Natural killer cell function is intact after direct exposure to infectious hepatitis C virions

Natural Killer Cell Function is Intact after Direct Exposure toInfectious Hepatitis C Virions

Joo Chun Yoon1, Masaaki Shiina2, Golo Ahlenstiel, and Barbara RehermannImmunology Section, Liver Diseases Branch, National Institute of Diabetes and Digestive andKidney Diseases, National Institutes of Health, Bethesda, MDJoo Chun Yoon: [email protected]; Masaaki Shiina: [email protected]; Golo Ahlenstiel: [email protected]

AbstractWhile hepatitis C virus (HCV) has been shown to readily escape from virus-specific T and B cellresponses, its effects on natural killer (NK) cells are less clear. Based on two previous reports thatrecombinant, truncated HCV E2 protein inhibits NK cell functions via crosslinking of CD81, it isnow widely believed that HCV impairs NK cells as a means to establish persistence. However, therelevance of these findings has not been verified with HCV E2 expressed as part of intact virions.Here we employed a new cell culture system generating infectious HCV particles with genotype1a and 2a structural proteins, and analyzed direct and indirect effects of HCV on human NK cells.Antibody-mediated crosslinking of CD16 stimulated and antibody-mediated crosslinking of CD81inhibited NK cell activation and IFN-γ production. However, infectious HCV itself had no effecteven at titers that far exceeded HCV RNA and protein concentrations in the blood of infectedpatients. Consistent with these results, anti-CD81 but not HCV inhibited NK cell cytotoxicity.These results were independent of the presence or absence of HCV-binding antibodies andindependent of the presence or absence of other peripheral blood mononuclear cell populations.

Conclusion—HCV 1a or 2a envelope proteins do not modulate NK cell function whenexpressed as a part of infectious HCV particles. Without direct inhibition by HCV, NK cells maybecome activated by cytokines in acute HCV infection and contribute to infection outcome anddisease pathogenesis.

Keywordsvirus; innate immune response; CD81; CD16

IntroductionNatural killer (NK) cells are an important component of the innate immune response againstmany viruses due to their ability to lyse virus-infected cells and to secrete cytokines thatinhibit viral replication and activate and recruit cells of the adaptive immune response. Therole of NK cells during the early antiviral immune response has been well described in mice.Murine cytomegalovirus (MCMV) activates myeloid dendritic cells through the engagementof toll-like receptor 9, which results in interferon (IFN)-α and interleukin (IL)-12 productionand activation of NK cells (1). Activated NK cells lyse virus-infected cells and produce IFN-γ (2) and thereby limit MCMV replication in liver and spleen (3). Thus, cytokine production

Contact Information: Address for correspondence: Barbara Rehermann, MD, Immunology Section, Liver Diseases Branch, NIDDK,NIH, 10 Center Dr, Room 9B16, Bethesda, MD 20892. E-mail: [email protected]; Phone: (301) 402-7144; Fax: (301) 402-0491.1current address: Department of Microbiology, Yonsei University College of Medicine, Yonsei University Health System, Seoul,Republic of Korea2current address: Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan

NIH Public AccessAuthor ManuscriptHepatology. Author manuscript; available in PMC 2010 January 1.

Published in final edited form as:Hepatology. 2009 January ; 49(1): 12–21. doi:10.1002/hep.22624.

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and cytotoxicity of NK cells contribute to the control of viral spread prior to thedevelopment of adaptive immune responses.

Viruses, however, have also developed mechanisms to escape from the antiviral response ofNK cells and to establish persistent infection (4). Human cytomegalovirus prevents NK-cellmediated lysis of infected fibroblasts by down-regulating the expression of moleculessuch as MICA (5) and CD155 (6), which are ligands for activating NK cell receptors.Hepatitis C virus (HCV) is characterized by an extraordinary ability to establish viralpersistence, which it achieves by evading intracellular responses of infected hepatocytes (7)and humoral and cellular responses of the adaptive immune system (8). However, HCV isalso induces type I interferons, which are detectable in the liver within the first weeks ofinfection (9) and are known as potent stimulators of NK cells. Activated NK cells have beenshown to recognize and lyse HCV-replicon containing hepatoma cells in vitro in a perforin-and granzyme-dependent manner (10). It has therefore been postulated that HCV may havedevelop strategies to persist in the face of early NK cell responses.

