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Natural killer cells and hepatitis C:action and reactionKuldeep Cheent, Salim I Khakoo

ABSTRACTIn 1989, hepatitis C virus (HCV) was first identified as theinfectious agent responsible for human non-A, non-Bhepatitis.1 Two decades later, HCV remains a globalpublic health problem with a suboptimal response rate totreatment and the absence of a protective vaccine.Recent work has highlighted the influence of the innateimmune system, and in particular natural killer cells, onthe outcome and pathology of HCV infection. These cellsare considerably more complex than was originallythought and their role in viral infections is currently beingunravelled. This review summarises our emergingunderstanding of natural killer cells in HCV infection.

INTRODUCTIONHepatitis C virus (HCV) is a small envelopedpositive-stranded RNA virus belonging to theHepacivirus genus of the Flaviviridae family.1 Thereare six major genotypes which vary by up to 30% innucleotide sequence. The global importance ofHCV is illustrated by the observation that world-wide there are currently 130e170 million peoplebelieved to be infected.2 The majority of individuals(50e85%) exposed to HCV develop chronic infec-tion, with the associated risks of cirrhosis, liverfailure and hepatocellular carcinoma.3 Therefore itis important to elucidate the mechanisms associ-ated with both viral clearance and disease progres-sion. Natural killer (NK) cells have been implicatedin all stages of HCV infection in both genetic andfunctional studies. This role may be either direct,by targeting infected hepatocytes, or indirect byinfluencing other key immunocytes such asdendritic cells (DCs) or T cells.

NK CELL RECEPTORS AND FUNCTIONSNK cells comprise 5e20% of peripheral bloodmononuclear cells but make up a substantiallygreater proportion (30e50%) of lymphocytes in theliver.4 They were originally described based on theirability to directly kill major histocompatibilitycomplex (MHC) class I-negative tumour targets, asdistinct from classical cytotoxic T cells, whichrequire the presence of MHC class I for target cellrecognition.5 The majority of NK cells are definedphenotypically by the presence of the surfacemarker CD56 and the absence of CD3. Dependingon the level of cell surface expression of CD56 theycan be divided into two main subsets, CD56bright

and CD56dim, each with distinct properties.6

CD56dim NK cells express a moderate level ofCD56, represent a differentiated subset and form

the majority (>90%) of the circulating NK cellpool. Compared to the CD56bright population,CD56dim NK cells express higher levels of killer cellimmunoglobulin-like receptors (KIRs), CD16 andperforin and are regarded to have marked cytotoxicpotential. The CD56bright subset, which expresseshigh levels of CD56, contribute up to 10% of theperipheral blood NK population and are regarded asa less mature subset with a potential to differen-tiate into CD56dim NK cells.7 CD56bright NK cellsexpress a high level of the inhibitory receptorCD94:NKG2A and generally do not express KIRs orCD16. The primary function of CD56bright NK cellsis cytokine production which enables them tohave an immunoregulatory function at sites ofinflammation where they may be expanded. Thefunctional distinction between the CD56bright andCD56dim populations is not absolute, andCD56bright NK cells can express the degranulationmarker CD107a while CD56dim NK cells canproduce cytokines. Indeed the total interferon g(IFNg) production from CD56dim NK cells canexceed that of the CD56bright fraction.8 SimilarlyCD56bright NK cells can be cytotoxic throughexpression of tumour necrosis factor-relatedapoptosis-inducing ligand (TRAIL).9

In contrast to T and B cells, NK cells do notrequire priming to recognise virus-infected cells,and can thus be rapidly activated by cytokinestimulation. They recognise potential target cellsusing a plethora of cell surface receptors thattransduce activating or inhibitory signals. Whenthe signals from the activating receptors exceedthose from inhibitory receptors NK cell effectorfunctions are initiated.10 These functions includesecretion of the T-helper 1 (Th1) cytokines IFNgand tumour necrosis factor a (TNFa), and directcytotoxicity of target cells (figure 1). Target cellkilling can be mediated by degranulation of cyto-toxic granules, and by surface expression of ligandssuch as Fas ligand (FasL) and TRAIL that activatedeath receptors on target cells. Additionally, theTh1 type cytokines can prime the adaptive immuneresponse and IFNg, in particular, can have a directantiviral effect.NK cell function is critically regulated by

combinations of stimulatory, co-stimulatory andinhibitory receptors (table 1). The net balance ofsignals derived from these receptors determineswhether the NK cell becomes activated. Activatingreceptors expressed on NK cells include the C-typelectin-like receptors NKG2D and CD94:NKG2C/E,natural cytotoxicity receptors NKp44, NKp30,NKp46, and CD16 (FC-g-RIII), which is the low-

Division of Medicine, ImperialCollege London, London, UK

Correspondence toSalim I Khakoo, 10th FloorQEQM Building, ImperialCollege, St Mary’s Campus,South Wharf Road, London W21NS, UK;[email protected]

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affinity Fc receptor and mediates antibody-dependent cytotoxicity. Key inhibitory receptors onNK cells are the killer cell immunoglobulin-likereceptors (KIR) and the CD94:NKG2A heterodimer.

