Immunology and Cell Biology (2002) 80, 84–92

Special Feature

Role of primary intrahepatic T-cell activation in the ‘liver tolerance effect’

P A T R I C K B E R T O L I N O , G E O F F R E Y W M c C A U G H A N a n d D A V I D G B O W E N

Liver Immunobiology Laboratory, The Centenary Institute Of Cancer Medicine and Cell Biology, Newtown, New South Wales, Australia

Summary There is accumulating evidence suggesting that hepatic permeability to both naive and activated Tlymphocytes may be unique among the solid organs. The possibility that the liver may act as a site of primaryactivation for CD8+ T lymphocytes is supported by experimental data and may contribute to some of the uniqueimmunological properties of this organ, particularly its ability to induce antigen-specific tolerance. This reviewdiscusses the nature of the liver APC inducing primary T-cell activation within the liver: Kupffer cells, liverdendritic cells, liver sinusoidal endothelial cells and hepatocytes are favourably located to allow physical contactwith circulating T lymphocytes. Here, we examine the capability of each cell type to act as APC for naive CD4+ orCD8+ T cells and to induce tolerance.

Key words: antigen presentation, hepatocytes, Kupffer cells, liver, liver sinusoidal endothelial cells, primary T-cellactivation, recirculation, T cells, tolerance, transplantation.

Introduction

Lymphocyte access to tissues is not random; transendothelialmigration is dependent on interactions between specificadhesion molecules expressed by endothelial cells and theirligands.1 In the absence of antigenic stimulation, T lympho-cytes do not generally express such molecules; thus in mostorgans, endothelial cells form a very effective physical barrierpreventing any contact between naive T lymphocytes andparenchymal cells.2,3 Adhesion molecule and chemokinereceptor expression by T cells are acquired upon activation, aprocess generally initiated in secondary lymphoid organs(lymph nodes and spleen), in which naive T cells meet ‘profes-sional’ antigen-presenting cells (APC), such as dendritic cells(DC), expressing relevant peptide/MHC complexes. PrimaryT-cell activation by DC allows blast formation, cytokinesecretion, proliferation and differentiation into cells endowedwith the capacity for transendothelial migration.

Although most organs do not support activation of naiveT cells, there is evidence that liver permeability to both naiveand activated lymphocytes may be unique among the solidorgans. Pioneering work from Chisari and colleagues hasdemonstrated that when antihepatitis B surface antigen(HBsAg) polyclonal cytotoxic T lymphocyte (CTL) lineswere injected intravenously into transgenic mice ubiquitouslyexpressing HBsAg, infiltration and tissue damage occurredonly within the liver, despite similar antigen expression inother sites.4 Cytopathic damage to renal tubules in the kidneyand choroid plexus epithelial cells in the brain did not occurunless CTL were injected beneath the kidney capsule or

intracerebrally.4 These results suggest that activated CD8+

T cells do not have easy access to the parenchyma of solidorgans other than the liver. We have recently obtained similarresults using naive CD8+ T cells. When naive H-2 Kb specificTCR transgenic CD8+ T cells were injected into transgenicmice expressing the relevant antigen ubiquitously, the major-ity of the donor T cells were preferentially retained by theliver within 1 h of transfer and underwent activation in situ.Other non-lymphoid organs did not retain or contain cells atthis early time point (DG Bowen et al., unpubl. data, 2001).We have hypothesized that the liver may be a site of primaryactivation for naive lymphocytes, in particular CD8+ T cells,but that activation by hepatic cells is abortive and results inthe death of the activated T cells, and this explains some ofthe unique tolerogenic properties of the liver (discussedbelow).

This review will examine the likelihood of naive T cellsbeing activated within the liver from both a physiological andimmunological viewpoint, the hepatic cell types that may actas APC, as well as the fate of liver cell-activated lym-phocytes. Primary T-cell activation within the liver leading totolerance does not exclude the participation of professionalAPC located in lymphoid tissues in the tolerogenic process,either by direct presentation or cross-presentation. In mostsettings, it is likely that both types of activation contribute toestablish tolerance; however, primary activation within lym-phoid organs will not be the focus of this review.