This concept appeared to be validated by two reports that a plate-bound, truncated,recombinant form of the HCV envelope 2 (E2) glycoprotein binds to the tetraspanin CD81expressed by NK cells (11,12). In those studies, plate-bound HCV E2 protein crosslinksCD81 molecules on the surface of NK cells and reduces the capacity of NK cells to respondto Fcγ receptor IIIA (CD16) ligation with IFN-γ secretion and cytotoxicity. Based on thesefindings, it has been stated in several reviews that HCV E2 impairs NK cell function viadirect contact as a means to establish persistence (13–15). The observed delay of adaptiveimmune responses (8) could be a consequence of an inhibitory effect of HCV on NK cellfunction early in the course of infection, because IFN-γ secretion by NK cells promotesdownstream adaptive immune responses (16).

However, it is yet to be established whether those effects observed with high concentration(1 – 10 µg/ml) of recombinant, plate-bound, HCV E2 protein (11,12) can be reproducedwith intact virions. In this scenario, the virus surface itself would provide the platform forrepetitive patterns of E2 proteins and thereby facilitate crosslinking of CD81 molecules.However, it is also possible that the configuration of the HCV E2 protein on the surface ofinfectious virions differs from that of truncated recombinant E2 used in the in vitro assays(11,12) and its concentration in the blood of infected patients is likely much lower.

The recently developed HCV culture system (17–20) enabled us to test the effect ofinfectious HCV particles on NK cells under controlled in vitro conditions. Specifically, weasked whether acute exposure to HCV affected activation, IFN-γ production andcytotoxicity of NK cells from healthy controls, who had never been exposed to HCV. NKcells, either purified or in the context of other cell populations were exposed to cell-cultureproduced hepatitis C virus for up to 18 hours, in the presence or absence of virion-specificantibodies and at HCV RNA and HCV protein concentrations similar to or higher than thoseobserved in the blood during acute HCV infection (21).

Materials and MethodsBlood Samples

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats or bloodsamples of healthy blood donors as described (22) and used fresh unless indicated otherwise.Sera of individuals who had cleared persistent HCV genotype 2 infection were preserved at−80°C. All subjects gave written informed consent for research testing under protocolsapproved by Institutional Review Boards of the National Institutes of Health.

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Monoclonal AntibodiesAnti-CD3-FITC(UCHT1), anti-CD3-PE-Cy5(UCHT1), anti-CD3-Alexa700(UCHT1), anti-CD14-APC(M5E2), anti-CD16-Pacific Blue(3G8), anti-CD19-PE-Cy5 (HIB19), anti-CD25-FITC(M-A251), anti-CD69-PE(FN50), anti-IFN-γ-PE-Cy7(B27) (all from BD Pharmingen,San Jose, CA), anti-CD56-PE(AF12–7H3), anti-CD56-APC(AF12–7H3) (all from MiltenyiBiotec, Berglisch Gladbach, Germany), and anti-CD14-PE-Cy5(TÜK4) (Serotec, Raleigh,NC) were used for flow cytometry. Anti-CD16(3G8) and anti-CD81(JS-81) (all from BDPharmingen) were used for NK cell stimulation and inhibition, respectively.

HCV cultureHCV JFH1 strain (genotype 2a) (18) and the chimeric H-NS2/NS3-J virus, which expressedHCV core to NS2 proteins of genotype 1a (H77) sequence and the remaining nonstructuralproteins of JFH1 sequence (23), were produced as described (24) by transfecting Huh7.5cells (Apath, St. Louis, MO) with linearized RNA from plasmids (provided by Dr. T.Wakita, National Institute of Infectious Diseases, Tokyo, Japan and Dr. S. Lemon,University of Texas Medical Branch, Galveston, TX, respectively). Transfected Huh7.5 cellswere cultured in DMEM with 10% fetal bovine serum (FBS; US Bio-Technologies,Pottstown, PA), 10 mM HEPES, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine (Mediatech, Herndon, VA). Cell culture supernatants at the peak of HCVproduction (typically days 12 through 16 after transfection) were used to infect naïveHuh7.5.1 cells (provided by Dr. F. V. Chisari, Scripps Research Institute, La Jolla, CA) in10 cm2 culture dishes at a multiplicity of infection of 0.006. The infected Huh7.5.1 cellswere transferred into T175 flasks after 1–2 days and passaged every 3.5 days. The viabilityof HCV-infected Huh7.5.1 cells was assessed by trypan blue staining and lactatedehydrogenase release using the CytoTox96 assay (Promega Corp., Madison, WI).

