antigen specific T cells. According to this theory, T cell receptor (TCR) triggering by cognate antigen on TCR transgenic T cells leads to activation and accumulation of those cells in the liver and undergoes depletion of mature T cells[20].

The theory was again proved by Mehal et al[21] by indicating that the normal liver is a “sink” for activated T cells. The liver was perfused by T cells showing retention of activated, but neither resting nor apoptotic T cells[21]. Liver as a graveyard for activated T cells theory forced to believe that all the immune response in the liver would be silent; in spite of this, the presence of an effective virus specific T cells in patients controlling hepatic viral infections[22,23] could challenge this theory. Nonetheless, the removal of activated T cells by the liver cannot be excluded, as evidenced by the ability of liver allograft to rescue rejecting skin grafts[21], in which lately tolerising capacity of the liver for activated allo-specific T cells occurs. In some cases, the limited capacity of the liver to induce tolerance could be due to large number of activated T cells[24].

Naïve T cells activation in the liver

The site of T cell activation is a determinant of the outcome of an immune response in the liver[22]. Tolerance will occur when T cells are activated in the liver. On the other hand, an effective immune response will be generated, when T cells are activated in the lymph nodes. This model put forward the theory that tolerance during viral hepatitis could be the result of early deletion of antigen-specific T cells from the T cell repertoire in the liver[22]. Usually, naïve T cells are activated in secondary lymphoid organs with consequent up regulation of adhesion molecules and integrins expression, which can bind to endothelial layer of the target organ and ultimately direct T cells to the parenchyma[25]. Moreover, T cells are not able to interact with parenchymal cells easily and thus they are not usually activated in the solid organs. In spite of this, the situation in the liver is slightly different. Fenestrated endothelial layer in the liver makes available interactions between naïve T cells and liver cells[26]. It has been showed by MHC class I-restricted, hepatitis B surface Ag-specific CD8+ polyclonal CTL adoptively transferred into wide-spread antigen expressing transgenic mouse model, leading to retention of those cells within the liver[26]. Moreover, it has been shown that primary antigen-specific T cell can be activated in the liver independently of lymphoid tissues[27].

Liver APCs in tolerance

Retention, activation and tolerance of naïve T cells in the liver is the result of the action of resident liver cells, including liver sinusoidal endothelial cells (LSEC), Kuffer cells (KC), liver dendritic cells (DC), hepatic stellate cells (HSC) and hepatocytes. Their collective function in induction of inflammatory response and tolerance has been illustrated in the Figure 1.

Figure 1 Collective illustration of the hepatic cells with inflammatory and tolerance activities by stimulation of different molecules or receptors. LSEC: Liver sinusoidal endothelial cells; KC: Kuffer cells; DC: Dendritic cells; HSC: Hepatic stellate cells; TNF: Tumor necrosis factor; IL: Interleukin; mDC: Myeloid dendratic cell; pDC: Plasmacytoid dendritic cell; PD-L1: Programmed death ligand-1; Bim: BCL-2 interacting mediator; Tim-3: T cell immunoglobulin mucin-3; CTLA-4: Cytotoxic T-lymphocyte antigen 4; TGF: Transforming growth factor; NO: Nitric oxide.

Endocytosis specialist-LSEC can express MHC class I and II, accessory CD80, CD86 and CD40 molecules. These features enable those cells to behave as potent APCs with the ability to activate both naïve CD4 and CD8 T cells as well as to cross-present exogenous antigen towards CD8 T cells[28]. However, LSEC primed naïve CD4+ T cells produce cytokines typical from Th0 rather than Th1 cells[29]. In addition, LSECs constitutively expressed ICAM-1, which helped in trapping of specific CD8+ T cells in the liver, resulting this process in activated T cell apoptosis[21]. Furthermore, the cross presentation of antigen by LSEC mainly leads to CD8+ T cells tolerance rather than immunity, demonstrating that LSEC-induced tolerance is an active and dynamic process[30].

Bone marrow derived and largest group of liver resident macrophages-KC mediate host resistance to infection. Interleukin (IL)-1, IL-6, IL-12 and tumor necrosis factor-α (TNF-α) pro-inflammatory cytokines released by KC[31] are involved in the inflammatory activities, whereas the nitric oxide, prostaglandin and IL-10 released by KC[29,32] down-regulate the production of pro-inflammatory cytokines and thereby may contribute to induction of hepatic tolerance. Furthermore, DC-induced antigen-specific T cell activation can be inhibited by KCs[29], which could also favor tolerance development. In addition, as in LSECs, KCs expressed ICAM-1 mediated trapping of specific CD8+ T cells in the liver resulting in activated T cell death[21].