Unique immunological properties of the liver

Until recently, the liver has been considered to be relativelyinert in its normal, uninflamed state, and essentially littledifferent to the other solid organs from an immunologicalviewpoint. However, this view has been challenged in recentyears, as immunologists have noted that the liver may possess

Correspondence: Dr P Bertolino, The Centenary Institute ofCancer Medicine and Cell Biology, Locked Bag No. 6, Newtown,NSW 2042, Australia. Email: P. Bertolino@centenary.usyd.edu.au

Received 9 October 2001; accepted 9 October 2001.

Primary T-cell activation within the liver 85

distinct properties.5–7 The liver is well placed to have animportant immunological function: it receives 25% of thecardiac output and is the largest source of lymph in the body,contributing 25–50% of the thoracic duct flow. It has beencalculated in the sheep that more lymphocytes migratethrough the liver via the lymph than any other non-lymphoidorgan.8 The majority of the hepatic blood supply is providedby the portal vein, which carries gut-derived nutrients, toxinsand antigens, as well as substances derived from the spleen.This unique location allows molecules absorbed by the intes-tine to transit the liver, where they are metabolized, ordegraded if toxic. This function also implies that the liver iscontinuously exposed to antigens and cells of the immunesystem, in particular lymphocytes. In agreement with thisargument, several observations suggest that the liver mayundergo unique interactions with lymphocytes; these will beonly briefly mentioned, as they are the subject of independentreview articles in this issue of Immunology and Cell Biology.

First, the liver contains unusual lymphocyte subsets; up to50% of intrahepatic lymphocytes in a normal mouse liverexpress intermediate levels of α/β T-cell receptor (TCR) andthe NK cell NK1.1 marker (NK T cells).9 An equivalentpopulation has also been described in humans.10 This intri-guing NK T-cell population is also found with a high fre-quency in the thymus and bone marrow. Most CD4+NKT cells are CD1-restricted and can readily secrete both Th2and Th1 cytokines upon stimulation.11 Recent reports haveshown that a lack of NK T cells favours autoimmunity;12

however, the function of these cells in the liver remainsunknown.

Second, the liver is a preferential site of T-cell apoptosis.This was first shown by Crispe and colleagues by use of TCRtransgenic mice injected with a specific peptide.13 Antigen-specific activation of peripheral T cells in these mice wasassociated with a massive accumulation of apoptotic TCRlow

CD8low B220+ cells within the liver: it was proposed that theliver functions as a graveyard for activated CD8+ T cells. Inagreement with these initial observations, accumulation ofapoptotic T cells in the liver has been demonstrated in a numberof other systems, including transplantation,14 transgenic modelsof peripheral tolerance15 and hepatitis C infection.16

Third, the liver is the site of some of the most importantpersistent viral infections. The pathological manifestations ofchronic hepatitis B and C appear to be mediated, at least inpart, by an ongoing immune response that fails to clear thevirus.17–19 However, the interplay between hepatitis virusesand the immune system is incompletely characterized and themechanisms of viral persistence remain poorly defined.

Finally, the liver possesses a unique ability to inducetolerance. Early reports have suggested that it may play animportant role in oral tolerance. Administration of antigensvia the portal rather than the systemic venous system wasshown to suppress delayed-type-hypersensitivity and anti-body formation on subsequent challenge (Chase–Sulzbergereffect).20–22 In agreement with these experiments, surgicalcreation of a mesenterico-caval shunt, which ‘short-circuits’the liver, has been shown to impair induction of oral toler-ance, suggesting that the intestinal venous drainage throughthe liver plays a role in oral tolerance induction.23 Thetolerogenic properties of the liver have also been illustrated intransplantation. In a variety of species, liver allografts are

accepted spontaneously across MHC incompatible straincombinations.24 Moreover, acceptance of a liver graft in therat induces specific tolerance to subsequent transplants ofother tissues that would otherwise be rejected.25 How thistolerant state is achieved remains poorly understood, andvarious mechanisms involving peripheral deletion and/or sup-pression of alloreactive T cells have been proposed.

Although several observations in various areas of hepaticresearch indicate that the liver possesses unique immunologi-cal properties, it remains unknown whether these are relatedand share a common basis. Hepatic permeability to T lym-phocytes and the ability of the liver to potentially function asa site of primary T-cell activation may provide a mechanismcontributing to these properties.

