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Journal of Virology, February 2004, p. 2152-2157, Vol. 78, No. 4
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.4.2152-2157.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cytotoxic T Lymphocytes Derived from Patients with Chronic Hepatitis C Virus Infection Kill Bystander Cells via Fas-FasL Interaction

Christel Gremion,1,{dagger} Benno Grabscheid,1,{dagger} Benno Wölk,2 Darius Moradpour,2 Jürg Reichen,3 Werner Pichler,1 and Andreas Cerny4*

Clinic for Rheumatology and Clinical Immunology/Allergology,1 Institute of Clinical Pharmacology, University of Bern, CH-3010 Bern,3 Department of Medicine, Ospedale Regionale di Lugano, CH-6903 Lugano, Switzerland,4 Department of Medicine II, University of Freiburg, D-79106 Freiburg, Germany2

Received 24 June 2003/ Accepted 27 October 2003


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ABSTRACT
 
The role of Fas-mediated lysis of hepatocytes in hepatitis C virus (HCV)-induced injury is frequently discussed. We therefore analyzed the effect of the number of HCV antigen-expressing cells, the mode of antigen presentation, and the number of cytotoxic T lymphocytes in a coculture system mimicking cellular components of the liver. Here, we show that endogenously processed HCV proteins are capable of inducing bystander killing. We further demonstrate that 0.8 to 1.5% of cells presenting HCV antigens suffice to induce lysis of 10 to 29% of bystander cells, suggesting that the mechanism may be operative at low fractions of infected versus uninfected hepatocytes in vivo. Our data underscore the role of the Fas pathway in HCV-related liver injury and support the exploration of Fas-based treatment strategies for patients with chronic hepatitis C virus infection.


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INTRODUCTION
 
The mechanisms of hepatic injury in acute and chronic hepatitis C virus (HCV) infection are not well understood (4). In acute infection, liver cell damage coincides with the development of the host's immune response and not with virus infection and replication, suggesting that HCV is a noncytopathic virus. This observation supports a major role of the cytotoxic T lymphocytes (CTL) in mediating liver cell damage in chronic hepatitis C virus infection (4, 31). Perforin-granzyme-mediated cytotoxicity and Fas-mediated cytotoxicity have been shown to be the major mechanisms of T-cell-mediated lysis (1, 3, 13, 14, 17), and both mechanisms, direct killing of infected cells and Fas-mediated killing (apoptosis) were demonstrated to be important in hepatic injury (1, 9). Fas (CD95) is a membrane protein member of the tumor necrosis family (26) which mediates apoptosis when bound by Fas ligand (FasL/CD95L). It is expressed predominantly on activated CD8+ and Th1 helper T cells (17). Fas is expressed on a variety of cells, but immunohistochemical analyses of normal liver sections detected no Fas or only low levels of it. In contrast, an up-regulation of Fas expression in chronically infected patients, especially near infiltrating lymphocytes, was described (7, 12).

Destruction of cells not directly targeted by effector cells has been termed (innocent) bystander killing. The role of bystander killing is controversial, especially to explain liver cell damage. HCV probably infects only a small fraction of hepatocytes in vivo: 1 to 10% of hepatocytes express core, envelope, or NS3 by immunohistochemical analyses (11), and 5% harbor HCV RNA in their cytoplasm (16). Some studies have shown that no or only a few (1 to 2.3%) liver-infiltrating CD8+ T cells respond to HCV epitopes, suggesting that the presence of HCV-specific CTL is sufficient to cause liver injury, a phenomenon probably increased by bystander killing, but not sufficient to clear the virus (10, 28).

In the present study, we investigated the impact of the number of CTL (reflecting antiviral activity), the type of antigen-presenting cells (APC) (lymphohematopoietic versus nonlymphohematopoietic) as well as their number (reflecting the fraction of hepatocytes presenting HCV antigens in a naturally infected liver), and the mode of antigen presentation (external peptide loading versus endogenous processing).


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Specific cytotoxic T cells recognize exogenously and endogenously synthesized proteins.
 
We first investigated whether CTL isolated from patients with chronic hepatitis C virus infection (blood was obtained after informed consent from the patients; the isolation method was described by Kammer et al. [15]), are able to recognize and kill via the perforin-granzyme pathway NS3 peptide-pulsed APC. Synthetic peptides of NS3 (amino acids [aa] 1073 to 1081) and core protein (aa 131 to 140) were obtained from Chiron Mimotopes (Clayton, Australia). For this investigation, a 4-h 51Cr release assay was used as previously described (15, 18). Percentage of cytotoxicity was calculated as follows: 100 x [(experimental release - spontaneous release)/(maximum release - spontaneous release)]. Maximum release was determined by lysis of the target with HCl (1 M). Spontaneous release was always <25% of maximal release in all assays. The results shown in Fig. 1A indicate that the CTL isolated efficiently lyse JY cells incubated with 10 µg of NS3 peptide/ml or with the core peptide (data not shown). All the data were confirmed with CTL lines isolated from other chronic HCV patients.