Liver DCs are primarily located within periportal areas and around central veins, which exert tolerogenic properties due to “immature” phenotype. The production of PD-1 and cytotoxic T lymphocyte antigen-4 (CTLA-4) by resting DCs, which are crucial negative co-stimulatory molecules, helps in inducing peripheral CD8+ T cell tolerance by inhibiting proliferation and cytokine production of liver infiltrating effector T cells[33]. In addition, liver generated DCs are more tolerogenic than DC in lymphatic tissue[34].

The role of HSCs in hepatic fibrosis includes stimulation of CD4, CD8+ T cells and NKT cells[35,36]. However, function of HSCs involves not only the inflammatory response[36], but also a tolerogenic role[37,38], which is the result of induction of T cell death[38] by intrinsic mechanism of immune inhibition. The HSCs regulate immune modulation by inducible B7-H1 expression, an inhibitor molecule of B7 family, resulting in T cell apoptosis induction.

Hepatocytes are also capable of activating naïve CD8+ T cells[39,40] and their interactions with CD8+ T cells may occur through LSEC fenestrations[38]. However, hepatocytes fail to promote activated CD8+ T cells survival, leading to an impaired T cell activation[39]. In addition, hepatocyte-activated T cells in vitro acquired activity and secrete cytokines but both levels are not constant and T cells consequently appeared to die by passive mechanisms[41]. Furthermore, infiltrating CD4+ T cells differentiate into a less inflammatory phenotype due to the interaction with MHC II-expressing hepatocytes, which also helps to abrogate antiviral CD8+ T-cell response and viral clearance[42], which conclude in the tolerance during infection. It has been already proved that T cells activated by hepatocytes undergo premature death[43], whereas naïve CD8+ T cells priming by DC in the lymph nodes acquired effector functions in the liver.

The site of primary T cell activation could also induce emperipolesis of CD8+ T cells in the liver[43], which leads to non-apoptotic, destruction of these CD8+ T cells after degradation by lysosomal proteolytic enzymes. This distinct form of emperipolesis has been termed as “suicidal emperipolesis” (SE)[44]. Benseler et al[44] suggested that SE is a significant mechanism by which death of activated naïve CD8+ T cells occur in the liver within the first few hours before T cells are able to divide and expand. It is also involved in maintenance of tolerance, which is reinforced by break of tolerance in immune-mediated liver damage by treatment of wortmannin[44], inhibitor of phosphoinositide 3-kinases that blocks emperipolesis. Therefore, SE is an extremely efficient mechanism, able to rapidly delete T cells.

T cell stimulation in the liver encourages tolerance by using mechanisms such as, immune divergence[45], generation of regulatory T cells[46], T cell anergy[47] and T cell death[1]. Undeniably, hepatic tolerance can explain the elevated frequency of viral persistence during hepatotropic virus infections[1]. Although there are evidences showing that most infectious microorganisms are promptly removed from the liver, a favorable situation for evading immune responses occurs in some viruses, leading to the triumph of certain pathogens such as HBV and HCV. Till date, there are two main mechanisms by which HBV and HCV could successfully escape from CTL action: escape mutant generation, and immunosuppressive effects exertion (effector T cell exhaustion and T cell death by apoptosis)[2,48-50]. Among these mechanisms involved in viral hepatitis persistence, new advances on the role of T cell death induction have been obtained recently and our review in the apoptosis role, paying special attention to the last new insights in this issue will be discussed in the following pages.

