Clinical Application of Basic Science

The molecular basis of the failed immune response in chronic HBV:Therapeutic implications

Mala K. Maini*, Anna Schurich

Department of Immunology & Molecular Pathology, University College London, UK

There is a pressing need to develop new immunotherapeutic loss of control of acute HBV infection in chimpanzees [3] and is

interventions in chronic hepatitis B virus (HBV) infection in orderto limit the high costs and risks of toxicity or viral resistanceassociated with maintenance antiviral treatment. Here we reviewrecent advances in our understanding of the molecular defectsunderlying the profound T cell depletion characteristic of thesepatients. We propose that T cells are driven to apoptosis by thecombination of persistent, high antigen load and excessive inhib-itory signals encountered in the hepatic microenvironment. Thefeasibility of boosting sustained antiviral control by targetedreversal of key tolerising mechanisms is discussed.� 2010 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

The case for an immunotherapeutic approach to HBV

HBV is a hepatotropic non-cytopathic DNA virus that continues tocause more than a million deaths annually from the complica-tions of persistent infection. Despite the advent of a new genera-tion of more potent antiviral agents such as Tenofovir andEntecavir, sustained off-treatment responses are still rarelyachieved [1]. Maintenance antiviral therapy carries substantiallong-term risks of viral resistance and drug toxicity, and unsus-tainable cost implications for many of the most heavily affectedcountries.

The case for developing an immunotherapeutic strategy as anadjunctive approach is supported by the fact that naturalimmune responses can control HBV in more than ninety percentof those infected as adults. Furthermore, bone marrow transplan-tation from an immune donor can mediate resolution of chronicHBV infection (CHB) in the recipient [2]. A better understandingof the defective antiviral responses present in chronic infectioncould be used to target specific therapeutic interventions, har-nessing the inherent ability of the immune system to controlHBV. CD8 T cells constitute a critical component of the responseto non-cytopathic viruses. This review will concentrate on HBV-specific CD8 T cell responses, the depletion of which results in

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Keywords: CD8 T cell exhaustion; Chronic hepatitis B virus infection;Immunotherapy.Received 12 November 2009; received in revised form 7 December 2009; accepted 8December 2009; available online 7 January 2010* Corresponding author. Tel.: +44 20 7679 9212; fax: +44 20 7679 9652.E-mail address: m.maini@ucl.ac.uk (M.K. Maini).

characteristic of chronic infection in humans [4]. However, it ispossible that alternative mechanisms to those required for thecontrol of an acute infection could usefully be exploited in thesetting of chronic infection. For example, chimpanzee [5] andhuman studies [6] have suggested that acute HBV infection canbe successfully controlled in the absence of a detectable inter-feron-alpha response, yet augmentation of this innate responseforms one of the most successful existing treatments for chronicinfection. Similarly, NK cells appear to have a more pathogenicthan protective role in chronic hepatitis B (CHB) [7], but may holdpromise for boosting antiviral control if T cell responses prove tobe beyond rejuvenation.

A number of attempts have already been made to boost anti-viral T cell responses by therapeutic vaccination in CHB [8]. Thelimited response induced by these initial interventions highlightsthe dysfunction of the immune system in CHB and underscoresthe importance of a more multifaceted approach. Careful moni-toring of the degree of immune constitution achieved with cur-rent antiviral therapy should direct the timing of futuretherapeutic vaccines and the need for these to be accompaniedby targeted reversal of tolerising mechanisms. In addition, suchmonitoring may identify immunological biomarkers that can beused to identify the limited number of patients in whom existingantiviral therapy can be successfully withdrawn and to predictwhen. Interferon alpha therapy has higher chances of achievingsustained responses after a finite course of treatment but is lim-ited by its toxicity [1]; a better understanding of the molecularmechanisms in those responding could improve pre-selection ofpatients. The goals of dissecting the molecular basis of immunedefects in chronic HBV infection can therefore be summarised as:

(A) To provide biomarkers for the selection of therapy, thewithdrawal of therapy or timing of therapeuticvaccination.

(B) To inform the development of targeted immunotherapeu-tic approaches.

Putative factors and mechanisms limiting the success ofexisting therapeutic approaches

The failure of existing therapeutic strategies to achieve sustainedviral control has been attributed to the profoundly depleted HBV-specific T cell response characteristic of patients with CHB

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LSEC

NK / NKT cells

KCT cell

HSC

Hepatocyte

Fig. 1. Antigen presentation in the liver. T cells circulate through the liver viathe extensive sinusoidal network, where they come in close contact with anumber of specialised cells capable of presenting antigen and shaping theimmune response. These include liver sinusoidal endothelial cells (LSEC) formingthe sinusoidal wall, Kupffer cells (KC), hepatic stellate cells (HSC) in the Space ofDisse and hepatocytes.