Filtered (0.45 µm) supernatant of HCV-infected Huh7.5.1 cells containing HCV was usedfor NK cell experiments. HCV RNA, infectious titer and HCV core protein concentrationwere determined exactly as previously described (24). To determine the HCV E2concentration cell culture supernatants were incubated with 0.1% NP-40 for 15 minutes at4°C followed by a Galanthus nivalis antigen (GNA) lectin capture EIA (25). Soluble JFH1E2 of known concentration (provided by Dr. J. McKeating, University of Birmingham, UK)was included as calibrant. In experiments reported in figure 1–figure 4 and supplementaryfigure 1 the JFH1 infectious titer was 4 × 103 ffu/ml (5 × 107 RNA copies/ml; 0.03 nmolHCVcore/l), comparable to those in the blood of infected patients (26). Supernatant fromuninfected Huh7.5.1 cells was used as control. To achieve an even higher viral titer (Fig. 5),500 ml supernatant from a separate batch of infected Huh7.5.1 cells was concentrated usinga Pellicon XL filter system (Millipore, Billerica, MA) with a pressure pump. This resulted inan increase of the JFH1 infectious titer to 2.39 × 105 ffu/ml (2 × 108 HCV RNA copies/ml,2.99 nmol HCVcore/l, 12 ng HCV E2/ml). The infectious titer of the chimeric H-NS2/NS3-Jvirus was 2.93 × 105 ffu/ml (2 × 108 HCV RNA copies/ml, 3.4 nmol HCVcore/l, 37 ngHCV E2/ml). Concentrated supernatant from uninfected Huh7.5.1 cells served as control.

Detection of Antibodies against HCV JFH1 Structural ProteinsEIA—96-well EIA plates (Dynex Technologies, Chantilly, VA) were coated with 1 µg/mlGNA lectin (Sigma, St. Louis, MO) overnight at 4°C and incubated with 1.8 × 107 RNAcopies HCV/well for 2h at room temperature. Fifty microliter HCV-RNA-negative serum ofindividuals who had cleared HCV genotype 2 or normal healthy donors were added at a 1:20dilution. After 1h at room temperature, plates were washed three times with PBS and thecaptured antibodies were detected with mouse anti-human IgG-horseradish peroxidase(Serotec) and tetramethylbenzidine super sensitive 1-C (BioFX Laboratories, Owings Mills,

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MD) (25). Optical density was determined at 450 nm with a microplate reader (Bio-Rad,Hercules, CA).

Neutralization Assay—Huh7.5.1 cells were incubated with 5 × 107 RNA copies/ml HCVin the presence of 20% human serum. The percentage of HCV-infected Huh7.5.1 cells wasassessed by flow cytometry using an anti-HCVcore antibody and a secondary, Alexa488-conjugated anti-mouse IgG (Invitrogen). Percent neutralization was calculated as (controlinfectivity – experimental infectivity) / control infectivity × 100.

NK Cell IsolationNK cells were negatively isolated from PBMCs with the NK Cell Isolation Kit andautoMACS separator (Miltenyi Biotec) after lysis of red blood cells with ACK lysing buffer(Biosource, Rockville, MD). The purity of isolated CD3−CD56+ NK cells as assessed withanti-CD3-FITC, anti-CD56-PE, and anti-CD14-APC antibodies by flow cytometry wasgreater than 91% in all experiments.

Stimulation and Inhibition of NK Cells106 cells/ml NK cells were cultured for 18h in RPMI medium containing 100 IU/mlpenicillin, 100 µg/ml streptomycin and 2 mM L-glutamine (Mediatech) and either 10% FBSor 20% human serum (complete medium) in (i) 96-well flatbottom culture plates (CorningInc., Corning, NY) with the indicated concentration of HCV in Huh7.5.1 supernatant or withsupernatant from uninfected Huh7.5.1 cells or in (24) 96-well flat-bottom EIA plates(Immulux HB; Dynex Technologies) that had been coated with 1 µg/ml anti-CD16 and/or10 µg/ml anti-CD81 in carbonate buffer (pH 9.5) overnight at 4°C. Human IL-12 (1.2 ng/ml,PeproTech, Rocky Hill, NJ), which is typically secreted by monocytes and DCs, was addedin all stimulation conditions. As previously described (27,28) and shown in figure 1, thissuboptimal concentration IL-12 alone did not induce significant activation of resting NKcells but primed them to respond to activating stimuli.

To examine possible inhibitory effects of HCV and anti-CD81 on NK cell stimulation, NKcells were incubated with the indicated concentration of HCV or plate-bound anti-CD81 2hbefore, at the same time or 2h after stimulation with plate-bound anti-CD16 or 50 µg/mlpoly(I:C) (InvivoGen, San Diego, CA) and 1.2 ng/ml recombinant human IL-12.