APOPTOSIS

A normal cellular process involving physiologically relevant cell death and deletion of unwanted cells is called apoptosis. Apoptosis is essential for cell selection, tissue homeostasis, morphogenesis, and host defense in multi-cellular organisms. A cell that undergoes apoptosis dies neatly, without damaging its neighbors. The apoptotic signals give rise to activate various proteins and follow a specific classical caspase chain reaction set activation[51]. Quickly and neatly dismantlement process includes membrane blebbing with shrinking of the cytoplasm and condensation of the nucleus. Phagocytic cells begin to pick up the apoptotic bodies, preventing the release of cellular content and ultimately avoiding inflammation[52] (Figure 2). Apoptosis occurs by two mechanisms: active and passive mechanism. No presence of antigen gives a signal for termination of immune response by passive apoptotic mechanism (intrinsic pathway). On the other hand, the ligation of Fas (CD95) and TNF receptors-“death receptors” triggered apoptosis lead to active mechanism of apoptosis (extrinsic pathway). Briefly, apoptosis mechanisms involve a family of cysteine proteases, called caspases. These molecules are synthesized in the cell as inactive precursors, or pro-caspases for self-protection against accidental death, which are usually activated after receiving proper trigger by cleavage (Figure 3). Structurally, pro-caspases contain three domains: N terminal prodomain, a large subunit and a small subunit. After activation, the active caspase enzyme is formed by heterodimerization of small and large subunits[43]. Moreover, active caspase molecules are ready to cleave target proteins such as structural or signaling proteins and other effector caspases, preventing other proteins cleavage randomly[52].

Figure 2 Apoptosis-programmed cell death.

Figure 3 Caspase activation: Inactive proenzyme (procaspase) activated by proteolytic cleavage by another member of caspase family and cleaved two fragments associate to form the active site of the caspase.

Extrinsic pathway

The extrinsic pathway initiates from outside the cell through triggering the activation of transmembrane “death receptors” that are members of the TNF receptor gene superfamily. Members of this receptor family bind to extrinsic ligands known as pro-apoptotic ligands[53] and transduce intracellular signals that ultimately result in the destruction of the cell[54,55]. To date the most well characterized ligands of these receptors are FasL, TNF-α, Apo3L and Apo2L and corresponding receptors are FasR, TNFR1, DR3 and DR4/DR5, respectively[55-57]. The signal transduction of active cell death process involves several caspases. Activated caspases have an effect on several cellular functions as part of the process that results in the death of the cells[53].

The signal transduction of mitochondrial-independent active cell death process involves binding of a pro-apoptotic ligand (such as FasL) with its receptors (Fas) on the surface of a target cell. The cytosolic tail of receptors contains a death domain, which when activated, binds to an adaptor protein, which in turn recruits the specific procaspase-8 and -10 and activates them by proteolytic cleavage[58] that finally initiates the proteolytic caspase cascade leading to apoptosis. Activated caspase 8 triggers the caspase cascade via two different pathways, leading to cell death. In type 1 apoptosis, such as in lymphocytes, caspase 8 activates caspase 3 whereas in type 2 apoptosis, like in hepatocytes and pancreatic cells, caspase 8 activate the pro-apoptotic molecule Bid and go ahead for apoptosis via the disruption of mitochondrial membrane and cytochrome C release[59] (Figure 4). The T cell death by type 1 and type 2 Fas induced apoptosis fate is decided by the ratio between proteolytically activated effector caspases, X-chromosome linked inhibitor of apoptosis protein and proto-typical effector caspase substrate inhibitor of caspase-activated DNase. Interestingly, HCV specific intrahepatic lymphocytes contribute to bystander killing via Fas-FasL interaction[60], which support the fact that the liver facilitates liver-trapped activated T cell apoptosis[61].

Figure 4 Extrinsic pathway. A: Mitochondria-independent extrinsic pathway: Fas-FasL ligation strikes to recruit pro-caspase 8 activation and induction of caspase cascade by caspase 3 leading to apoptosis; B: Mitochondria-dependent extrinsic pathway: Fas-FasL ligation trigger to activate the pro-caspase 8, which cleave Bid (pro-apoptotic Bcl-2 family molecule) to form truncated Bid (tBid). Then, mitochondrial dependent cell death begins with tBid.

Intrinsic pathway

The intrinsic or mitochondrial pathway is initiated within the cell, involving non-receptor-mediated intracellular signals and inducing activities in the mitochondria that initiate apoptosis. DNA damage, loss of cell-survival factors or other types of severe cell stress causes the induction signal for the intrinsic pathway. This passive death process pivots on the balance of activity between pro- and anti-apoptotic signals of the B cell lymphoma 2 (Bcl-2) family proteins[62]. This balance is maintained by regulation of the permeability of the mitochondrial membrane and by the pro- or anti-apoptotic signal that will be released inside the cell[63]. Following mitochondrial permeabilization, the intrinsic pathway divides into two pathways: Apoptosis protease-activating factor-1 (Apaf-1) dependent and Apaf-1 independent pathway. In Apaf-1 dependent pathway, release of cytochrome c from mitochondria, by triggering the pro-apoptotic Bcl-2 family member[64], and ATP activate monomer inactive Apaf-1 proteins by a conformational change, leading to form a heptamer of Apaf-1 molecules called apoptosome[65]. Apoptosome allows activation of pro-caspase 9, which consequently triggers the caspase cascade[66]. On the other hand, in Apaf-1 independent pathway, permeabilization of mitochondrial membrane release DIABLO like proteins, which activates effector caspases by provoking inhibitors of apoptosis proteins[67] and triggers caspase cascade[68] (Figure 5).