NK CELLS AND THE LIVERThe liver is relatively enriched in NK cells. Thisintrahepatic population is embedded in the endo-thelial lining of the liver sinusoids and was origi-nally described as ‘pit’ cells.11 These cells are largegranular lymphocytes that are capable of sponta-neous cytotoxicity against an MHC deficient cellline without prior sensitisation, a defining NKcell characteristic.12 13 They contain characteristicgranules and, uniquely, rod-cored vesicles.14 Theycan be sub-classified based on the density and sizeof their granules which are either low density andsmall granules; or high density and large granules,with NK cells that contain the latter resemblingperipheral blood NK cells. It has been shown in ratsthat peripheral blood high-densityelarge granularcells migrate to the liver and differentiate intoliver-specific low-densityesmall granular NK cells.15

Intrahepatic NK cells may behave differently toNK cells in other areas due to the ‘tolerogenic’environment in the liver. Murine intrahepatic NKcells are hyporesponsive. They are less cytotoxicand have an altered cytokine profile producinglower levels of IFNg and greater levels of immu-noregulatory cytokines, such as interleukin-10(IL-10), compared to peripheral blood and splenicNK cells.16 This hyporesponsive state has beendescribed in the early stages of hepatitis B virusinfection and may contribute to the establishmentof chronic viral infection.17

In addition to their role in protection againstpathogens and tumour transformation, intra-hepatic NK cells have been demonstrated to haveanti-fibrotic functions via inhibition of hepaticstellate cells (HSCs). They are capable of directlyinducing HSC apoptosis, and producing IFNg

which inhibits HSC activation.18e20 Interestingly,greater levels of peripheral blood NK cell cytotox-icity have been associated with less liver fibrosis inpatients with chronic HCV consistent with thelysis of activated hepatic stellate cells.21

NK CELLS AND THE OUTCOME OF HCV INFECTIONUnderstanding the mechanisms by which HCV issuccessfully eradicated is especially important fortherapeutic and vaccination strategies. NK cellswere originally implicated in determining theoutcome of HCV infection in an immunogeneticstudy of the KIR genes and their human leucocyteantigen-C (HLA-C) ligands.22 The KIR genes area highly polymorphic group of receptors, showingdiversity at the levels of both the locus and allele.23

Different individuals have different combinationsof KIR genes. Their MHC class I ligands are alsohighly polymorphic, therefore there is a substantialpopulation diversity in this receptoreligandsystem. This diversity makes the KIR genes idealcandidates for modulating disease outcomes. Bycomparing the presence or absence of KIR genesand their HLA ligands in over 1000 individualsexposed to HCV it was shown that the specificcombination of the inhibitory receptor KIR2DL3and its group 1 HLA-C ligand (HLA-C1) wasprotective against chronic HCV infection.22 Theprotective effect of this gene combination waslimited to individuals infected by intravenous druguse or accidental needle-stick injury. No protectiveeffect was observed in subjects infected by trans-fusion of blood products in whom the innateimmune response was thought to be overwhelmedby the higher infecting inoculum. The associationof KIR2DL3 and group 1 HLA-C was subsequentlyconfirmed in a smaller study of intravenous drugusers of Puerto-Rican origin, indicating that thisprotective effect is consistent across differentpopulations.24 It has been postulated that this genecombination is protective because the KIR2DL3

Figure 1 Effector mechanisms of natural killer (NK) cells. Effector mechanisms of NK cells include: degranulation ofcytotoxic granules resulting in direct cytotoxicity to target cells; induction of apoptosis via surface upregulation of Fasligand (FasL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL); and secretion of both inteferon g(IFNg) and tumour necrosis factor a (TNFa), which have a direct antiviral effect and induce the adaptive immuneresponse. DC, dendritic cell T, Tcell.

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binds HLA-C with a lower avidity than otherinhibitory KIR, and thus NK cells expressing thisspecific inhibitory receptor have a lower thresholdfor activation.25 26 Consistent with this, NK cellsexpressing KIR2DL3 respond more rapidly in vitroto influenza infected cells.27

NK cells are also inhibited by the heterodimericreceptor CD94:NKG2A, which has the oligomor-phic MHC class I molecule HLA-E as is its ligand.In a genetic study homozygosity for the HLA-ER

allele has been shown to be protective againstchronic infection with HCV genotypes 2 and 3.28

This protective effect was thought to be due to aneffect on HLA-E restricted T cells, although theHLA-ER allele may have a lower affinity forpeptides and hence be expressed at lower levels.This could therefore lead to less inhibition of NKcells via the CD94:NKG2A receptor.29

Similarly, a number of cytokines involved in NKactivation or function have been implicated in theoutcome of HCV infection. These include the NKcells associated cytokines IL-12, IL-18 andIFNg.30e34 The type III interferon IL-28B (IFNl3)has received much attention as a mediator ofclearance of HCV.35 36 It is not known whetherIL-28B has a similar or complementary functions tothe type I interferon, IFNa, which is involved inNK cell activation. However, as these moleculesshare common signalling pathways there is likelyto be at least some overlap in function.