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  		FIG. 1. Comparison of peptide-specific perforin-granzyme-mediated killing with Fas-mediated bystander killing using exogenously and endogenously processed HCV antigens. (A to C) The CTL line isolated from patient P04 was tested for its ability to lyse via perforin-granzyme secretion peptide-pulsed JY cells (APC+p) (2,500 JY cells/well) (A), JY cells infected with recombinant vaccinia virus SC59NNRd (wild-type WR used as a control) (B), and transfected U-2 OS cells UNS3-4A-24 and UNS3-4A/A2-19 in the presence (+Tet) or absence (-Tet) of tetracycline. Data shown in panels A and B were confirmed with core peptide aa 131 to 140 and with CTL lines isolated from other chronic HCV patients. (D to G) The CTL line isolated from donor P04 is able to induce Fas-mediated killing of L1210/Fas cells. (D) NS3-pulsed JY cells (2,500/well) were used as APC. (E) JY cells infected with recombinant vaccinia virus SC59NNRd (wild-type WR used as a control). (F) transfected U-2 OS cells UNS3-4A-24 and UNS3-4A/A2-5 in the presence or absence of tetracycline. These results were confirmed with CTL lines isolated from other chronic HCV patients (data not shown). (G) Fas-mediated bystander killing also occurs with a CTL line isolated from donor S99 specific for core peptide aa 131 to 140. Core-pulsed JY cells (1,250/well; & JY+p) were used as APC. JY cells alone were used as the control (& JY w/o p; 2,500 L1210/Fas cells/well; bystander cells).

Keeping in mind the in vivo situation where infected hepatocytes endogenously produce HCV proteins and present processed peptides associated with major histocompatibility complex class I (MHC-I) molecules, we examined if the CTL lines are also able to lyse cells presenting endogenously synthesized peptides. For this, we used JY cells infected with vaccinia virus SC59NNRd, generated as previously described (15), expressing aa 364 to 1619 of HCV and, as a control, JY cells infected with the wild-type WR vaccinia virus (the viruses were kindly provided by Michael Houghton, Chiron Corp., Emeryville, Calif.). Vaccinia virus infection of target cells was performed as previously described (5, 15). Briefly, vaccinia virus-infected targets were prepared by infection of 106 cells at a multiplicity of infection of 10:1 to 100:1 on a rocking platform at room temperature for 1 h, followed by a single wash and overnight incubation at 37°C. Under these experimental conditions, target cells were efficiently lysed by the CTL (Fig. 1B). To refine our model, we generated a new type of APC based on cells recently described, the U-2 OS human osteosarcoma-derived, HLA-A2 positive cell line (UNS3-4A-24) (30). These UNS3-4A-24 cells allow tetracycline-regulated expression of the HCV NS3-4A complex. These cells were stably transfected with a cytomegalovirus promoter-driven genomic HLA-A2 construct to augment MHC-I expression. In brief, UNS3-4A-24 cells were cotransfected with pCMV/HLA-A2 and pTK-Hyg (Clontech, Palo Alto, Calif.), followed by selection with 500 µg of G418/ml (for the selection of the tetracycline-regulated transactivator [tTA]), 1 µg of puromycin/ml (for the selection of the NS3-4A expression construct driven by a tTA-dependent promoter), and 100 µg of hygromycin/ml (for the selection of the HLA-A2 expression construct). Transfectants were cloned and screened for high-level HLA-A2 expression by flow cytometry using fluorescein-5-isothiocyanate (FITC)-labeled monoclonal antibody (MAb) BB7.2 (produced by hybridoma BB7.2 [American Type Culture Collection, Manassas, Va.]; ATCC HB-82). Purified MAb BB7.2 was labeled with FITC (Molecular Probes, Eugene, Oreg.) as specified by the manufacturer. Tightly regulated expression of the NS3-4A complex was confirmed by Western blot analyses using MAb 1B6 against HCV NS3 as previously described (30). Several independent clones which showed a high level of HLA-A2 molecules and tightly regulated expression of the HCV NS3-4A complex were obtained. Clones UNS3-4A/A2-5 and -19 were used in this study (Fig. 2). These cells were maintained in continuous culture for more than 12 months with stable characteristics. Clones UNS3-4A/A2-5 and -19 were used as target cells in 51Cr release assays. For this purpose, 5 x 105 cells were cultured in Easy Grip petri dishes (60 by 15 mm; Falcon 1016) in the presence or absence of tetracycline (1 µg/ml) for 3 days at 37°C to not induce or induce, respectively, the production of the NS3-4A complex. On day 3, cells were collected and used as target cells. Figure 1C shows the results obtained for the clone UNS3-4A/A2-19 and clearly demonstrates that lysis was induced only when NS3-4A was expressed by the withdrawal of tetracycline. Similar results were obtained with clone UNS3-4A/A2-5 (data not shown). As expected lysis of inducible cell lines constitutively expressing high levels of HLA-A2 was more efficient than that of the parental U-2 OS-derived inducible cell line.