Analysis of NK cell Activation and IFN-γ ProductionAfter 18h culture supernatants were frozen at −20°C and later tested with the Human IFN-γQuantikine EIA Kit (R&D systems, Minneapolis, MN). Cells were washed, stained withanti-CD25-FITC, anti-CD69-PE, anti-CD3-PE-Cy5, and anti-CD56-APC antibodies andanalyzed on a flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA) usingCellQuest v3.3 (BD Biosciences) and FlowJo v6.4.7 software (Tree Star, Ashland, OR).

Analysis of NK Cell CytotoxicityThawed PBMCs were incubated in complete medium at 37°C in a CO2 incubator for 8hprior to NK cell isolation. Two thousand 51Cr-labeled (GE Healthcare, Piscataway, NJ)MHC class I negative K562 cells/well (ATCC, Manassas, VA) were plated in 96-wellround-bottom plates (Nunc, Roskilde, Denmark) and in parallel in 96-well round-bottomEIA plates (Immulux HB; Dynex Technologies) coated or not coated with 10 µg/ml anti-CD81. NK cells were added at effector to target (E:T) ratios of 30:1, 15:1, 7.5:1, and 3.75:1with or without 5 × 107 RNA copies/ml HCV in complete RPMI medium supplementedwith 20% human serum with or without HCV JFH1-neutralizing antibodies. Virus wasincubated with serum for 30 minutes prior to the use in NK cell assays. The amount of 51Cr

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released in the culture supernatant was determined after 3h, 5h and 7h using a scintillationcounter (PerkinElmer, Wellesley, MA). Percent specific cytotoxicity was calculated as(experimental release – spontaneous release) / (maximum release – spontaneous release),and the mean cytotoxicity of at least three cultures per E:T ratio was calculated.Spontaneous release was less than 30% of maximum release in all assays.

Analysis of NK Cells in Stimulated PBMCsPBMCs were stimulated at 2.5 × 106 cells/ml as described for NK cells. Brefeldin A (1 µl/ml, Golgi Plug; BD Biosciences) was added after 1h. PBMCs were stained with 50 µg/mlethidium monoazide (EMA; Invitrogen Molecular Probes), anti-CD3-Alexa700, anti-CD14-PE-Cy5, anti-CD16-Pacific Blue, anti-CD19-PE-Cy5, and anti-CD56-APC antibodies after18h. After fixation and permeabilization with BD Cytofix/Cytoperm solution (BDBiosciences), cells were stained with anti-IFN-γ-PE-Cy7, anti-CD25-FITC, and anti-CD69-PE antibodies and analyzed on a flow cytometer (LSR II; BD Biosciences) using FACSDivav4.1.2 (BD Biosciences) and FlowJo v6.4.7 software. NK cells were identified by gating onsinglets (FSC-A vs. FSC-H plot), lymphocytes (FSC-A vs. SSC-A plot) and finally onCD3−CD56+ NK cells after exclusion of CD14+, CD19+, and EMA+ (dead) cells.

Statistical AnalysisPaired Student t test analysis was performed with GraphPad Prism (San Diego, CA). A two-sided P value of less than 0.05 was considered statistically significant.

ResultsAnalysis of NK Cell Activation after Exposure to HCV

Freshly isolated, peripheral blood NK cells of healthy blood donors responded to stimulationby plate-bound anti-CD16 in the presence of IL-12 with a significant upregulation of theactivation markers CD25 (IL-2 receptor α chain, Fig. 1A,B) and CD69 (Fig. 1A,C) andsecretion of IFN-γ (Fig. 1D). Most responding NK cells were CD56dim (Fig. 2A). Thiscorrelated well with the observation that nearly all CD56dim NK cells are CD16 (Fcγreceptor IIIA)bright (not shown) and that CD56bright NK cells are typically CD16− orCD16dim. Similar results were observed using poly(I:C) for stimulation (not shown). Incontrast, neither HCV, derived from the culture supernatant of HCV JFH1 infected Huh7.5.1cells, nor plate-bound anti-CD81 stimulated CD25 and CD69 expression (Fig. 2B–D) orIFN-γ production (Fig. 2E). These results show that IL-12-primed primary human NK cellscan be activated by anti-CD16, but neither by HCV nor by anti-CD81.

Analysis of NK Cell Inhibition after Exposure to HCVCrosslinking of CD81 by plate-bound anti-CD81 resulted in significant inhibition ofactivation marker expression (Fig. 2A–D) and IFN-γ secretion (Fig. 2E) by NK cells inresponse to anti-CD16. Inhibition of NK cell activation was observed for all NK cells, butstronger for the CD56dim than CD56bright subset (Fig. 2A).