The hepatic sinusoids and liver cells

One very important feature of liver architecture that may becrucial for its permeability is the structure of the hepaticsinusoid (Fig. 1). These channels have an average diameter ofapproximately 10 µm and are lined by endothelial cells. Dueto the size of the sinusoids, interactions between T lym-phocytes and liver endothelial cells occur under conditions oflow velocity blood flow, a property that may be important infavouring lymphocyte activation within the liver. Flow ofblood in the sinusoids is also intermittent due to the contrac-tion of contractile smooth muscle sphincters in the walls ofhepatic arterioles.8 Blood flow in the sinusoid may also bepartially obstructed by Kupffer cells patrolling the sinu-soids.26,27 Liver sinusoidal endothelial cells (LSEC) possessunusual properties; unlike other endothelial cells, they do notform junctions with adjacent endothelial cells, but rather forman incomplete barrier between the sinusoidal lumen and theperivascular parenchymal tissue8 (Fig. 1). Furthermore, thesecells are perforated by several 120 nm diameter holes. These

Figure 1 Schematic structure of the liver sinusoidal endothe-lium. Liver sinusoids are lined by liver sinusoidal endothelial cells(LSEC), which separate the sinusoid lumen from hepatocytes.Kupffer cells patrol the sinusoids and bind to LSEC and occasion-ally hepatocytes through the gaps of two adjacent LSEC. Stellatecells are located in the space of Disse.

86 P Bertolino et al.

fenestrae are grouped in clusters forming sieve plates andbehave as dynamic structures that dilate or contract to facili-tate solute exchange between the plasma and hepatocytes.The unique structure of hepatic sinusoids, combined with thelack of basement membrane, defines a perisinusoidal spacebetween LSEC and hepatocytes, the space of Disse (Fig. 1),which is in continuity with the sinusoidal lumen. Stellate cellsare found in the space of Disse, while Kupffer cells andintrahepatic lymphocytes line the lumen of the hepaticsinusoids8 (Fig. 1). The unique structure of hepatic sinusoidsand conditions of low velocity blood flow may be theprincipal properties responsible for the singular ability of theliver to be permeable to both naive and activated CD8+ cellsand to induce T-cell activation.

Primary T-cell activation within the liver

Primary T-cell activation in lymphoid tissues is generallymediated by DC. These cells are known as professional APC,

as they meet all the requirements for optimal antigen presen-tation to both CD4+ and CD8+ T cells. These requirementsinclude the ability to efficiently process antigens, the presenceof a specialized endocytic compartment in which nascentMHC class II molecules bind antigenic peptides, high expres-sion levels of MHC class I and class II molecules, invariantchain, CD80 and CD86 costimulatory molecules, as well asseveral other adhesion molecules involved in T-cell activa-tion, including CD54 [intercellular adhesion molecule-1(ICAM-1)].28 During their maturation some DC also acquiremigratory ability; DC migration into regional lymph nodes,where naive T cells recirculate, optimizes antigen presenta-tion efficiency.28

Although most liver cells are not migratory, the highproportion of cardiac output traversing the liver suggests thatthe hepatic milieu would be continuously exposed to themajority of naive T cells recirculating via the blood. The mainrequirements for liver cells to act as APC for naive Tlymphocytes are, therefore, the ability to induce primaryactivation in the absence of inflammation or exogenous help,as well as a location that allows physical contact withcirculating T lymphocytes. Liver cells that may contact lym-phocytes in the non-inflamed state are those located in hepaticsinusoids: Kupffer cells, LSEC, DC and hepatocytes. Biliaryepithelial cells (BEC) will not be discussed in this review;although BEC may function as APC in vitro,29 they arelocated in portal tracts and are likely to be accessible tolymphocytes only in the presence of prior inflammation.

Role of Kupffer cells

Kupffer cells are hepatic macrophages that play a major rolein clearance of particulate material and endotoxin from theblood, antigen sequestration by phagocytosis, clearance ofimmune complexes and release of IL-1, IL-6 TNF-α andIFN-γ.8 Kupffer cells constitute 80–90% of total tissue macro-phages in the body. They are ideally located to encountercirculating lymphocytes in the sinusoidal lumen, in particularin the periportal sinusoids, as they rest on endothelial cellslining the sinusoidal vessels without forming junctional com-plexes (Fig. 2a). Some Kupffer cells are also found within thegap separating adjacent endothelial cells.8 Kupffer cellspossess migratory capacity and continuously patrol the sinu-soids at 2 µm/min.27 Like macrophages, Kupffer cells arederived from bone marrow, belong to the mononuclearphagocytic lineage and have phagocytic potential; they do,however, express markers distinguishing them from extra-hepatic macrophages.8 Kupffer cells express MHC class IImolecules, ICAM-1,30 as well as low levels of CD80 andCD86.31,32 Data showing that Kupffer cells may induceactivation of naive CD4+ or CD8+ T cells is lacking. However,in vitro experiments suggest that although they appear lessefficient than splenocytes and extrahepatic macrophages,31,33,34