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  		FIG. 2. UNS3-4A/A2 cells express high levels of HLA-A2 and inducibly express HCV NS3-4A. (A) Flow-cytometric analyses of HLA-A2 expression of UNS3-4A-24 and two representative UNS3-4A/A2 cell clones (solid line, UNS3-4A/A2-5; checkered line, UNS3-4A/A2-19). FITC-conjugated MAb BB7.2, specific for HLA-A2, was used for staining. In the histograms fluorescence intensities are shown. (B) Western blot analysis of inducible NS3 expression. UNS3-4A-24, UNS3-4A/A2-5, and UNS3-4A/A2-19 cells were cultured for 24 h in the presence (+) or absence (-) of tetracycline (tet) and subsequently processed for immunoblotting using MAb 1B6, specific for NS3. Arrow, position of NS3. Molecular mass markers are on the left.


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Bystander cytotoxic activity is dependent on specific antigen recognition and is predominantly Fas ligand mediated.
 
CTL have been reported to kill mainly antigen-bearing cells through perforin-granzyme secretion (14) but also to kill bystander cells via the production and expression of FasL on the cell surface (1). We therefore decided to determine if the CTL lines that we isolated were capable of mediating damage to noninfected bystander cells. As both human and mouse recombinant FasL can cross-interact and induce apoptosis of the cells expressing either human Fas or mouse Fas (27), 51Cr-labeled Fas-transfected L1210 mouse cells (L1210/Fas), expressing the mouse Fas receptor, were used as bystander target cells (21, 29) (fluorescence-activated cell sorter analysis showed that 84.6% of cells expressed Fas receptor [data not shown]) and peptide-pulsed JY cells (Epstein-Barr virus-transformed B-cell line) were used as the APC (all the concentrations are shown in the legends of the figures). Pulsed JY cells were incubated with peptide-specific CTL and 51Cr-labeled L1210/Fas cells for 18 h at 37°C. Figure 1D demonstrates that L1210/Fas cells are efficiently lysed after this period of incubation. Moreover, a very low number of specific CTL are able to kill significant numbers of both APC and noninfected bystander cells. The same experiment performed after 8, 11, 14, 19, and 24 h of incubation indicates that lysis due to FasL expression does not occur at a significant level before 8 h of incubation (data not shown). Therefore the killing observed in Fig. 1A to C is most likely perforin-granzyme mediated and not Fas or FasL dependent. The same results were obtained with a CTL line specific for HCV core aa 131 to 140 and JY cells pulsed with the same peptide, indicating that the effect is not confined to the NS3 epitope (Fig. 1G). Similar results were obtained with HepG2 target cells (ATCC HB-8065), which showed 25 and 10% specific lysis at 10:1 and 3:1 effector-to-target cell (E:T) ratios, respectively, in a typical experiment using effector cells derived from patient P04. Interestingly the supernatant of the coculture JY cells and specific CTL is not able to induce the lysis of the bystander cells; from this we conclude that the killing observed is due to the interaction of membrane-bound FasL and not the soluble form of this ligand (data not shown) (19, 22, 23).


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Endogenously produced HCV proteins induce bystander killing.
 
Keeping in mind the in vivo situation, we decided to test if bystander killing also occurs when HCV proteins are endogenously produced. We investigated two different modalities of endogenous antigen production: recombinant vaccinia virus-driven HCV protein expression in Epstein-Barr virus-transformed B-cell lines and in UNS3-4A/A2 cells which express HCV proteins in a tetracycline-regulated fashion. First, assays were performed using JY cells infected with recombinant vaccinia virus SC59NNRd as the target cells; JY cells were infected by coincubation with recombinant vaccinia virus for 5 h. The APC were then collected and used in a cytotoxic assay with specific CTL and 51Cr-labeled L1210/Fas cells for 18 h at 37°C. Wild-type WR vaccinia viruses were used as a negative control. As shown in Fig. 1E, endogenously produced HCV proteins also provoke killing of the Fas-bearing cells at low numbers of viral-antigen-expressing cells. The same results were obtained with UNS3-4A/A2-5 (Fig. 1F) and UNS3-4A/A2-19 (data not shown). This demonstrates that cells endogenously producing and presenting HCV peptides, as the infected hepatocytes do, are also able to activate CTL to lyse Fas-expressing bystander cells. In the absence of HCV expression, there was no bystander killing, suggesting the need for T-cell receptor-dependent T-cell activation.