NK CELLS IN ACUTE HCV INFECTIONIn addition to genetic studies, NK cells have alsobeen implicated in the acute phase of HCV infec-tion (figure 2). Nowadays, these patients rarelycome to the attention of clinicians, as they areoften asymptomatic and therefore do not seekmedical assistance. Amadei et al recently reportedan increase in CD56bright NK cells (with an associ-ated reduction in the CD56dim subset) in acuteHCV patients compared to healthy individuals.37

Individuals who spontaneously cleared the virusshowed a decline in the CD56bright population,with levels comparable to healthy control individ-uals after 1e3 months, indicating a return tobaseline which was not observed in those thatwent on to have a chronic infection. Expression ofthe activating receptor NKG2D was also increased

in the acute phase of infection. Functional experi-ments showed augmented IFNg production andcytotoxicity in these patients and a trend for moreNK cell degranulation in individuals expressingHLA-C1 specific KIR receptors, which wasmaximal in those with self-limiting infection. Thusin the acute phase of HCV infection there is acti-vation of NK cells indicating their role in theimmune response at this stage. Pelletier et al havealso studied individuals in the acute phase ofHCV infection.38 They also found increasedactivity of NK cells as determined by a degranula-tion assay, but found that the NK cells fromintravenous drug users had generally lower levels ofIFNg secretion as compared to healthy controls,and suggest that this may be related to opioid use.They found that the levels of the inhibitoryreceptor NKG2A, declined on CD56bright NK cellsduring the follow-up phase in those spontaneouslyresolving infection only. Furthermore they wereable to correlate NK cell activity with T cellactivity, implying a coordinated innate and adap-tive immune response to acute HCV infection.Thus there is activation of NK cells in the acutephase of HCV infection, which declines in thoseclearing HCV and persists in those remainingchronically infected.These findings contrast with acute HBV infec-

tion in which it was recently shown that there maybe relatively little secretion of the innate cytokinesIFNa, IFNl1 and IL-15.17 Associated with this, anattenuation of NK cell IFNg secretion has beendemonstrated, although this has not been univer-sally reported.39 A full description of the innateimmune response in HBV infection is beyondthe scope of this article, and has recently beencomprehensively reviewed.40 However, in summary,HBV and HCV appear to have distinct effects onNK cell activation in the acute phase of infection,underpinning key differences in the pathobiologyof these hepatotrophic viruses.

NK CELLS IN CHRONIC HCV INFECTIONMore amenable to study are NK cells fromindividuals with chronic HCV infection wherecomparisons with healthy donors have shownperturbations in NK cell frequency, phenotypeand function (figure 3). Peripheral blood NKcell frequencies, both absolute number andpercentage of total lymphocyte population, arereduced in chronic HCV compared to healthyindividuals.21 41e44 The reduction in NK frequencymay be a consequence of HCV infection, ora predisposing factor to chronic HCV infection, andboth explanations have some support. In individ-uals with chronic HCV infection, NK cell frequencyincreases following successful antiviral therapywhile a reduction in peripheral blood NK cellfrequency in individuals with chronic HCV ascompared to spontaneous resolvers has also beennoted.42 45e47 IL-15, a pivotal cytokine for NK celldevelopment, proliferation and function, may berelevant to this observation. Meier et al showeda significant reduction in IL-15 levels in HCV

Table 1 Key receptors for natural killer cells

Function Family Receptor Ligand

Activating C-type lectinreceptor

NKG2DCD94:NKG2CCD94:NKG2E

MIC-A/B, ULBPsHLA-E

Natural cytotoxicityreceptor

NKp30NKp44NKp46

BAT-3, B7-H6, CMV pp65Viral haemagglutininViral haemagglutinin

Killer cell immunoglobulinreceptor

3DS1 HLA-Bw4

Others CD16 IgG

Toll-like receptors Pathogen-associated molecularpatterns (PAMPs)

Inhibitory Killer cell immunoglobulinreceptor

2DL12DL2/3

Group 2 HLA-CGroup 1 HLA-C

C-type lectin receptor CD94:NKG2A HLA-E




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patients as compared to healthy controls anddemonstrated that exogenous IL-15 rescued HCVNK cells from apoptosis, increasing ex vivo prolif-eration and function.43 Furthermore, DCs are animportant source of IL-15 and have been shown tocross-talk with NK cells. In chronic HCV infectionIL-15 production by IFNa-stimulated DCs is defi-cient.48 Thus a downstream consequence of thisdendritic cell dysfunction could be inadequateproduction or proliferation of NK cells.