Figure 5 Intrinsic pathway. Death stimulation up regulates Bcl-2 interacting mediator leading to the separation from Bcl-2, favoring the activation of Bax, Bak, which form pores in the mitochondrial membrane leading to release of cytochrome c. Cytochrome c with Apaf-1 and procaspase 9 participate in the formation of apoptosome, which activate caspase 9. Caspase-9 activates caspase 3 after cleavage of pro-caspase-3. That caspase-3 triggers to induction of caspase cascade and cell death. Apaf-1: Apoptosis protease-activating factor-1. Bim: Bcl-2 interacting mediator.

The balance of pro- and anti-apoptotic proteins maintains the apoptotic activity[69]. The Bcl-2 family members regulate mostly neglect or intrinsic pathway. This family is subdivided into three groups of proteins on the basis of their functions and the number of Bcl-2 homology (BH) motifs included in their primary structure; first group: “anti-apoptotic multidomain” members, such as Bcl-xL, have four BH domains (BH1 to BH4) which inhibits apoptotic process. Other two groups of “pro-apoptotic multidomain” members, which are Bax-like proteins and “BH3-only” proteins[70]. Bax-like proteins possess threee BH domain (BH1 to BH3), including Bax, Bak, and Bok, which are referred as death effector members. BH3-only members contain BH3 domain, including Bim, Bad, Bik, Puma, Noxa and Bid and are known as messengers of death. In addition, C-terminal transmembrane (TM) fragment is thought to confer anchorage to mitochondrial membranes, which is also possessed by most multi-BH members and several BH3-only proteins.

Three models (Figure 6) have been postulated by which the BH3 family promotes passive cell death in which Bax and Bak bind directly or indirectly with cell death sensitizer (e.g., Bad, Bik) and activators of cell death (e.g., Bim, tBid). The direct activation model proposes that sensitizer BH3-only proteins displace the activator BH3-only proteins from the anti-apoptotic proteins to promote apoptosis. Anti-apoptotic proteins inhibit the activator BH3-only proteins but not Bax and Bak to suppress apoptosis. In the displacement model, Bax and Bak are sequestered by anti-apoptotic proteins for cell survival and constitutively active in cells. BH3-only proteins play the sensitizer role and inhibit their respective anti-apoptotic proteins to promote apoptosis. The third model, called embedded together model, highlights the interactions occurring in and on membranes, which were not explained by direct activation and displacement model. In embedded together model, Bcl-2 family proteins insert into and change their conformations according to their functions in membrane[71]. The predominantly studied messenger death molecule, Bcl-2 interacting protein (Bim) will be focused further.

Figure 6 Models for intrinsic cell death. A: Direct activation model postulates Bcl-2 interacting mediator (Bim) is required for activating Bax and Bak. Anti-apoptotic proteins inhibit BH3-only proteins to suppress apoptosis, but not Bax or Bak. Replacement of Bim to sensitizer BH3-proteins from the anti-apoptotic proteins occurs to promote apoptosis; B: The displacement model proposes that anti-apoptotic proteins for cell survival must sequester constitutively active Bax and Bak in cells. Bim inhibits their respective anti-apoptotic proteins by playing sensitizer role to promote apoptosis; C: Embedded together model highlights the active role of the membrane, which is not defined in direct activation model and displacement model. Bcl-2 family proteins insert into and change their conformations that dictate their functions at the membrane. Sensitizer BH3-only proteins relocate the activator BH3-only proteins and Bax/Bak from the anti-apoptotic proteins to endorse apoptosis. Activator BH3-only proteins recruit Bax to the membrane to induce mitochondrial outer membrane permeabilization and apoptosis. These reversible interactions are directed by equilibrium constants that are depended on the concentrations and interactions of the proteins with each other and with membranes.