Kupffer cells can function as APC for allo CD4+ T cells31,35–37

and Th1 clones.38

Role of liver sinusoidal endothelial cells

Liver sinusoidal endothelial cells are strategically located in thesinusoids to enable the extraction of blood-born material, asdemonstrated by their ability to clear low density lipoprotein

Figure 2 Cellular interactions that may mediate activation ofnaive T lymphocytes within the liver. Naive T lymphocytes recir-culating within the sinusoids may be activated by contact with (a)Kupffer cells (1) or LSEC (2) located within the lumen of thesinusoids, or by (b) contact with hepatocytes. Interaction of T cellswith hepatocytes may either occur through the gap between twoadjacent LSEC (3) or through LSEC fenestrae (4). This type ofinteraction may be facilitated by the low blood flow and thenarrow diameter of the sinusoids.

Primary T-cell activation within the liver 87

(LDL).39 Antigen uptake by LSEC, in particular in the form ofimmune complexes, has been demonstrated in several sys-tems. In some cases, antigen uptake by LSEC is moreefficient than by Kupffer cells.40 As with DC, antigen uptakeis mediated by specific receptors expressed on LSEC, includ-ing mannose receptor. Although vascular endothelial cellshave been shown to differentiate from bone marrow progeni-tor cells in vitro,41 experiments utilizing bone marrow chi-meric mice have suggested that LSEC are not derived frombone marrow.40 However, they do constitutively express bothMHC class I and class II molecules, low levels of CD8631 aswell as adhesion molecules including ICAM-1,30 CD106[vascular cell adhesion molecule-1 (VCAM-1)] and DC-specific ICAM-3-grabbing, non-integrin (DC-SIGN),42 sug-gesting that they can potentially function as APC to bothCD4+ and CD8+ T cells.33,40 Like Kupffer cells, they are alsoideally located to interact with T lymphocytes in the sinusoids(Fig. 2a). While early experiments by Rubinstein et al. indi-cated that guinea pig liver sinusoidal lining cells, a mixture ofLSEC and Kupffer cells, were very poor stimulators of naiveCD4+ T cells in MLR, despite expressing allo-MHC class IImolecules on their surface,33 more recent in vitro experimentshave shown that purified mouse LSEC induced activation andexpression of IFN-γ by naive CD62Lhigh TCR transgenicCD4+ T cells, as well as activation of TCR transgenic CD8+

T cells.43 Liver sinusoidal endothelial cells have also beenshown to be capable of cross-presentation of soluble antigenson MHC class I molecules to CD8+ T cells.44 Such contra-dictory results between early and recent reports may resultfrom species differences, or from the high affinity TCR usedin the transgenic mouse model. Although T-cell activation bymouse LSEC appears to be much less efficient than activationby DC or other macrophages,33,34 this property is unique toLSEC in comparison to other endothelial cells, which areunable to induce activation of naive T cells in the absence ofpro-inflammatory cytokines. As for most APC, antigen pres-entation by LSEC is sensitive to various cytokines that maybe released by neighbouring Kupffer cells; in particular, bothIL-10 and TNF-α downregulate LSEC-dependent activationof CD4+ T cells.31,40

