Stable human lymphoblastoid cell lines constitutively expressing hepatitis C virus proteins

Benno Wölk1,{dagger},{ddagger}, Christel Gremion2,{dagger}, Natalia Ivashkina1,§, Olivier B. Engler2, Benno Grabscheid2, Elke Bieck1,||, Hubert E. Blum1, Andreas Cerny3 and Darius Moradpour1,||

1 Department of Medicine II, University of Freiburg, Hugstetter Str. 55, D-79106 Freiburg, Germany
2 Clinic for Rheumatology and Clinical Immunology/Allergology, Inselspital, University of Bern, CH-3010 Bern, Switzerland
3 Department of Medicine, Ospedale Regionale di Lugano, Via Tesserete 46, CH-6903 Lugano, Switzerland

Correspondence
Darius Moradpour
Darius.Moradpour{at}hospvd.ch


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The cellular immune response plays a central role in virus clearance and pathogenesis of liver disease in hepatitis C. The study of hepatitis C virus (HCV)-specific immune responses is limited by currently available cell-culture systems. Here, the establishment and characterization of stable human HLA-A2-positive B-lymphoblastoidxT hybrid cell lines constitutively expressing either the NS3–4A complex or the entire HCV polyprotein are reported. These cell lines, termed T1/NS3-4A and T1/HCVcon, respectively, were maintained in continuous culture for more than 1 year with stable characteristics. HCV structural and non-structural proteins were processed accurately, indicating that the cellular and viral proteolytic machineries are functional in these cell lines. Viral proteins were found in the cytoplasm in dot-like structures when expressed in the context of the HCV polyprotein or in a perinuclear fringe when the NS3–4A complex was expressed alone. T1/NS3-4A and T1/HCVcon cells were lysed efficiently by HCV-specific cytotoxic T lymphocytes from patients with hepatitis C and from human HLA-A2.1 transgenic mice immunized with a liposomal HCV vaccine, indicating that viral proteins are processed endogenously and presented efficiently via the major histocompatibility complex class I pathway. In conclusion, these cell lines represent a unique tool to study the cellular immune response, as well as to evaluate novel vaccine and immunotherapeutic strategies against HCV.

{dagger}These authors contributed equally to this work.

{ddagger}Present address: Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10021, USA.

§Present address: Department of Immunology, University of Göttingen, D-37075 Göttingen, Germany.

||Present address: Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 44, CH-1011 Lausanne, Switzerland.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chronic infection with hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide (National Institutes of Health, 2002). Therapeutic options are limited and a vaccine does not exist. In recent years, it has become clear that the vigour and breadth of early CD4+ and CD8+ T-cell responses are crucial for recovery from acute HCV infection (Cerny et al., 1995; Diepolder et al., 1995; Missale et al., 1996; Cooper et al., 1999; Lechner et al., 2000; Thimme et al., 2002; reviewed by Shoukry et al., 2004). Patients who have cleared the virus after acute infection sustain an HCV-specific T-cell response, even after a period of 20 years, whereas circulating HCV-specific antibodies often become undetectable with time (Takaki et al., 2000). However, the mechanisms that lead to virus clearance or allow HCV to establish and maintain a persistent infection are not fully understood (Cerny & Chisari, 1999).