BIM

Bim/Bod is a pro-apoptotic protein belonging to the BH3-only group of Bcl-2 family members and is being called the “ghost” molecule or “suicide” molecule, which enables cells to expire gracefully. Two independent studies discovered Bim as a Bcl-2 binding protein and Mcl1-binding protein in 1998[72,73]. Bim induces apoptosis by binding to and antagonizing anti-apoptotic members of the Bcl-2 family. The Bim interactions have been observed with Bcl-2 family members, such as Bcl-2, Bcl-xL, Mcl-1, Bcl-w, etc[72,73].

Bim is a well-known pivotal initiator of apoptosis in thymocyte-negative selection[74]. Bim has 19 Bim isoforms including three major isoforms, which have distinct sizes and pro-apoptotic activities in the mammals, caused by alternative splicing: BimEL (extra long), BimL (long) and BimS (small)[73]. The shortest form, BimS, is the most potent and is generally only transiently expressed during apoptosis[73]. The other two isoforms are sequestered to the dynein motor complex, and apoptotic activity of these longer isoforms is regulated by phosphorylation[75,76], which is triggered by environmental stress, resulting in its dissociation from the dynein complex and increasing apoptotic activity.

Expression of Bim is up regulated in human T cells in response to TCR-triggering by protein kinase C and calcineurin pathways[77]. Nevertheless, there are other mechanisms involved in Bim up-regulation during chronic infection, such as the effect of certain cytokines. In fact, in a persistent viral infection animal model, Bim-mediated apoptosis correlates with low IL-7 receptor expression on specific T cells[78].

The regulation of Bim expression at transcriptional level in growth factor deprivation and in endoplasmic reticulum stress has observed by the class O fork-head box transcription factor (FOX03A) and transcriptional factor CEPB-α respectively[79,80]. Post-transcriptional phosphorylation of Bim can also regulate its function. Phosphorylated Bim is targeted for proteasomal degradation and avoid its interaction with Bax, thus maintaining cell existence[81,82]. The signaling adaptor TNFR-associated factor 1 (TRAF1) negatively correlates with Bim and it contributes to CD8 T cell-mediated control of chronic viral infections. In addition, linking between survival effects of TRAF1 and TRAF1-dependent Bim down-modulation has been shown in CD8 T cells[83-85]. TRAF1 is particularly vanished from virus-specific CD8 T cells during the chronic human immunodeficiency virus and lymphocytic chorio-meningitis virus (LCMV) infection[86].

Bim plays a vital role in the immune system, in bone biology and in tumor-genesis by inducing apoptosis[87]. Bim in T cells, B cells, neurons and many other cell types can trigger apoptosis[87]. Gene targeting in mice for the important region for apoptosis, BH3 region, uncovered the important physiological role in Bim[88]. In fact, in the absence of Bim leukocytes in blood as well as in LNs, thymus, spleen were high in number[88]. The role of Bim in apoptosis has been revealed in Bim-/- thymocytes, which were more resistant to apoptosis after different apoptotic treatment such as ionomycin, taxol, γ irradiation[88].

DEATH OF ACTIVATED T CELLS BY BIM

The liver is having a property that might explain its role in inducing tolerance due to its recognition as an alternative primary activation of CD8 T cells site. The phenotype of activated CD8 cells in the liver was the same as in lymph nodes. However, liver-activated CD8 T cells displayed poor effector functions and a unique CD25^low CD54^low phenotype, which was associated with increased expression of the Bim and caspase-3, demonstrating that these cells are programmed to apoptosis following intrahepatic activation. Strikingly, Bim deficient T cells survived following intrahepatic activation[1]. Therefore, the phenotype and fate of naïve CD8 T cells activated by hepatocytes in vivo could explain the death penalty role of Bim in chronic hepatotropic viral infection[1]. The distinct phenotype can be due to the lack of co-stimulatory molecule expression on hepatocytes[43]; however the treatment with IL-2 or anti-CD28 antibodies could rescue hepatocyte-activated cells from death[41].

Lymphocyte fate deciding pathways synergize to kill activated T cells in chronic herpes simplex viral immune responses, whereas death of activated T cells in acute immune responses relies only on the mitochondrial pathway involved only Bim with no contribution by Fas, which showed critical overlapping roles for Fas and Bim in T cell death during immune response shutdown, leading to immune tolerance[23].