Two previous reports (11,12) described crosslinking of CD81 on NK cells by highconcentration of plate-bound, recombinant HCV E2 protein, which resulted in inhibition ofNK cell responsiveness to anti-CD16 (11). To examine whether HCV E2 also inhibits NKcells when expressed as a part of intact, infectious HCV particles, we stimulated NK cellswith anti-CD16 and IL-12 in the presence or absence of HCV. Infectious HCV JFH1 washarvested as supernatant of JFH1-infected Huh7.5.1 cells and used at 5 × 107 RNA copies/ml, a concentration 10 to 100-fold higher than typically observed in chronic HCV infection(26) and comparable to HCV RNA titers in acute HCV infection (29). However, in contrastto previous reports with high concentrations of plate-bound, recombinant HCV E2 protein

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(11,12), infectious HCV JFH1 did neither inhibit the expression of the activation markersCD25 and CD69 (Fig. 2A–D) nor the production of IFN-γ (Fig. 2E) by anti-CD16-stimulated NK cells. These results held true, whether NK cells were incubated with HCV 2hprior to (Fig. 2C–E), at the same time or 2h after stimulation with anti-CD16 (not shown).Similar results were also obtained using poly(I:C) rather than anti-CD16 for NK cellstimulation (not shown). In contrast, plate-bound anti-CD81 inhibited activation markerexpression and IFN-γ secretion of NK cells over a wide concentration range and as low as40 ng/ml (not shown) and irrespective of whether it was administered 2h prior to, at thesame time or 2h after NK cell activation. These results demonstrate that HCV E2 as a part ofintact, infectious HCV JFH1 virions does not efficiently crosslink CD81 and does not inhibitNK cell function.

Effect of HCV on NK Cell Activation and IFN-γ Production in the Presence of other PBMCSubpopulations

To investigate the possibility that HCV affects NK cells indirectly via interaction with othercell populations, HCV was directly added to PBMCs. As shown in supplementary Fig. 1,HCV did not induce CD69 expression (Suppl. Fig. 1A,B) and IFN-γ production by NK cells(Suppl. Fig. 1A,C). Likewise, HCV did not inhibit CD69 expression and IFN-γ productionof anti-CD16 stimulated NK cells. Thus, infectious HCV JFH1 virions affect NK cellfunction neither directly nor indirectly via interaction with other peripheral blood cells.

Effect of Antibody-Coated HCV on NK Cell Activation and FunctionAnother potential mechanism for HCV to interact with NK cells is via antibodies bound toits surface. In infected patients, antibodies against HCV structural proteins cover HCVvirions and thereby create repetitive patterns of extruding Fc fragments, which may engageand crosslink CD16 (FcγRIIIA) on NK cells. To evaluate whether HCV modulates NK cellfunction via this mechanism, we had to identify antibodies against genotype 2a JFH1structural proteins. For this purpose, we screened sera from 11 subjects who had previouslyrecovered from HCV genotype 2 infection and who tested HCV RNA negative by RT-PCR.As shown in Table 1, sera of subjects 4 and 6 contained antibodies that bound to HCV JFH1in an EIA and neutralized in vitro infection of Huh7.5 cells by 79.4% and 88.9%,respectively.

The NK cell activation (Fig. 1) and inhibition (Fig. 2) experiments were then repeated in thepresence of serum from subject 4 or control serum from a healthy uninfected subject. Asshown in Fig. 3, HCV JFH1 did not stimulate NK cell activation and IFN-γ production anddid not inhibit anti-CD16-mediated activation of NK cells in the presence of antibodies. Inaddition to activation marker expression and IFN-γ secretion, we also investigatedcytotoxicity as a readout of NK cell activation and effector function. As shown in Fig. 4A,HCV JFH1 did not significantly enhance or inhibit NK cell cytotoxicity in the presence ofvirus-specific antibodies. In contrast, crosslinking of CD81 on NK cells by anti-CD81substantially inhibited NK cell cytotoxicity (p<0.05, Fig. 4B).

Effect of high concentrations of HCV that encode either genotype 2a or genotype 1astructural proteins on NK cells

Because a plate-bound, truncated HCV E2 protein of genotype 1a sequence was used toinhibit NK cell activation and function in the two previously published reports (11,12) weasked whether a potential effect of HCV on NK cells could be genotype-specific. For thispurpose we used the recently described chimeric H-NS2/NS3-J virus that expressed the core,E1, E2, p7 and NS2 proteins of the HCV genotype 1a (H77) sequence and the remainingnonstructural proteins of genotype 2a (JFH1) sequence [Fig. 5A, (23)]. The HNS2/NS3-Jvirus replicated well in infected Huh7.5.1 cells, but HCV RNA titers in the cell culture

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supernatant reached maximum titers 3 days later than the JFH virus (Fig. 5B) consistent witha 3.5-day delayed cytopathic effect evidenced by an increase in lactate dehydrogenaserelease (Fig. 5C) and a decrease in the percentage of viable Huh7.5.1 cells (Fig. 5D).