Role of liver dendritic cells

The liver contains a very small subset (<1% of normal liver-non-parenchymal cells) of DC, which has been proposed to bethe major subset responsible for tolerance following livertransplantation.45 Liver DC reside around the portal tracts andcentral veins. It is now well documented that DC can undergoa blood–lymph translocation via the hepatic sinusoid, passingthrough the space of Disse before entering the regionalhepatic lymph nodes;46–48 thus, it remains unclear whether DCfound in the normal liver are organ-specific or whether theyare recruited from the blood. Freshly isolated normal liver DCare phenotypically and functionally immature.49,50 As in lym-phoid tissues, two distinct subsets, one of lymphoid origin,the other of myeloid origin, have been described in the liver.In mice, both subsets express CD11c and low levels ofCD11b, CD40, CD80, CD86 and MHC class II.50,51 However,only mouse liver lymphoid DC express the α chain of theCD8 molecule.50 Consistent with the low level of expressionof MHC and costimulatory molecules observed, recent

studies have demonstrated that liver DC appear unable toinduce efficient proliferation of allo T cells, in contrast tosplenic DC.45,50,51 Liver DC allostimulatory activity was notinducible by the addition of the pro-inflammatory cytokinesIFN-γ and TNF-α, but was induced by antigens51 or byovernight culture.50 Upon activation, both liver DC subsetsacquired the phenotype of mature DC, expressing high levelsof MHC class II, CD80 and CD86, and became capable ofinducing allogeneic T-cell proliferation in primary MLR.50,51

Given their immature phenotype and apparent lack ofallostimulatory capacity when unmanipulated, it has beensurmised that liver resident DC are unlikely to play a signifi-cant role in primary T-cell activation within a non-inflamedliver. However, it has been hypothesized that liver DC mayplay a role in the generation of liver allograft tolerance, amechanism possibly enhanced by the presence of inflamma-tion in the post-transplant setting. The putative role of liverDC in tolerance will be discussed further below.

Role of hepatocytes

Hepatocytes are the principal cell of the liver, comprising80% of hepatic cells. They are polyhedral, often polynucle-ated, cells involved in the synthesis and secretion of a widevariety of biologically crucial molecules, as well as themetabolism and excretion of an extensive range of bothendogenous and xenobiotic substances. Hepatocytes possessthe capacity for very efficient replication; if injured, the livercan regenerate the majority of its mass within a few days.52

Mathematical calculations based on serial mouse hepatocytetransplantation experiments have established that one hepato-cyte has the potential to divide at least 69 times, giving rise to6 × 1020 hepatocytes, without compromising their metabolicfunction.53 This astonishing growth potential is similar to thatof haematopoietic stem cells. Recent reports suggest thathepatocytes may be derived from bone marrow residentprecursors in certain circumstances.54–56 We have used radia-tion-induced bone marrow chimeric mice in order to test thishypothesis and have found that the contribution of donor bonemarrow to the hepatocyte compartment, two months follow-ing bone marrow reconstitution, was minor. The proportion ofhepatocytes bearing the transgenic antigen was less than 2%,even following partial hepatectomy, indicating that the major-ity of liver regeneration was derived from non-bone marrowresident cells (DG Bowen, unpubl. data, 2001).

Resting hepatocytes express MHC class I molecules,57

CD1 and ICAM-1 (P Bertolino, unpubl. data, 2001); MHCclass II molecules, CD40L and costimulatory molecules suchas CD80 are not constitutively expressed, however they areupregulated following inflammation. Because they normallylack MHC class II expression, resting hepatocytes may,therefore, only act as APC for CD1 or MHC class I restrictedT cells. Using TCR transgenic T cells, we have shown thathepatocytes can act as very efficient APC for high aviditynaive CD8+ T cells in vitro.58 Moreover, with the exception ofDC, other antigen-expressing cells were not able to induceproliferation of transgenic T cells in the absence of exoge-nously added IL-2, despite high levels of antigen expression. Itis likely that the unique ability of hepatocytes to activate naiveCD8+ T cells results from high levels of ICAM-1 expression. Inagreement with this hypothesis, hepatocyte-induced T-cell