The study of HCV-specific cellular immune responses has been limited by the lack of a robust cell-culture system. Analyses of the HCV life cycle are based on model systems, including heterologous expression or the replicon system (Pietschmann & Bartenschlager, 2003). These systems, however, are not optimal for the study of HCV-specific immune responses, as they are not based on professional antigen-presenting cell (APC) lines, which characteristically have high major histocompatibility complex (MHC) class I expression levels. External peptide loading of B-lymphoblastoid cells is an alternative and widely used method to obtain target cells for functional CD8+ T-cell assays. However, the choice of peptide sequences is based on MHC class I epitope-prediction algorithms or the use of overlapping peptide libraries (Rotzschke et al., 1991; Cerny et al., 1995; Lauer et al., 2002; Wertheimer et al., 2003). Both approaches neglect MHC class I epitope-restriction mechanisms originating upstream of MHC class I peptide loading (Engelhard et al., 2002; Hanada et al., 2004) and they fail to detect possible virus-induced alterations of antigen presentation (Park et al., 2004). As a result, predicted epitopes may not be identical in sequence to naturally processed ligands. Expression of HCV proteins in APCs resembles a more natural model, allowing endogenous antigen processing, restriction and presentation. Yet transient-expression systems lead to artificially high expression levels of HCV proteins and do not allow analyses of immune responses in the context of steady-state protein expression. Moreover, heterologous viral shuttle systems may interfere with processing and presentation of HCV-derived epitopes. Stable transfection systems overcome these limitations and have been employed successfully, e.g. for analyses of cellular immune responses against cytomegalovirus and Plasmodium falciparum circumsporozoite proteins (Kumar et al., 1997; Retière et al., 2000). The aim of this study, therefore, was to establish professional human APC lines that express HCV proteins constitutively, display a common human leukocyte antigen (HLA) type and can be used reliably to study human HCV-specific CD8+ T-cell responses.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression constructs.
Plasmid pEFNS3-4A was constructed by ligation of the EcoRI–XbaI fragment of pCMVNS3-4A (Wölk et al., 2000), encoding the NS3–4A complex derived from a prototype HCV H clone (Grakoui et al., 1993), into the EcoRI–XbaI sites of pEF1/V5-HisA (Invitrogen). Plasmid pEFHCV(H)con was prepared in two steps by ligation of the 1200 bp EcoRI–AflII and 7803 bp EcoRI–EcoRI fragments of pBRTM/HCV1-3011con (Kolykhalov et al., 1997) into the EcoRI–XbaI sites of pEF1/V5-HisA. This construct allows expression of the entire open reading frame (ORF) derived from a functional HCV consensus cDNA, with authentic translation-initiation and stop codons. Finally, the EcoRI–XbaI fragment of pEGFP-N1 (Clontech), encoding enhanced green fluorescent protein (EGFP), was ligated into the EcoRI–XbaI sites of pEF1/V5-HisA to yield plasmid pEFEGFP. All constructs were under the transcriptional control of the elongation factor-1{alpha} promoter of the pEF1/V5-HisA backbone and contained a neomycin phosphotransferase expression cassette, allowing G418 selection of stably transfected cells.

Antibodies.
Monoclonal antibodies (mAbs) C7-50 against HCV core protein (Moradpour et al., 1996), 1B6 (Wölk et al., 2000) and 1878 (ViroStat) against NS3, and 5B-12B7 and 5B-3B1 against NS5B (Moradpour et al., 2002) have been described previously. mAb A11 against E2 was a kind gift of Jean Dubuisson, Institut Pasteur de Lille, Lille, France, and Harry Greenberg, Stanford University, Stanford, CA (Dubuisson et al., 1994). mAbs 8N and 11H (Brass et al., 2002) against NS4A and NS5A, respectively, were kindly provided by Jan Albert Hellings, bioMérieux, Boxtel, the Netherlands. mAb BB7.2, specific for the {alpha}2 domain of HLA-A2, was obtained from ATCC (HB-82) and conjugated with fluorescein as described previously (Gremion et al., 2004). Alexa Fluor 488-labelled F(ab')2 fragment of goat anti-mouse IgG (Molecular Probes) was used as secondary antibody in indirect immunofluorescence analyses. Sheep anti-mouse IgG horseradish peroxidase-linked whole antibody (Amersham Biosciences) was used as secondary antibody in Western blot analyses.