Parallel to testing the effect of H-NS2/NS3-J virus on NK cells, we repeated some of theprevious JFH1 experiments at a very high concentration which typically exceeds that ofchronically HCV-infected patients (26). For this purpose, supernatant of JFH1-infectedHuh7.5.1 cells was concentrated to 2 × 108 RNA copies/ml. H-NS2/NS3-J virus was used atthe same titer and concentrated supernatant from uninfected Huh7.5.1 cells served ascontrol. As shown in Fig. 5E–G, anti-CD16 stimulated NK cell activation and IFN-γproduction. In contrast, neither high titer HCV H-NS2/NS3-J virus nor high titer HCV JFH1virus modulated NK cell activation or effector functions (Fig. 5H–J).

DiscussionIn this study, we examined the hypothesis that NK cell functions are inhibited by short-termexposure to HCV E2. Previous reports used high concentrations of truncated, recombinant,platebound, HCV E2 protein to crosslink CD81 on NK cells (11,12) and reported inhibitionof NK cell activation and IFN-γ production. The recently developed tissue-culture systemfor HCV allowed us to study the effects of HCV E2 as part of a complete, infectious HCVparticle. Consistent with the previous reports (11,12), crosslinking of CD81 with plate-bound monoclonal antibodies inhibited NK cell activation and effector functions in ourexperimental setting. However, HCV virions with either genotype 2a or 1a structuralproteins did not affect NK cell activation or effector functions. These results suggest thatHCV E2 does not efficiently crosslink CD81 on the surface of NK cells when it is displayedas part of infectious virions. Likewise, HCV-antibody complexes did not affect NK cellfunctions via crosslinking of FcγRIIIA (CD16) receptors.

The discrepancies between our findings and the previous reports (11,12) are most likely dueto differences in the configuration of natural E2 on the surface of HCV virions as comparedto that of plate-bound, recombinant E2. In addition, differences in the HCV E2concentration may play a role. Whereas very high concentrations of 1 and 10 µg/ml HCV E2protein were used previously, the E2 concentrations in our study were much lower eventhough RNA titers were comparable to those in the blood of HCV-infected patients (21).

We also considered potential indirect effects of HCV on NK cells that may involve otherPBMC subpopulations, such as dendritic cells and monocytes. Dendritic cell (24) andmonocyte functions (21) have been shown to be affected by HCV and HCV core protein,respectively. To investigate whether HCV affected NK cell function via an immune networkwith other cell populations, HCV was added to the complete PBMC population and NK cellfunction was assessed by multicolor flow cytometry. However, HCV virions did not inhibitNK cells in this setting. Likewise, the presence of HCV-specific antibodies on the surface ofvirions did not modulate NK cell function.

A potential limitation of the current and previous studies on NK cells in HCV infection isthe use of peripheral blood NK cells. NK cells constitute a large population of liver-residentlymphocytes and have never been studied in acute infection. Virions could for examplebecome enriched on liver resident cells such as sinusoidal lining cells which express HCV-binding proteins DC-SIGN and L-SIGN. Also, hepatocytes may upregulate MHC class I inresponse to viral proteins and thereby avoid recognition by NK cells (30). The specificinflammatory micromilieu, and specifically cytokines released by virus-infected hepatocytesmay play an important role in this context. These effects cannot yet be analyzed in vitro,because the HCV-permissible cell lines Huh7.5 and Huh7.5.1 are impaired in their type I

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IFN response (31). It also remains possible that other NK cell functions other thanactivation, IFN-γ production and cytotoxicity are altered.

It is important to note that our results are most relevant for the early acute phase of HCVinfection because we analyzed direct short-term exposure of NK cells to HCV, mostly in theabsence of HCV-specific antibodies. In this context, Khakoo et al reported in animmunogenetic study (32) that specific KIR/HLA compound haplotypes, which have beenshown to be associated with more rapid and more vigorous NK cell activation and effectorfunction (33), are associated with a higher likelihood of HCV clearance and lower likelihoodof chronic hepatitis C. As regards to chronic HCV infection, there are multiple reports thatNK cells are activated and display effector functions (34–37). The questions how the earlyinnate immune response impacts the development of adaptive immunity and to which extentactivated NK cells contribute to the pathogenesis of liver disease in chronic infectiontherefore require further analysis in detailed prospective studies.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

List of Abbreviations

HCV hepatitis C virus

NK cell natural killer cell

DC dendritic cell

PBMC peripheral blood mononuclear cell

IFN interferon

IL interleukin

TLR toll-like receptor

FcR Fc receptor

EIA enzyme immuno-assay

AcknowledgmentsWe thank Drs. Takashi Wakita, Stanley Lemon and MinKyung Yi for providing HCV expression constructs, Dr.Jane McKeating for advice and reagents for the E2 EIA, and Drs. Takanobu Kato, Sukanya Raghuraman and Eui-Cheol Shin for discussion.