BIM IN HEPATITIS

Bim has been shown to be important for CD8 T cell viability during chronic LCMV infection in mice[89]. In this study, in Bim mutated mice, Bim mutation almost completely blocked the deletion of cognate antigen specific CD8 T cells in liver during chronic viral infection. Bim has a critical role in maintaining naive and memory T cells in LCMV infection[90]. In another study, it has been shown that a defect in apoptosis dramatically not only enhances the antigen-specific memory T cells but also increased the number of virus-specific CD4+ T cells in the lymph nodes following acute LCMV infection, compared to the parental genotypes or wild type mice[91]. Therefore, the loss of both Bim and Fas caused the increase in memory T cells in acute LCMV infection[91]. The Bim role has been demonstrated in the development of LCMV-induced, T cell-mediated hepatitis by controlling the apoptosis of both T cells and hepatocytes[92].

Bim attrition of virus specific CTLs during HBV infection has also been confirmed[3,93]. The gene expression profile in HBV infection showed different patterns of gene expression on HBV-specific CD8+ T cells according to viral control. Bim was one of the up-regulated genes in HBV-specific CD8+ T cells from patients with chronic HBV infection. Blocking Bim-mediated apoptosis improved recovery of HBV-specific CD8+ T cell function[3]. Furthermore, the elevated apoptosis has been observed not only with Bim tolerogenic phenotype, but also with co-inhibitory signals through CTLA-4[93] or T cell-intrinsic transforming growth factor-β[94].

As discussed earlier, robust CD8 responses are essential to control HCV infection. However, in HCV chronic infection, HCV specific CD8 are depleted by Bim mediated attrition, and remaining cells are functionally exhausted. The cell survival factor CD127 counteracts the induction of apoptosis after antigen encounter through myeloid cell leukemia sequence-1 (Mcl-1) expression and Bim down-regulation[95] after the cognate antigen recognition by TCR. Similarly, our group has shown in previous work, HCV-specific CTLs displayed a high Bim expression in persistent infection respect to resolved infection patients[2], suggesting a similar apoptotic mechanism to the one described in chronic HBV infection.

The procedure of T cell death during chronic viral infection is determined by a carefully balanced and complex group of pro- and anti-apoptotic proteins of the Bcl-2 family, such as Bim and Mcl-1[96] (Figure 7). Interestingly, persistent hepatotropic viral infection is characterized by continuous TCR triggering and CD127 down-regulation on viral-specific CTLs[97], which could favor Bim up-regulation. In addition, it is well known that Bim is clearly involved in intrahepatic specific-CTL apoptosis in animal models[1]. Furthermore, Bim pro-apoptotic effect is blocked by the action of Bcl-2 family anti-apoptotic proteins such as Mcl-1 and Bcl-2[78,98], clearly pointing out that T cell death also depends on the anti-apoptotic protein expression. Bearing in mind all these facts, recently our group has suggested a model to explain specific CTL deletion during persistent hepatotropic viral infection (Figure 8). This model shows that CD127 phenotype modulates Bim and Mcl-1 expression on virus-specific CTLs, leading to Mcl-1/Bim imbalance during persistent infection, which impairs T cell reactivity and suggesting that restoration of T cell function could occur by correcting the levels of Mcl-1 and Bim expression.

Figure 7 Balance between co-stimulatory/apoptotic molecules and viral-specific cytotoxic T lymphocytes reactivity according to infection outcome. Neg.: Negative; Pos.: Positive; CTLs: Cytotoxic T lymphocytes; (+): Possible molecules induced by viral infection; (-): Possible molecules down-regulated by viral infection; BIM: Bcl-2 interacting mediator; Mcl-1: Myeloid cell leukemia sequence-1.

Figure 8 Cell survival marker CD127 modulates Bim and myeloid cell leukemia sequence-1 expression on hepatitis C virus-specific cytotoxic T lymphocytes after cognate antigen stimulation. Misbalance of Mcl-1/Bcl-2 interacting mediator (Bim) triggers to apoptosis of hepatitis C virus specific cytotoxic T lymphocytes. TCR: T cell receptor; Mcl-1: Myeloid cell leukemia sequence-1.