Skewing of subset distributionA number of studies have documented a relativeincrease in circulating CD56bright, but not CD56dim

NK cells, in chronic HCV compared to healthyindividuals and spontaneous resolvers.21 41 47 49e51

Bonorino et al demonstrated that the relativeproportions of CD56dim and CD56bright NK cells inthe liver are altered in chronic HCV infection.41

They found that 80.5% of intrahepatic NK cellswere CD56dim as compared to 94% in peripheralblood and 19.5% intrahepatic NK cells wereCD56bright as compared to 6.0% in peripheral blood.This implies that the decreased frequency ofCD56dim NK cells in the periphery is not related totheir sequestration in the liver, although there is no

clear data on the relative proportions of CD56dim

and CD56bright NK cells in the healthy liver.CD56�CD16+ NK cells appear to be more

terminally differentiated NK cells and there is anexpansion of this subset in chronic HCV infec-tion.52 These cells have reduced perforin expressionas compared with CD56dim NK cells and have beenshown to be hypofunctional, particularly in theirinteractions with dendritic cells.53 In HCV,chemokine production by this subset was skewedtowards MIP-1b, and there was also a reduction inIFNg and TNFa secretion compared to the CD56+NK population. Thus, overall, there is a skewing ofNK cells away from the CD56dim CD16+ subset,which is thought to be the main cytotoxic subset ofNK cells. This may be an effect of IFNa, as there isa strong IFNa response to HCV infection, andtherapy with pegylated IFNa and ribavirin leads toan increase in CD56bright and a decline in CD56dim

NK cells.42 54

Alterations in phenotypeChanges in phenotype may reflect changes in subsetdistribution and also the effect of cytokines onspecific subsets of NK cells. CD56bright NK cells are

Figure 2 Natural killer (NK) cells in acute hepatitis C virus (HCV) infection. NK cells are activated in the acute phase ofHCV infection. Downregulation of major histocompatibility complex (MHC) class I on virus-infected hepatocytes mayreduce the inhibitory signal to NK cells, shifting the balance towards NK cell activation. Dendritic cells (DCs) engagewith NK cells via the NKp30 receptor, and produce cytokines which boost NK cell proliferation towards an ‘NK1’phenotype. Activated NK1 cells produce cytokines such as interferon g (IFNg) and tumour necrosis factor a (TNFa)which suppress HCV replication, reciprocally activate DCs, and prime naive CD4 T cells inducing a T-helper (Th)-1response. IL, interleukin.




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KIR-negative and NKG2A-positive, and the mostconsistent finding has been an increase in NKG2Aexpression in chronic HCV infection.49 55e58 Thisoccurs on both intrahepatic and peripheral bloodNK populations,41 with some studies reportinga decrease in KIR-expressing NK cells.42 44 54 59

Overall, there is also an increase in activatingreceptor expression. Such receptors include NKG2C,NKp44, NKp46 and NKp30.49 59 60 Initial reports ofdecreased expression of NKp46 have not beensubsequently confirmed.57 Similarly there isconflicting evidence with respect to NKG2Dexpression which has been reported as being upre-gulated, downregulated and also unchanged.49 55 58 59

Such contrasting data may relate to genuinedifferences in NK cell phenotype and function, ortechnical issues such as sample preparation, cytokinestimulation or freezing. There is also substantialpopulation diversity in both NK cell phenotype andfunction which may account for these conflictingfindings, especially if the study populations aresmall.61 62 Thus consistent with the findings fromlongitudinal studies of individuals with acute HCVinfection, NK cells from those with chronic HCVinfection appear to be chronically activated.

Altered functionInitial reports suggested diminished natural cyto-toxicity in chronic HCV that was restored bysuccessful HCV clearance with IFNa and ribavirintherapy.4 46 63 64 However, the number of cytotoxicCD56dim NK cells in the peripheral blood isdepressed and hence, recent studies which take intoaccount cytotoxicity per NK cell have demon-strated normal or increased NK cell cytotoxicity inchronic HCV.21 44 47 49 60 65 66 There is greaterexpression of activation markers such as CD122(a subunit of IL-2 receptor which is crucial for IL-2and IL-15 signalling), CD69, and NKp44.44 60

Upregulation of TRAIL on NK cells may also be animportant mechanism underlying the anti-HCVeffect of NK cells. Liver NK cells expressing TRAILkill autologous hepatocytes in mice,67 and maytherefore contribute to liver injury. NK cell TRAILexpression is increased in chronic HCV, and thesecells have a phenotype consistent with IFNastimulation.9 60