Financial Support

This study was supported by the NIDDK, NIH intramural research program. GA was supported by grantAH173/1-1 from Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.

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19. Yi M, Villanueva RA, Thomas DL, Wakita T, Lemon SM. Production of infectious genotype 1ahepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci U S A.2006; 103:2310–2315. [PubMed: 16461899]

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21. Dolganiuc A, Chang S, Kodys K, Mandrekar P, Bakis G, Cormier M, Szabo G. Hepatitis C virus(HCV) core protein-induced, monocyte-mediated mechanisms of reduced IFN-alpha andplasmacytoid dendritic cell loss in chronic HCV infection. J Immunol. 2006; 177:6758–6768.[PubMed: 17082589]

22. Takaki A, Wiese M, Maertens G, Depla E, Seifert U, Liebetrau A, Miller JL, et al. Cellularimmune responses persist and humoral responses decrease two decades after recovery from asingle-source outbreak of hepatitis C. Nat Med. 2000; 6:578–582. [PubMed: 10802716]

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23. Yi M, Ma Y, Yates J, Lemon SM. Compensatory mutations in E1, p7, NS2, and NS3 enhanceyields of cell culture-infectious intergenotypic chimeric hepatitis C virus. J Virol. 2007; 81:629–638. [PubMed: 17079282]

24. Shiina M, Rehermann B. Cell culture-produced hepatitis C virus impairs plasmacytoid dendriticcell function. Hepatology. 2008; 47:385–395. [PubMed: 18064579]

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26. Bouvier-Alias M, Patel K, Dahari H, Beaucourt S, Larderie P, Blatt L, Hezode C, et al. Clinicalutility of total HCV core antigen quantification: a new indirect marker of HCV replication.Hepatology. 2002; 36:211–218. [PubMed: 12085367]

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28. Sivori S, Falco M, Della Chiesa M, Carlomagno S, Vitale M, Moretta L, Moretta A. CpG anddouble-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine releaseand cytotoxicity against tumors and dendritic cells. Proc Natl Acad Sci U S A. 2004; 101:10116–10121. [PubMed: 15218108]

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37. Kawarabayashi N, Seki S, Hatsuse K, Ohkawa T, Koike Y, Aihara T, Habu Y, et al. Decrease ofCD56(+)T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in theirsusceptibility to hepatocellular carcinoma. Hepatology. 2000; 32:962–969. [PubMed: 11050046]

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Fig. 1. Anti-CD16 stimulates NK cell activation and IFN-γ production, but HCV does not(A) Contour plots show the percentage of activated (CD25+ and/or CD69+) NK cells afterexposure to 5 × 107 RNA copies/ml HCV (JFH1 strain) for a representative subject.Numbers indicate the percentage of events in each quadrant.(B-D) Percentage of CD25+ (B) or CD69+ (C) NK cells or amount of secreted IFN-γ (D) forall subjects. 1.2 ng/ml human IL-12, which is typically secreted by monocytes and DCs, wasadded in all conditions (A-D) as previously described (27,28).

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Fig. 2. Crosslinking of CD81 by anti-CD81 antibodies inhibits anti-CD16-mediated NK cellactivation and IFN-γ production, but HCV does not(A) Histograms show the effect of anti-CD16 alone (grey line), anti-CD16 and anti-CD81(black line), and anti-CD16 and HCV (dotted line, overlapping with grey line) on theexpression of CD25 by CD56bright and CD56dim NK cell subsets. NK cells were exposed toplate-bound anti-CD81 and to 5 × 107 RNA copies/ml HCV (JFH1 strain) 2h prior tostimulation with plate-bound anti-CD16. The grey area represents unstimulated NK cells.(B) Contour plots show the percentage of activated (CD25+ and/or CD69+) NK cells for arepresentative subject. Stimulation conditions are indicated above the graphs and numbersindicate the percentage of events in each quadrant.(C-E) Percentage of CD25+ (C) or CD69+ (D) NK cells (after gating on all CD3-CD56+cells) or amount of secreted IFN-γ (E) for all subjects.1.2 ng/ml human IL-12, which is typically secreted by monocytes and DCs, was added in allconditions (A–E) as previously described (27,28). The results were obtained in the sameexperiment as those in figure 1 and negative (no stimulation) and positive (anti-CD16)controls are therefore identical.