88 P Bertolino et al.

proliferation was ICAM-1 dependent. We have shown thatT-cell activation induced by hepatocytes is as efficient asactivation induced by mature splenic DC, in terms of thenumber of APC required to obtain the equivalent degree ofT-cell proliferation.58 T cells activated by hepatocytes under-went most early activation associated events, includingexpression of early activation markers, blast formation, prolif-eration and acquisition of CTL activity. Although hepatocytesmay act as efficient APC in vitro, the question remains as towhether this phenomenon occurs in vivo. We have recentlyexplored this hypothesis by using Met-Kb transgenic mice, inwhich H-2 Kb antigen expression was mostly restricted tohepatocytes. When anti-H-2 Kb-specific Des-TCR T cellswere adoptively transferred into Met-Kb mice, transgenicCD8+ T cells were found to localize to the liver within minutesfollowing transfer,59 and to undergo activation within the next2 h. This study was complicated by the fact that transgeneexpression in Met-Kb mice is ‘leaky’, and consequently H-2Kb is also expressed at very low levels in bone marrowderived APC in lymphoid tissues. Concomitant activation inthe lymph nodes of Met-Kb mice was, therefore, also notedwithin 2 h following transfer; at this time point, however,CD69+ cells were not detected in the blood, suggesting thatactivation occurred independently in both liver and lymphnodes. Activated CD8+ T cells within the liver underwent celldivision after 24 h, acquired some CTL activity, and began torecirculate within this time period. This was in contrast to thesituation observed when antigen was restricted to the lymphnodes, where activated T cells were retained within thelymphoid tissue for at least 2 days before recirculating via theblood.59 Our results also indicated that TCR affinity for theantigen was important in T-cell activation by hepatocytes. InTCR transgenic mice, differences in TCR affinity are mim-icked by differences in TCR avidity resulting from variabilityin transgenic TCR expression levels due to incomplete exclu-

sion of endogenous alpha chains.60 Only high avidity CD8+

transgenic T cells were activated and proliferated within theMet-Kb liver (DG Bowen et al., unpubl. data, 2001). Incontrast, most low avidity T cells were not efficiently acti-vated, did not undergo intra-hepatic proliferation, and contin-ued to recirculate. These results suggest that hepatocytes mayactivate high avidity naive CD8+ T cells directly, without T-cell priming in the lymph nodes.15 It remains unclear howantigen presentation by hepatocytes may occur. It is unlikelythat hepatocyte protocytoplasmic extensions extend into thesinusoid lumen through the gaps between two adjacent LSECor through the fenestrae, as this is rarely seen in electronmicroscopy pictures; it is possible, however, that when lym-phocytes find their way through the narrow sinusoidal ves-sels, their cytoplasmic extensions penetrate these gaps or,alternatively, fenestrae in a similar fashion to Kupffer cells.8

Given the small diameter of the sinusoids, it is likely thatlymphocytes are squeezed between LSEC resulting in cyto-plasmic protuberances penetrating the space of Disse, andallowing such contact with hepatocytes (Fig. 2b). It has beenproposed that this ‘endothelial massaging’ by blood cells isimportant in causing fluid movement in the space of Disse;61

it is possible that this is also important for T-cell activationby hepatocytes.

T-cell activation by liver cells leads to tolerance

There is considerable data indicating that immune responsesto liver antigens result in tolerance rather than immunity. Thefact that the liver is a preferential site of T-cell apoptosis maybe the end result of the tolerance process. However, themechanisms leading to tolerance remain unknown. Toleranceto liver antigens results from a specific process rather thangeneralized immunosuppression; thus, tolerogenic mechan-isms are likely to involve specific interaction of the TCR with

Figure 3 T cells activated byhepatocytes undergo death byneglect in vitro. CD8+ T cellsactivated by hepatocytes or spleendendritic cells (DC) experiencedifferent fates in vitro. TransgenicCD8+ T cells activated by antigen-expressing DC survived up to5 days in coculture, while hepato-cyte activated T cells died within3 days. Premature death was dueto a low level of activation, withlow levels of cytokine production,and low expression levels of bcl-xL

survival genes, and could be pre-vented by adding exogenous IL-2or anti-CD28 monoclonal anti-body.63

Primary T-cell activation within the liver 89

peptide/MHC complexes. Although tolerance is probablydetermined by multiple events targeting both CD4+ and CD8+

T cells, primary T-cell activation within the liver by at leastone of the liver cell types discussed above may play animportant role in this process. The fate of CD4+ or CD8+

T cells activated by different liver APC has been examined indifferent systems, and has shown to be dependent on eitherimmune deviation for CD4+ T cells or deletion or ignorancefor CD8+ T cells.