A change in the cytokine profile of NK cells inchronic HCV may be relevant to the persistence ofHCV infection. Failure of NK cell production ofIFNg in chronic HCV has been reported42 44 60

Figure 3 Natural killer (NK) cells in chronic hepatitis C virus (HCV) infection. In chronic HCV infection, changes occurin NK cell frequency, phenotype and function associated with chronic stimulation by interferon a (IFNa). HCVcore-encoded peptides upregulate HLA-E, binding to the inhibitory receptor CD94:NKG2A and there is skewing of the NKcell cytokine profile towards the immunoregulatory cytokines IL-10 and transforming growth factor b (TGFb). These NK2cytokines influence DC and T cell function, leading to further production of immunoregulatory cytokines and generationof a T-helper (Th)-2 response. Upregulation of NK cell tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)contributes to liver injury while increased TGFb and attenuated IFNg production promotes activation of hepatic stellatecells (HSCs) leading to fibrosis. Regulatory T cells (Treg) may suppress the tumour surveillance function of NK cells,increasing the risk of hepatocellular carcinoma. DC, dendritic cell.




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IFNg has potent direct anti-HCV properties,blocking HCV replication in a dose-dependentmanner.68e70 IFNg also indirectly suppresses HCVactivity by polarising T cell differentiation towardsa virus specific Th1 phenotype. Additionally,increased HCV-NK cell production of Th2 cyto-kines such as IL-10 and transforming growth factorb (TGFb), and the chemokine IL-8, may skew thecytokine profile towards an environment which ismore permissive for HCV.44 49 55 69 71 This pheno-type, which is infrequently found in healthy indi-viduals, is similar to the NK2 phenotype asdescribed by Peritt et al in which NK cells secretethe Th2 cytokines IL-5 and IL-13.72 In vitro NKcells can be polarised towards this phenotype underthe influence of IL-4. Thus the Th2 environmentfound in chronic HCV infection may affect thedifferentiation and maturation of NK cells, towardsthis NK2 phenotype which further contributes tothe Th2 environment in a positive feedback loop.This polarisation may occur either in the periphery,or within the liver microenvironment in which IL-10 secreting T cells have been described in chronicHCV infection localised to specific areas of T cellinfiltration.73 74

Thus the cytokine microenvironment may affectNK cell phenotype and also function. In chronicHCV infection the dominant effect on NK cellsappears to be of IFNa, but the roles of IL-4, IL-10and IL-13 warrant further investigation, as theprofile of NK cells in chronic HCV is consistentwith a reduced maturation of CD56bright NK cells,and also enhanced differentiation of CD56dim NKcells towards a CD56� CD16+ phenotype (figure 4).NK cells may also be modulated by direct cellular

interactions, especially with DCs. All mature NKcells express the activating receptor NKG2D, the

ligands for which are MHC class I chain-related(MIC) proteins. In HCV infection there is animpairment of MIC-A/B expression which resultsin lower levels of NK cell activation.48 NK cellactivation of DCs may be reciprocally perturbed.When co-cultured with human hepatic cells, NKcells enhance maturation and activation of DCs topromote a Th1-polarised CD4 T-cell response.55 NKcells from HCV-infected individuals have a reducedcapacity to activate DCs, due to NK cell inhibitionby the CD94:NKG2A receptor and a consequentincrease in NK expression of the immunoregulatorycytokines IL-10 and TGFb, which promote Th2type differentiation. Interestingly, inhibition ofNKG2A restored the ability of HCV-NK cells toactivate DCs, and also the production of the Th1cytokines IFNg and TNFa. This may be importantas HCV can upregulate HLA-E, the ligand forNKG2A, in vivo and so represents a mechanism bywhich HCV may modulate the NK cell response.56

The activation status of NK cells correlates withliver inflammation. Increased expression ofNKG2A, CD69 and CD107a (a marker of NK celldegranulation) on peripheral blood NK cells haveall been linked with disease activity, and in thestudy of Oliveiro et al an inverse correlation ofperipheral NK cell NKG2D expression with alanineamino transferase (ALT) was found.9 41 44 60

Furthermore, NK cells have been localised tonecrotic areas in liver biopsy specimens in chronicHCV, but not chronic HBV.41 These intrahepaticNK cells express higher levels of TRAIL and theactivating receptor NKp46.60 Bonorino et al alsofound an inverse correlation between NKG2A-positive NK cells and viral load.41 However, al-though correlations with disease and RNA levelshave significant p values, they have relatively low

Figure 4 A model of abnormal natural killer (NK) cell differentiation in chronic hepatitis C virus (HCV) infection. TheCD56bright CD16� NK cell population is expanded in chronic HCV as a consequence of a Th2 type cytokinemicroenvironment, either in the periphery or within the liver, leading to a failure of their maturation to the CD56dim CD16+ subset. Chronic stimulation by activating receptors and cytokines within the liver may then lead to accelerateddifferentiation of CD56dim CD16+ subset towards the functionally defective CD56� CD16+ subset, which is alsoexpanded. These processes lead to a reduced frequency of the functionally mature, cytotoxic CD56dim CD16+ subsetand the observed relative increase in CD56bright CD16� NK cells.