JOURNAL OF HEPATOLOGY

[4,9,10]. There is some increase in HBV-specific T cell responsesin the first few months after starting antiviral therapy [11,12],but these wane again rapidly [13]. Therefore it seems likely thatspecific interventions will be required to reverse T cell exhaustionin the majority of patients, necessitating a better understandingof the precise mechanisms driving and mediating it.

Insights into the nature of the CD8 T cell exhaustion develop-ing in persistent viral infections have been derived from elegantstudies in mice persistently infected with high levels of lympho-cytic choriomeningitis virus (LCMV). These studies have defined ahierarchical loss in T cell effector function driven by the degreeand duration of antigenic stimulation [14–16]. This is pertinentto the situation in CHB, where T cells are exposed over severaldecades to extremely high levels of viral replication. In addition,the production of sub-viral particles results in microgram quanti-ties of circulating surface antigen that, along with eAg (the secre-tory form of core antigen), could further contribute to T cellexhaustion. The fact that production of these proteins is rarelyswitched off by antiviral drugs, even when viral replication is effi-ciently suppressed, supports their putative role in maintainingthe residual T cell paralysis. Another factor to take into accountwhen considering the nature of T cell response in CHB is the roleof the liver in biasing and perpetuating mechanisms of tolerance[17], as highlighted by some of the examples we will discuss.

There have been a number of recent advances in our under-standing of the precise mechanisms mediating T cell exhaustion.They can be broadly divided into T cell intrinsic and T cell extrin-sic mechanisms, although there is considerable overlap, sinceextrinsic mechanisms can promote intrinsic defects.

T cell intrinsic defects

Using an unbiased gene expression profiling approach, we haverecently identified Bim-mediated apoptosis as a key mechanismmediating deletion of HBV-specific CD8 T cells. This pro-apopto-tic mediator was specifically upregulated in the HBV-specific CD8T cells of patients with CHB; blocking it downstream, allowedrescue of multispecific functional responses directly ex vivo[18]. Elucidation of pathways resulting in the induction of thispro-apoptotic phenotype could potentially allow upstream block-ing in future immunotherapeutic trials. We postulate that thisapoptotic propensity, which has also been observed in HCV infec-tion [19], is imposed by tolerogenic activation by the array ofpotential antigen presenting cells in the liver (Fig. 1, [17,20]). Thisis supported by the fact that antigen presentation by hepatocytesresults in T cells prone to premature death [21], which wasrecently shown to be mediated through Bim; T cells deficientfor Bim survived intrahepatic activation [22]. We hypothesisethat when T cells encounter HBV antigens in the context of anintrahepatic APC, they receive insufficient co-stimulation, out-weighed by an excess of co-inhibitory signals, which drives themto exhaustion (Fig. 2).

One such co-inhibitory signal is mediated through the PD-1pathway, which has already been shown to be critical for intrahe-patic tolerance [23–25] and to contribute to the failure of some Tcell specificities in CHB [9,26]. PD-1 is increased on both HBV[9,26] and HCV-specific [27,28] CD8 T cells, whilst the expressionof PD-1 ligands is upregulated on a number of intrahepatic celltypes in inflammatory liver diseases including CHB [29]. Defec-tive antigen presentation has been shown to induce T cell toler-

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ance through the combination of both PD-1 and CTLA-4 [30];the latter is another candidate mechanism in CHB that hasalready been implicated in being able to synergise with PD-1 inT cell exhaustion in HCV [31]. Although in vivo trials of PD-1blocking have shown promise in SIV [32] and are ongoing inHCV, it is likely that a carefully tailored approach will be neces-sary, requiring a more complete understanding of the degree ofsynergy and redundancy of the multiple candidate co-inhibitorypathways. This has been recently highlighted in the LCMV model[33] and in HIV, where another co-inhibitory molecule, Tim-3,was found to be expressed on largely non-overlapping T cell pop-ulations to those expressing PD-1 [34]. It is also plausible thatblocking of co-inhibitory signals will need to be supplementedby enhanced co-stimulatory signals, such as through 41BB [35],which may hold the potential to rescue virus-specific CD8 fromBim-mediated attrition by restoring intracellular levels of TRAF[36,37]. A schematic representation of the molecular basis forreversing intrinsic T cell deletion is shown in Fig. 2.