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Bystander cytotoxic activity is Fas and FasL dependent.
 
To examine whether bystander cytotoxic activity was due to Fas-FasL-induced killing, we compared the killing of the L1210/Fas cells with that of Fas-negative L1210 cells. The results presented in Fig. 3A clearly show that a killing is observed only when Fas is expressed on the surface of the bystander target cells, demonstrating that the killing is Fas-FasL mediated. This finding was substantiated by a second series of experiments in which anti-FasL antibodies (NOK-1; BD PharMingen, Basel, Switzerland) were added at various concentrations. We found that inhibition was antibody dose dependant and that inhibition was almost complete at 40 µg of antibody/ml (Fig. 3B). An unrelated immunoglobulin G1 (IgG1) isotype antibody (107.3; BD PharMingen) was used as a control. To confirm FasL expression, we performed a flow cytometry analysis of the CTL line P04 after 18 h of incubation with NS3-pulsed APC or APC alone. As a positive control cells were stimulated with phorbol 12-myristate acetate (50 ng/ml) and ionomycin (1 µM). Using a mouse anti-FasL antibody (NOK-1; BD PharMingen) revealed by a fluorochrome-labeled polyclonal goat anti-mouse antibody (catalog no. R0480; Dako Diagnostics AG, Zug, Switzerland), we were able to demonstrate that 20% of CTL were FasL positive (fixed E:T ratio of 30:1) as shown in Fig. 3C. Samples were analyzed on an EPICS XL-MCL flow cytometer (Beckman Coulter Inc., Hialeah, Fla.). These results also confirm a T-cell receptor-dependant T-cell activation, as APC alone are not able to induce FasL expression on the CD8+ T cells. A low number of peptide-bearing cells are sufficient to initiate an important bystander cytotoxic activity. To mimic the presumed low in vivo ratio of infected to uninfected hepatocytes (20), we used graded numbers of peptide-loaded cold target cells (representing the infected hepatocytes) with a constant number of 51Cr-labeled Fas-transfected L1210 cells (representing the bystander uninfected hepatocytes) and a constant number of CTL. As shown in Fig. 4, the NS3-specific CTL line P04 is able to mediate significant FasL-mediated killing even at the lowest concentration of peptide-loaded cold target cells. Thus, 0.8 to 1.5% of cells presenting the NS3 peptide suffice to induce the lysis of 10 to 29% of noninfected cells.



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  		FIG.3. Bystander killing is dependent on Fas expression on the bystander cells and FasL expression on the effector cells. (A) Bystander cytotoxic assay compared Fas-negative L1210 cells with the same cells expressing Fas (L1210/Fas) (2,500 cells/well). A CTL line from patient P52 was used as effector cells, and 2,500 JY cells/well were used as APC; these cells were pulsed with NS3 peptide aa 1073 to 1081 (& JY+NS3) or were used without the peptide (& JY w/o NS3). (B) The anti-FasL antibody was added to a bystander cytotoxic assay to block Fas-FasL-mediated lysis. CTL clones isolated from patient P04 were used as effector cells (fixed effector/APC ratio of 3:1), and L1210/Fas cells (2,500/well) were used as bystander cells (APC: 2,500 JY cells/well; pulsed with NS3 peptide). A mouse IgG1 antibody was used as an isotype control. (C) FasL expression on the surface of CTL line P04 was assessed by flow cytometry analysis. Cells were incubated with phorbol 12-myristate acetate (PMA) and ionomycin (top row), with 2,500 JY cells (APCs) pulsed with NS3 peptide aa 1073 to 1081 (middle row), or with 2,500 JY cells alone (bottom row) (E:T of 30:1). FasL expression was detected with an anti-FasL antibody revealed by a fluorochrome-labeled goat anti-mouse antibody (2nd Antibody-PE). PE, phycoerythrin. As control, a mouse IgG1 isotype and the fluorochrome-labeled goat anti-mouse antibody alone were used. Numbers indicate percentages of cells in quadrants.