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values for the correlation coefficient r, indicatingthat much of the variability in inflammation andRNA levels is accounted for by factors other thanthose specifically studied.

NK CELLS AND INTERFERON TREATMENTIFNa is a potent NK cell activating cytokine that isthe cornerstone of treatment for HCV infection.NK cells have been implicated as determining theoutcome of treatment for HCV in genetic, pheno-typic and functional analyses. Genetic studies haveindicated that a similar combination of KIR andHLA-C genes (KIR2DL3 and group 1 HLA-C) areassociated with a beneficial response to treatmentwith IFNa based regimens, as has been observed forspontaneously resolving HCV infection.75 76 Thisdemonstrates a consistency in the protective effectsof these genes in HCV infection, which has alsobeen extended to multiply exposed intravenousdrug users.75

Pre-treatment peripheral blood NK cell pheno-type may also predict the response to pegylatedinterferon and ribavirin therapy. An 80% sustainedvirological response (SVR) rate was observed inindividuals in whom the frequency of CD56�CD16+ NK cells was in the normal range,compared to only 25% SVR in individuals whereCD56� CD16+ frequency was significantly abovethe normal range.52 Additionally, in a small study,individuals with an SVR were found to have moreKIR-negative NKG2A-positive CD56dim NK cellsthan non-responders.59 Thus both KIR-positive andKIR-negative NK cells may have a role to play inthe outcome of HCV treatment.During IFNa therapy there is an increase in NK

cell cytotoxicity4 46 63 64 which may be due toaugmented degranulation and also the capacity toinduce apoptosis. TRAIL induces apoptosis of cellsexpressing the death receptors DR4 and DR577 andhas emerged as an important marker associatedwith the response to IFNa.9 In vitro, HCV infectionsensitises hepatocytes to TRAIL-mediatedapoptosis and TRAIL on NK cells has been associ-ated with liver inflammation in HBV infection.78

Baseline levels of TRAIL on NK cells in chronicHCV may be normal or elevated.9 60 However,upon IFNa stimulation NK cells express higherlevels of TRAIL, and upregulation of TRAIL onCD56bright NK cells is observed in individualsundergoing anti-viral therapy. Additionally, analysisof paired liver biopsy samples has shown that SVRis associated with an increase in the frequency andtotal number of intrahepatic NK cells followingIFNa and ribavirin treatment.79

POTENTIAL FOR HCV TO ESCAPE NK CELLSURVEILLANCEHCV has a number of strategies to evade the hostimmune response and these may specifically affectNK cells. The virus has multiple mechanisms tointerfere with secretion of type I interferons, whichmay attenuate anti-viral NK cell activity.80 HCVcore protein has been reported to substantially

upregulate the expression of MHC class I on Hep3Bcells with an associated downregulation of NKcytotoxicity.81 Similarly, a peptide derived fromHCV core (HCVcore 35e44) is capable of upregu-lating HLA-E, leading to CD94:NKG2A mediatedinhibition of NK cells.56 This peptide is alsopresented by HLA-A2 to cytotoxic T lymphocytes(CTL), and the presence of CTL specific to thispeptide in individuals with chronic HCV impliesthat it can be endogenously processed and pre-sented.82 Thus in vivo it could also be loaded ontoHLA-E and so represents a tenable mechanism bywhich HCV may escape NK cell activity. Con-versely, the NS4A/B protein of HCV has beenshown to downregulate MHC class I as a conse-quence of an inhibition of its trafficking throughthe ER and this could lead to NK cell activation.83

Thus the effects of HCV on MHC class I arecomplex and the net effect that these changes mayhave on NK cells in vivo are not clear at present.HCV-E2 has been shown to reduce NK cell IFNg

secretion by binding to CD81 on NK cells.69 Thereduction in NK cytotoxicity was partly attributedto a direct effect of the HCV envelope protein E2cross-linking tetraspanin CD81 receptors on NKcells, a theory supported by in vitro evidence fromtwo groups.63 84 Both of these studies used a highconcentration of truncated, plate bound HCV-E2protein, rather than complete infectious virions,which have been developed relatively recently.These earlier studies have now been challenged.Although anti-CD81 antibody inhibited NK cells,HCV-E2 protein when part of complete solubleinfectious particles is unable to do so.66 69 Inter-estingly, Crotta et al found that immobilised HCVvirions inhibited IFNg production by IL-12-activatedNK cells and this effect was due to engagement ofcellular CD81 by HCV-E2. However, given theobservations of Yoon et al and also that NK cellsappear to be activated in acute HCV infection, itappears that the direct effects of HCV-E2 on NKcells are unlikely to be significant in vivo.37 38 66