T cell extrinsic defects

Reversal of T cell intrinsic defects will fail to reconstitute ade-quate T cell responses if they remain subject to ongoing mecha-nisms of extrinsic T cell suppression. In particular, it will be nouse generating robust T cell responses in the periphery if thesecannot retain functionality at the site of HBV replication becauseof extrinsic factors at play in the liver microenvironment. Onesuch influence may be the nutrient milieu, including the levelsof conditionally essential amino acids such as arginine [38] andtryptophan [39]. Our data suggest that depletion of arginine inthe inflamed HBV-infected liver could downregulate the CD3fcomponent of the TCR signalling complex and reduce the abilityof T cells to produce IL-2 and proliferate [38]. A deficiency in IL-2

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hepaticAPC

MHC I

CD80CD86

PD-L1

CD8 T cell

PD-1

TCR

CTLA-4

Bim

Bim

Bim

CD28

Tim-3

galectin 9

+

+

_

_

_

ExhaustionImmunity

co-stimulation

co-inhibition

CD8 T cells

hepaticAPC

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galectin 9

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co-stimulation

co-inhibition

IFNγ

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T cell reconstitution

+ anti-viral therapy+ therapeutic vaccination

Fig. 2. Altering the balance of co-stimulatory versus co-inhibitory signals to restore T cell antiviral function. We postulate that excessive co-inhibitory signals fromintrahepatic antigen presentation drive Bim-mediated apoptosis of CD8 T cells in CHB. Strategies to reverse the balance of co-stimulation versus co-inhibition (incombination with antiviral suppression and a therapeutic HBV vaccine) may restore effective antiviral T cell responses.

Clinical Application of Basic Science

production by intrahepatic T cells could further drive apoptosis ofthose T cells already poised to die through the intrinsic mecha-nisms described above [40].

Extrinsic regulation of T cells by a number of suppressivemechanisms, including classical FoxP3 + Tregs [41], may contri-bute to T cell tolerance in the HBV-infected liver. A variety ofintrahepatic cell types can produce the immunosuppressive cyto-kine IL-10 and we have found this to be induced in close temporalcorrelation with HBV replication [6]. This may synergise with theeffects of TGF-b, which can be produced by HCV and HBV-specificT cells [18,42], resulting in their auto-suppression. The cellintrinsic production of TGF-b has recently been shown to mediateBim-dependent apoptosis of virus-specific CD8 T cells; selectiveattenuation of TGF-b signalling restored sufficient numbers ofLCMV-specific T cells to eradicate the virus [43]. Altering the ba-lance of cytokine signals in CHB, by enhancing levels of IL-2 and/orblocking the immunosuppressive cytokines TGF-b or IL-10, maytherefore constitute an additional therapeutic approach for rescu-ing T cells from deletion. Blocking IL-10 in the context of CHBcould act as a double-edged sword because of the importantanti-inflammatory role of this cytokine, which may help to limitliver pathology [6]. By contrast, blocking TGF-b may constitute amore appealing therapeutic approach as this could contribute toT cell reconstitution whilst having the additional benefit ofreducing the pro-fibrogenic activity of this cytokine in the HBV-infected liver [44].

Can T cell responses be safely augmented in CHB?

Reversal of the tolerising mechanisms described above couldresult in a generalised reconstitution of T cells of many specifi-cities, with the attendant risks of autoimmunity [45]. The reversalof T cell tolerance would need to be directed towards HBV-spe-cific T cells by combining any proposed immunotherapeuticintervention with a therapeutic vaccination for HBV. This strategyhas already been applied in the LCMV model, showing synergisticenhancement of virus-specific T cell responses when therapeuticvaccination was combined with PD-1 blockade [46,47]. Morework is needed to optimise the HBV vaccine to be used, including

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defining whether it should target the core protein, sinceresponses to this protein have been associated with the controlof HBV [10]. Alternative approaches to overcoming the profoundtolerance in CHB include the application of chimeric antigenreceptors [48] or T cell receptor gene transfer [8,49], allowingexisting T cells to have their specificity re-directed towardsHBV. Again, these re-directed T cells may need to be protectedfrom becoming rapidly deleted by the incorporation of additionalgenetic modifications to reverse one or more of the key molecularmechanisms described here.

Assuming that the boosting of T cell responses through theblockade of tolerising mechanisms could be restricted to thosedirected against HBV, would this be a safe approach? If suchattempts at reconstitution were carried out in patients who werealready on effective antiviral suppression, this may result in a si-tuation analogous to that observed in low-level chronic carriersof HBV. These patients can have high frequencies of intrahepaticHBV-specific CD8 T cells without resultant liver damage [4], inline with the maintenance of viral control through non-cytolyticeffector function [50]. However, there remain concerns that suchan approach could trigger a significant hepatic flare, since HBV-specific CD8 have the potential to initiate liver damage throughthe killing of infected hepatocytes [51]. Extending our under-standing of the molecular mechanisms mediating and amplifyingliver damage in this setting [4,7,52] will allow the developmentof new strategies to limit any toxicity.

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