CD4+ T cells recognize peptides associated with MHCclass II molecules; therefore, only Kupffer cells, LSEC andDC may act as APC for CD4+ T cells in the absence ofinflammation. Knolle et al. have shown that CD4+ T-cellactivation by LSEC results in immune deviation: TCR trans-genic T cells activated by LSEC do not differentiate towardsTh1 cells expressing IFN-γ and IL-2, but rather into regula-tory cells expressing IL-4 and IL-10, but not TGF-β.43 Thisparticular differentiation pathway appears isolated to LSECand may be due to constitutive levels of TGF-β expressed byLSEC.62

The situation is more complex for CD8+ T cells, as all livercells express MHC class I molecules and hence are potentialAPC. As mentioned above, we have recently reported thathepatocytes induce efficient in vitro activation of CD8+ TCRtransgenic T cells. However, T cells activated by hepatocytesdied sooner than those activated by DC (Fig. 3). Prematuredeath was Fas and TNFR independent, but was contingent onlow expression of cytokine and bcl-xL survival genes.63 Mono-clonal antibody crosslinking of CD28 molecules during T-cellactivation by hepatocytes resulted in enhanced cytokine andbcl-xL expression and protected hepatocyte-activated T cellsfrom early death in culture.63 These experiments suggest thatT cells activated by hepatocytes died by neglect due toinsufficient expression of IL-2 and survival genes resultingfrom lack of efficient costimulation during activation. Ifhepatocytes contribute to tolerance of CD8+ T cells to liverantigens, the end result of T-cell activation mediated byhepatocytes would be peripheral deletion. In this event,peripheral deletion would result from death by neglect, ratherthan exhaustion and/or activation-induced cell death. Periph-eral T-cell deletion was indeed observed in our transgenicmodel in which anti-H-2 Kb-specific TCR transgenic CD8+

T cells were injected into Met-Kb transgenic mice expressingH-2 Kb in the liver.15 Deletion occurred following a transienthepatitis peaking at day 5–6 after adoptive transfer. Theseexperiments suggest that hepatocyte-activated T cells inducehepatitis, but undergo deletion within the liver. However, dueto low level transgenic expression on recipient lymph nodeAPC, we are unable to exclude the possibility that T cellsactivated by hepatic cells died in situ, before the peak ofhepatitis, while those activated in lymph nodes inducedhepatitis and underwent apoptosis later on. This hypothesiswould be compatible with the kinetics of death by neglectobserved in vitro, which occurred 3 days following initialactivation, and may also explain why hepatitis was notobserved in similar transgenic mice expressing the relevantalloantigen in the liver under the control of the hepatocyte-specific albumin promoter (Alb-Kb). In Alb-Kb mice injectedwith Des-TCR lymph node T cells, hepatitis only occurredwhen inflammation was induced within the liver.64

Hepatocytes may not be the only liver cells involved inCD8+ T-cell tolerance. Recent reports have shown thatLSEC can cross-present exogenous antigens to transgenicCD8+ T cells in vitro and in vivo. Anti-ovalbumin TCRtransgenic mice challenged with ova-pulsed LSEC wereunable to reject a tumour expressing the same antigen.44

Although the authors of this study suggest that LSEC wereable to induce specific tolerance in the CD8+ T-cell compart-ment, they did not examine other liver cells to test whetherthis property is unique to LSEC. Interestingly, when studiedin vitro, the phenotype of CD8+ T cells activated by LSECin vitro was very similar to that we have described forhepatocyte-activated T cells.63 Although these authors did notexamine apoptosis in vitro, T cells cocultured with LSEC for5 days secreted significantly lower amounts of IL-2 andIFN-γ and were poorer CTL than T cells activated by spleno-cytes. Similarly, CTL activity was restored when IL-2 wasadded to LSEC/T-cell cocultures.44 These results suggest thatdeath by neglect may be a general mechanism of tolerance forCD8+ T cells activated by liver APC.

In a recent report, Crispe and colleagues hypothesized thattolerance to hepatic antigens may well involve a combinationof liver cells.65 Using bone marrow chimeric mice in whichantigen presentation was restricted to bone marrow-derivedAPC (Kupffer cells and DC) or other hepatic cells, this studysuggests that antigen presentation by hepatocytes or LSECcontrols intrahepatic T-cell trapping while bone marrow-derived APC promote T-cell apoptosis within the liver.