NK CELLS IN HCV-ASSOCIATED CIRRHOSIS ANDHEPATOCELLULAR CARCINOMAApproximately 20% of patients with chronic HCVinfection will develop cirrhosis after 20 years ofinfection. NK cells may impact on this both bycontrolling infection and by directly affecting thefibrogenic process. This is because NK cells cancause apoptosis of the fibrogenic stellate cells byNKG2D and TRAIL mediated signalling, and alsobecause the pro-inflammatory cytokine IFNg isanti-fibrogenic.85 86 Reduction in intrahepatic NKfrequency also correlates with the progression ofHCV and the development of cirrhosis.87 Hepato-cellular carcinoma (HCC) is a common complica-tion of cirrhosis, and reductions in peripheral andintrahepatic NK cell frequency, cytotoxicity andIFNg production have been reported in individualswith HCC as compared to healthy controlsubjects.88 89 Depressed NK cell activity may bemediated by regulatory T cells, which suppress theanti-tumour functions of NK cells.90 An anti-




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tumour effect of NK cells is supported by animmunogenetic study in which the combination ofthe activating NK cell receptor KIR3DS1 andits HLA-Bw4180 ligand was under-represented inHCV-positive patients who developed HCC, ascompared to those who remained tumour free.91

TRAIL has been implicated as one importantmolecule in this anti-tumour function of NKcells,92 with another being NKG2D. NKG2Drecognises the stress ligands MICA/B, which havea restricted expression on healthy cells but areupregulated by viral infection and tumour trans-formation thereby providing a strong signal for NKcell activation via NKG2D. Shedding of MICA/Binto the circulation leads to downregulation ofNKG2D on T and NK cells and provides a mecha-nism of tumour escape.93e95 This may happen inHCC and the anti-HCC drug sorafenib has beenshown to inhibit this proteolytic cleavage ofMICA/B from HCC cells, thus enhancing NK cellmediated cytolysis of tumour cells.96

NK CELLS AND LIVER TRANSPLANTATIONHCV-related liver disease is a frequent indicationfor orthotopic liver transplantation (OLT) in theWestern world. As NK cells express inhibitoryreceptors for polymorphic HLA class I moleculesand liver transplants are not HLA matched, there isa strong potential for alloreactivity of recipient NKcells against the donor liver, especially if the recip-ient does not have a ligand for the donor inhibitoryreceptors. This model is supported by two retro-spective analyses of patients who underwent OLTfor a variety of causes.97 98 In the latter study of 416UK patients, individuals who received donor allo-grafts expressing one group 2 HLA-C allele had lessgraft loss and a 13.6% improvement in survival at10 years compared to those receiving group 1 HLA-C homozygous allografts. The outcome figureswere even better for those receiving a group 2 HLA-C homozygous allograft with 26.5% reduction ingraft loss and lower frequencies of both chronicrejection and recurrent cirrhosis. This is consistentwith the group 2 HLA-C:KIR interaction beingstronger than that of group 1 HLA-C:KIR and thusresulting in greater NK cell inhibition.99 Theauthors suggest that group 2 homozygous allo-grafts may be better allocated to high risk recipients

(eg, second transplants) to maximise the chances oflong-term graft function. Unfortunately, thesefindings were not replicated in a large Europeanstudy.100 Population diversity may play a role inthese differences as individual HLA alleles withinthe group 1 and group 2 HLA-C ‘supertypes’ mayhave different interactions with their KIR ligands.6275

Recurrence of HCV post-transplantation isuniversal and often has an accelerated course withallograft cirrhosis in up to 30% of individuals at5 years post-transplant.101 Predicting which indi-viduals are more likely to develop aggressive HCVrecurrence post-transplant may be a useful toolgiven the shortage of donor organs. In OLT recipi-ents expressing KIR2DL3, the mismatching ofHLA-KIR ligands correlated with the progression toliver fibrosis.102 Therefore KIR2DL3 recipients maybe better suited to HLA-KIR matched allografts.A prospective study looking at phenotypic

changes of peripheral NK cells pre- and post-transplant has also been reported.103 A reduction inperipheral blood NK frequency was noted in thefirst week after transplant returning to pre-transplantlevels after 1 month. This was attributed tohoming of NK cells to the liver, possibly due tograft re-population or de novo graft infection. Aprogressive increase in NKG2C expression was alsonoted post-OLTand appeared to be related to HCVrecurrence. Increased NK cell natural cytotoxicityreceptor expression correlated with ALT levelssupporting the theory that NK cells contribute toliver inflammation. Thus the effects of NK cells inpost-transplant HCV are complex, with weakerinhibitory signals leading to a more pronouncedanti-viral effect and carrying an increased risk ofrejection.