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  		FIG. 4. A low number of peptide-bearing cells are sufficient to initiate bystander cytotoxicity. In this experiment a constant number of CTL and a constant number of L1210/Fas cells were mixed together with a titrated number of APC. A CTL line from patient P04 specific for the NS3 peptide was used as effector cells (5 x 104/well) with various concentrations of APC (JY cells) pulsed with the peptide (& JY+NS3); JY cells alone were used as the control (& JY w/o NS3). L1210/Fas cells were used as bystander cells (2,500/well).

What is the evidence to support the importance of the Fas-FasL system in chronic HCV infection? As described previously, perforin-granzyme-mediated lysis and Fas-FasL-mediated lysis, the two major mechanisms of T-lymphocyte cytotoxicity, are involved in liver destruction (1, 13, 14). A first piece of evidence is the finding of an up-regulation of Fas expression on chronically HCV-infected hepatocytes (3, 7, 9, 12, 17) in the vicinity of CTL, suggesting that Fas-mediated apoptosis may play an important role in liver cell injury. Second, hepatocyte destruction starts when CTL activity initiates but not with viral replication, suggesting that the immune process is probably responsible for cell death and not the virus. Third, a recent study (2) describes the correlation between the degree of inflammation of the liver and the number of apoptotic cells, as determined by detection of activated caspases, including caspase 3, a hallmark of Fas-FasL activation.

The efficient lysis of bystander cells by CD8+ T cells is in accord with studies of other systems, for example, studies of papillomavirus (CD8+ T cells) and dengue virus (CD4+ T cells), as well as of autoimmunity (8, 24, 25). Ando et al. (1) reported that Fas-FasL- and tumor necrosis factor alpha-induced mechanisms are responsible for bystander killing by HCV-specific human CTL. In the present study, we take the argument further using newly developed tools to demonstrate that CTL lines isolated from various patients with chronic HCV infection recognize and kill HCV protein-expressing cells probably via perforin-granzyme secretion. Bystander killing was found with both NS3 and core peptides exogenously loaded or endogenously produced. Trying to mimic the in vivo situation, we performed a CTL assay with a titrated number of HCV peptide-bearing cells. By this means, we were able to determine that a minimum of 0.8 to 1.5% of cells presenting HCV antigens suffice to induce the lysis of 10 to 29% of the bystander cells. It was found that a median of 5% of the hepatocytes present HCV RNA in the cytoplasm (16), and, according to Bantel et al. (2), 7 to 20% of hepatocytes show caspase 3 activation typical of that for apoptotic cells. Although we present here in vitro results, it is remarkable that this proportion of infected versus apoptotic hepatocytes is very similar to the results we obtained.

A significant fraction of intrahepatic lymphocytes, which presumably include non-HCV-specific CD8+ T cells, are CD69 positive. These cells could contribute to bystander killing and are not present in our model. However, the importance of this phenomenon is moderated by the fact that the liver is probably also a site of apoptosis for activated T cells, which are trapped in this organ and undergo cell death (6).

While the coculture system has its limitations in representing the complex three-dimensional architecture of the liver by a mixture of cell types in suspension, it underscores the role of the Fas pathway in HCV-related liver injury. Our data clearly show that the Fas-FasL mechanism induced by CTL from chronic HCV patients may be responsible for a high level of lysis of bystander cells, supporting recent data demonstrating extensive caspase 3 activation during apoptotic liver cell damage in chronic HCV infection and encouraging the exploration of Fas-based treatment strategies for patients with chronic HCV infection.


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ACKNOWLEDGMENTS
 
This work was supported by the Swiss National Science Foundation, Switzerland (SNF Proposal 32-059564.99), the Department of Clinical Research of the University of Bern, Switzerland, the Deutsche Forschungsgemeinschaft, Bonn, Germany (Mo 799/1-2 and Mo 799/1-3), the Bundesministerium für Bildung und Forschung, Bonn, Germany (01 KI 9951), and the European Commission, Brussels, Belgium (QLK2-CT1999-00356,QLK"-CT-2002-01329, BBW no. 99.0363, and BBW no. 99.0365).

We thank Michael Houghton (Chiron Corporation) for providing us with the recombinant HCV-vaccinia virus constructs used in this study and Thomas Brunner for helpful comments on this work.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Medicine, Ospedale Regionale di Lugano, CH-6903 Lugano, Switzerland. Phone: 41 91 811 60 46. Fax: 41 91 811 60 45. E-mail: andreas.cerny{at}bluewin.ch. Back

{dagger} C.G. and B.G. contributed equally to this work. Back


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Journal of Virology, February 2004, p. 2152-2157, Vol. 78, No. 4
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.4.2152-2157.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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