Recent studies have suggested that liver DC may alsocontribute to tolerance under certain conditions,66 via theinduction of Th2 and IL-10-producing regulatory Tr1 cellexpansion.45 However, antigen presentation to T cells by liverDC appears to occur in lymphoid tissues, where mature DCmigrate upon hepatic inflammation, rather than in the liveritself. The role of hepatic DC in liver tolerance is derivedfrom the original observation by Starzl and coworkers thatsuccessful liver transplantation was associated with migrationof donor leucocytes into recipient lymphoid tissues.67 Thisassociation is not absolute, however, and it is difficult toassess whether this microchimerism is a cause or conse-quence of the tolerance induced by the liver transplant.Indeed, the increased rejection observed when mice trans-planted with an allogeneic liver were treated with the potentDC-inducing haematopoietic cytokine Flt3 ligand68 does notsupport this model. Furthermore, some reports have sug-gested that liver DC induce immunity rather than tolerance,51

leading to the proposal that liver DC may represent theimmunogenic APC of the liver, while other hepatic APC maybe tolerogenic.51 Consistent with this model, it would not beunreasonable to hypothesize that the ability of hepatic DC toemigrate the tolerogenic intrahepatic milieu, and induceimmunity upon activation within lymph nodes, may be crucialto generating immune responses to liver antigens. The possi-bility exists, however, that there are several DC subsets withvarying characteristics within the liver, only some of whichare tolerogenic.66 This question is very difficult to address asDC function and phenotype change during purification andare highly dependent on cytokines present in their micro-environment. Therefore, the role of DC in liver toleranceremains unclear (see review by Doherty and O’Farrelly69).

90 P Bertolino et al.

Conclusion

There is accumulating data demonstrating that T-cell activationagainst hepatic antigens induces tolerance. However, the mecha-nisms involved in this process remain ill defined. It is likely thatin the absence of inflammation, the cells residing within a normalliver may induce tolerance by directly activating naive T cellscirculating via the sinusoids. This process may involve livercells endowed with the ability to activate naive T cells: LSEC,Kupffer cells, hepatocytes, or a combination of these. The slowblood flow in the sinusoids and the unique structure of the liversinusoidal endothelium would favour contact between T cellsand hepatic cells; this activation would result either in alteredcytokine production by CD4+ T cells or deletion, by neglect, ofCD8+ T cells. Primary T-cell activation within the liver may playa major role in establishing peripheral tolerance to intrahepaticantigens by autoreactive T cells that have escaped negativeselection during thymic differentiation. It may also play a role inthe development of oral tolerance to antigens absorbed throughthe gut or injected intravenously. The role of liver DC in thisprocess, mediated either by direct presentation or by crosspresentation, remains unknown, although hepatic DC may play arole in the setting of inflammation, such as occurs following livertransplantation. In this situation it is possible that both antigenpresentation by DC in lymph nodes, and primary T-cell activa-tion within the liver occur concomitantly to induce T-cell toler-ance. By selectively activating and deleting high avidity CD8+

T cells shortly after transplantation or viral infection, the livermay dispose of the majority of host cells responsible for earlyacute rejection or viral clearance. In the case of viral hepatitis,peripheral deletion of high avidity T cells might leave cells ofinsufficient avidity to clear the virus, but still capable of mediat-ing ongoing immune injury.

In all these settings, tolerance is a non-passive processinvolving T-cell activation. This may explain why livertransplant tolerance is associated with greater T-cell activa-tion and cytokine production than rejection at early timepoints.70 Such activation leading ultimately to tolerance, maycause transient liver damage: Met-Kb transgenic mice injectedwith TCR transgenic T cells developed hepatitis that resolvedover a few days, and was associated with peripheral T-celldeletion. This may be due to the unique ability of the liver toregenerate upon injury, providing continuous antigen supply:such regenerative capacity suggests that immune responsestowards liver antigens occur under very different conditionsto that in the other solid organs. In the majority of other solidorgans, this type of injury would have caused irreversibledamage to the tissue and would have been classified asautoimmunity rather than tolerance. In this context, thefactors enabling the occurrence of chronic immune-mediatedliver injury associated with both autoimmune hepatitis andchronic viral hepatitis remain to be defined.

Acknowledgements

This work was supported by the National Health and MedicalResearch Council of Australia (NHMRC). P Bertolino wassupported by a grant from the NHMRC. DG Bowen wassupported by an NHMRC Medical Postgraduate ResearchScholarship. The authors wish to thank Dr Caroline Deman-gel for helpful comments on the manuscript.

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