In our work, Bim up-regulation has been observed on CD127^low-expressing HCV-specific CTLs but not on CD127^high cells after antigen encounter, suggesting that TCR triggering can only lead to Bim up-regulation in absence of IL-7 stimulation on HCV-specific CTLs. Nevertheless, Bim level is not enough to lead to T cell apoptosis. Our data also showed the Mcl-1/Bim ratio could decide the fate of the activated T cells by sequestration of experienced CD127^low/Mcl-1^low-expressing T cells to the liver and subsequent Bim up-regulation after antigen encounter due to the absence of IL-7 stimulus[99]. Finally, Bim would be released freely to activate Bax, due to the low level of the anti-apototic protein Mcl-1 during chronic HCV infection. Consequently, CD127 level play a central role in hepatotropic virus-specific CD8+ T cell apoptosis by regulation of Mcl-1 expression in vivo and by Bim modulation after antigen encounter, which is checked by T cell reactivity restoration and Mcl-1/Bim phenotype on CD127^low specific CTLs after apoptosis blockade (Figure 9), that suggested a link between apoptosis after TCR triggering and low CD127 expression on experienced specific CTLs during persistent infection that could be related to Mcl-1/Bim imbalance.

Figure 9 Figure illustrated the FACSan^® dot plots and histograms from peripheral blood lymphocytes from two hepatitis C virus patients with different CD127 expression on hepatitis C virus-specific cytotoxic T lymphocytes (CD8+/Pentamer+ cells). The different plots show the Bcl-2 interacting mediator (Bim) and myeloid cell leukemia sequence-1 (Mcl-1) expression directly ex vivo and after specific stimulation on peripheral CD8+/pentameter+ cells according to CD127 level. The figure on the top of the dot-plots represents the frequency of penatmer+ cells out of total CD8+ cells. The figure in the upper right corner in the histogram plot represents the MFI for Bim and Mcl-1 staining. The continuous and dashed line in the dot-plots and histograms represents the cut-off point to consider a staining positive according to the negative control. ND: Not done due to lack of pentamer+/CD8+ proliferation after specific stimulation.

Therefore, CD127 phenotype modulates Bim and Mcl-1 expression on specific CTLs and this affect to T cell reactivity through apoptosis regulation. Specifically, during chronic infection, Mcl-1/Bim imbalance could be involved on CD127^low specific CTL hyporeactivity, but it could be overcome by blocking apoptosis.

For control of hepatotropic viral infection is essential to develop a robust viral-specific cellular response. However, during chronic infection this response is altered, showing a pro-apoptotic phenotype due to the deprivation of IL-7 secondary to the low expression of CD127. Recently, it has been investigated that TRAF1 is a signal adapter for positive co-stimulatory receptors whose level depends on the action of IL-7 and inhibits the expression of the pro-apoptotic molecule Bim[86]. Therefore, in situations of deprivation of IL-7, action of TRAF1 could be impaired, favoring an imbalance between anti- and pro-apoptotic molecules. On the other hand, in an experimental model, IL-7 deprivation during stressing conditions leads to Mcl-1 down-regulation on T cells, conducting to T cell death that could be avoided by IL-7 treatment[100]. Consequently, strategies directed to block the pro-apoptotic effect of IL-7 deprivation should be designed to increase the effectiveness of CTL response restoration, by enhancing the TRAF1 and Mcl-1 expression level that could restore Bim/Mcl-1 balance. On of those strategies could be short-term use of cyclosporine-A or FK506 could block the induction of the pro-apoptotic molecule Bim on CD127^- cells[77]. This strategy could favor specific-CTL restoration during anti-viral treatments in combination with the standard of care. Another possible strategy to restore hepatotropic virus-specific CTL reactivity during chronic infection could be the administration of IL-7, in order to increase the stimulation of the reduced number of IL-7R molecules on specific CTLs, to modulate the balance between Bim and Mcl-1. In fact, in an animal model of cytotoxic T cell exhaustion, IL-7 treatment resulted in amplified cytokine production, increased T cell effector function, and viral clearance[101].

CONCLUSION

The deletion of hepatitis virus-specific CD8+ T cells is likely to represent the deregulation of the Bim pro-apoptotic pathway. The balance between pro- and anti-apoptotic molecules is critical for cell survival. The unavailability of appropriate survival marker modulates Bim and Mcl-1 expression on virus hepatitis-specific CTLs and this affect to T cell reactivity through apoptosis regulation. The level of those molecules is regulated by CD127 (IL-7R) expression, which is down-modulated during persistent infection. Consequently, Mcl-1/Bim imbalance could be the reason for the deletion of virus hepatitis-specific T cells, but it could be overcome by interruption of apoptosis. The interruption of this tolerizing mechanism may provide a new strategy to restore the balance between apoptotic molecules in order to achieve viral specific T cell immunity, as a future treatment strategy of chronic viral hepatitis.