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Fig. 3. Antibody-coated HCV does not activate or inhibit NK cells5 × 107 RNA copies/ml HCV (JFH1 strain) in the presence of neutralizing, HCV-specificantibodies did not stimulate CD25 expression (A), CD69 expression (B) and IFN-γ-production (C) by NK cells or inhibit anti-CD16 stimulated CD25 expression (A), CD69expression (B) and IFN-γ-production (C). HCV was pre-incubated with HCV JFH1-neutralizing antibody-containing serum for 30 minutes prior to the use in these assays. 1.2ng/ml human IL-12, which is typically secreted by monocytes and DCs, was added in allconditions (A–C) as previously described (27,28).

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Fig. 4. HCV does not inhibit NK cell cytotoxicity but crosslinking of CD81 by antibodies does(A) NK cell-mediated killing of K562 cells was evaluated in the presence (open squares) orabsence (filled circles) of 5 × 107 RNA copies/ml HCV (JFH1 strain) in 3h, 5h, and 7hassays. Mean and standard error of mean of the results of 4 subjects are indicated.(B) NK cell-mediated killing of K562 cells was evaluated in the presence (open squares) orabsence (filled circles) of plate-bound anti-CD81. Mean and standard error of mean of theresults of 3 subjects are indicated.

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Fig. 5. Fig. 5: High concentrations of HCV encoding either genotype 2a or genotype 1a structuralproteins does neither activate nor inhibit NK cells(A) The genome of HCV H-NS2/NS3-J chimeric virus as published by Yi et al. (23)compared to the genome of the HCV JFH1 strain. Proteins encoded by JFH1 are shaded. H-NS2/NS3-J chimeric virus encodes core to NS2 of the HCV H77 (genotype 1a) sequenceand NS3-NS5 proteins of the HCV JFH1 (genotype 2a) sequence.(B-D) HCV RNA titer in the supernatant of Huh7.5.1 cultures infected with either JFH1 orH-NS2/NS3-J chimeric virus (B). Both viruses exert cytopathic effects as evidence by anincreased release of lactate dehydrogenase (LDH) (C) and a decrease in the percentage ofviable, trypan blue positive cells in the culture (D). The results for JFH1 have previously

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been published (24) and are shown for reference purposes to allow a comparison to theinfection kinetics with H-NS2/NS3-J chimeric virus.(E-G) Crosslinking of CD16 by antibodies stimulated NK cell activation [CD25 expression(E) and CD69 expression (F)] and IFN-γ production (G). In contrast, neither antiCD81antibodies nor high titer HCV JFH1 with genotype 2a proteins nor high titer HCV H-NS2/NS3-J with genotype 1a structural proteins stimulated NK cell activation [CD25 expression(E) and CD69 expression (F)] and IFN-γ production (G). Both viruses were concentratedfrom the supernatant of infected Huh7.5.1 cultures and concentrated supernatant fromuninfected Huh7.5.1 cells was used for the unstimulated control and the stimulation withanti-CD16 and anti-CD81.(H–J) High titer HCV JFH1 with genotype 2a structural proteins or high titer HCV H-NS2/NS3-J with genotype 1a structural proteins did not inhibit anti-CD16-mediated NK cellactivation [CD25 expression (H) and CD69 expression (I)] or IFN-γ production (J). 1.2 ng/ml human IL-12, which is typically secreted by monocytes and DCs, was added in allconditions (B-J) as previously described (27,28).

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Table 1

Selection of Human HCV-RNA-Negative Sera with HCV-JFH1-Specific Antibodies

Subject HCV Genotype Years after Cessation of Therapy Binding (OD) Neutralization of Viral Infectivity (%)

1 2 6 0.256 n.t.b

2 2a 6 0.250 n.t.

3 2 3 0.196 n.t.

4 2a 8 0.557a 79.4

5 2 11 0.240 n.t.

6 2b 2 0.844a 88.9

7 2a 2 0.307 n.t.

8 2b 1 0.337 n.t.

9 2b 2 0.283 n.t.

10 2b 1 0.221 n.t.

11 2b 1 0.262 n.t.

aabove cut-off (1.5 × mean OD of two independent negative controls = 0.489)

bn.t., not tested


Natural killer cell function is intact after direct exposure to infectious hepatitis C virions

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