THERAPEUTIC PROSPECTS AND FUTUREDIRECTIONSThere are several potential mechanisms of modi-fying NK cell function to improve their anti-viraland anti-tumour properties. These would includemanipulating NK cell receptor expression (eg,blocking NK inhibitory receptors), cytokinetherapy (eg, IL-2, IL-12, IL-15), and infusion of exvivo expanded autologous NK cell populations. NKcells can be successfully expanded ex vivo to upre-gulate TRAIL, FasL and NKG2D expression witha consequent increase in anti-tumour activity.104 Aphase I clinical trial based on these findings usingadoptive infusion of ex vivo expanded autologousNK cells in patients with metastatic tumours iscurrently in progress. Furthermore, adoptive trans-fer of TRAIL-expressing NK cells may representa potential therapy for reducing recurrence ofHCC following partial hepatectomy and livertransplantation.105 106 Ishiyama et al reportedthat IL-2-stimulated NK cells extracted from donorliver graft perfusate have marked anti-tumouractivity (via increased TRAIL expression) againsta hepatocellular carcinoma cell line.105

While adoptive immunotherapy may have a rolein HCC, targeted NK therapies have not been used

Key points 1: Natural killer cells in hepatitis C virus infection

< NK cells are effector cells of the innate immune system that can directly lyseinfected cells and modulate adaptive immune responses.

< In acute HCV infection NK cells are activated, displaying increasedcytotoxicity and increased IFNg secretion.

< In chronic HCV infection, NK cell frequency is reduced, with changes inphenotype towards NK2 type cytokines (IL-10 and TGFb).

< NK cell abnormalities in chronic HCV infection are distinct from those inchronic HBV infection.

< HCV core encoded peptides may upregulate MHC-class I and inhibit NK cells.< In vitro NK cells are capable of inducing apoptosis of HCV infected cells.




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for the treatment of HCV; although it is notablethat IFNa is a potent NK activating cytokine. Inorder to target NK cells, deeper insights into theirrole in HCV need to be gained. It will be importantto translate genetic insights into functional ones; inparticular, why KIR2DL3 and group 1 HLA-Calleles are protective. The recruitment of NK cellsto the liver and their role in the acute phase ofinfection has received little attention to date due todifficulties in obtaining patient derived material.However these cohorts will be key to under-standing mechanisms by which NK cells areinvolved in the eradication of HCV. The potentialfor NK cells to interface with dendritic cells, T cellsand also IL-28B in a synergistic response to acuteHCV infection requires further exploration.Bringing together genetic and functional observationsto understand mechanisms by which theKIReHLA interactions can alter the cross-talkbetween these cells may be particularly importantto understanding eradication of HCV. Whilst thereis now a substantial body of information on NKcells in chronic HCV infection, relatively littleprogress has been made as to how they may bemanipulated in order to generate a successful anti-viral immune response. Finally, our understandingof NK cell biology continues to evolve and this willcontinue to generate novel ideas related to viraleradication and disease progression.

CONCLUSIONOur emerging understanding of NK cells is givingus new insights into their role in HCV infection.Critically, HCV is the prototypic infection forwhich inhibitory KIR determine outcome againstchronicity. In chronic HCV infection NK cellfunction is attenuated under the influence ofcytokines, and probably inefficient receptor-medi-ated stimulation. A weak NK cell response is onefactor in the failure to generate an adequate adap-tive immune response. Addressing both the innateand adaptive immune systems will hold key to thedevelopment of successful vaccination strategies forHCV.

Funding KC and SIK are supported by The Wellcome Trust

Competing interests None to declare.

Provenance and peer review Commissioned; externally peerreviewed.

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Key points 2: KIR and MHC genetics in hepatitis C virus infection

< NK cells are regulated by polymorphic inhibitory receptors (KIR) withpolymorphic MHC class I ligands.

< These receptors are crucial for both maturation and regulation of NK cellactivity.

< The combination of the inhibitory receptor KIR2DL3 and its group 1 HLA-Cligand is beneficial in spontaneous and treatment-induced resolution of HCVinfection.

< Recipientedonor KIR/HLA interactions may influence the outcome of livertransplantation. Associations have been made with graft and patient survivaland HCV recurrence post-transplant.

< Allelic diversity of KIR and MHC genes may account for differences in findingsfrom genetic association studies.




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Natural killer cells and hepatitis C: action and reactionreceptor CD94:NKG2A, which has the oligomor-phic MHC class I molecule HLA-E as is its ligand. In a genetic study homozygosity