Cell lines and transfections.
Human professional APC line 174xCEM.T1, also termed T1, was obtained from ATCC (CRL-1991). This cell line represents a cloned hybrid of B-lymphoblastoid cell line (LCL) 721.174 and T-LCL CEMR.3 and expresses high levels of HLA-A2 (DeMars et al., 1984; Salter et al., 1985). Cells were grown in RPMI 1640 medium supplemented with 10 % fetal calf serum. Electroporation conditions were optimized as described previously (Baum et al., 1994). In the final protocol, 107 cells were resuspended in 400 µl culture medium, mixed with 20 µg plasmid DNA and pulsed in a 4 mm gap cuvette by using an Electroporator II (Invitrogen) set to 240 V, 1000 µF capacitance and infinite load resistance. Stably transfected cells were selected by G418 2 days after transfection and were later cloned and subcloned in 0·3 % soft agar. Clones were screened by indirect immunofluorescence microscopy and immunoblot analyses. UNS3-4A-24 and UHCVcon-57.3 cells, which inducibly express the NS3–4A complex and the entire HCV polyprotein, respectively, have been described previously (Moradpour et al., 1998; Wölk et al., 2000; Schmidt-Mende et al., 2001). Generation of cytotoxic T-lymphocyte (CTL) lines specific for the previously described HLA-A2 core131–140 epitope (ADLMGYIPLV, aa 131–140 of the HCV H polyprotein) and the NS31073–1081 epitope (CINGVCWTV, aa 1073–1081) have also been described previously (Cerny et al., 1995; Kammer et al., 1999).

HLA typing and CD4+ T-cell proliferation assays.
The complete haplotype of T1 cells was determined by PCR sequence-specific primer typing using the Cyclerplate system (Protrans). Tetanus toxoid (TT)-specific CD4+ T-cell clone AP TT 9.04 (Serum and Vaccine Institute, Bern, Switzerland) was used in thymidine-incorporation assays to evaluate the functional MHC class II-restricted presentation capacity of T1-derived cell lines. In these assays, 5x104 TT-specific CD4+ T cells were mixed with 1x105 T1-derived cells, which were irradiated with 8000 rad, with or without 10 mg TT ml–1. After 48 h, 0·5 µCi (18·5 kBq) [3H]thymidine was added. Cells were harvested 12 h later and incorporated radioactivity was determined by using a {beta}-counter. Autologous Epstein–Barr virus (EBV)-immortalized B cells were used as a positive control and the mouse lymphoblast cell line L1210 (CCL-219; ATCC) was used as an HLA-A2 negative-control APC.

Immunoblotting, immunofluorescence and confocal laser-scanning microscopy (CLSM).
Immunoblotting was performed as described previously (Moradpour et al., 1996, 1998). For immunofluorescence staining, cells were transferred to adhesion slides (Marienfeld), fixed with 4 % paraformaldehyde for 20 min at 20 °C, permeabilized with 0·05 % saponin for 20 min and incubated in blocking buffer (3 % BSA in PBS) for 30 min. Cells were subsequently incubated with primary and secondary antibodies diluted in blocking buffer and washed with PBS. CLSM was performed with a Zeiss LSM 510 Meta system (Carl Zeiss). The manufacturer's LSM 510 software was used for image processing and analyses.

Detection of HCV-specific mRNA by RT-PCR.
Total cellular RNA was isolated by using an RNeasy Mini kit (Qiagen) with on-column DNase I digestion. RT-PCR was performed by using the SuperScript First-strand Synthesis system (Invitrogen) as recommended by the manufacturer, with random-hexamer primers for the reverse-transcription reaction and specific primers flanking the entire HCV core (primer pair: core-fwd, 5'-GAGAATTCCGTGCACCATGAGCACGAATCCTAAACC-3', and core-rev, 5'-GCTGTCTAGATTAGGCTGAAGCGGGCACGGTCAGGC-3') or the NS3 protease domain coding region (primer pair: NS3-fwd, 5'-GCACGAATTCACCATGGCGCCCATCACGGCGTACGCCCAGCAGAC-3', and NS3P201-rev, 5'-GCTGTCTAGATTAGTGGGCCACCTGGAAGCTCTGGGGCACTGC-3').

51Cr-release assays.
CTL activity was measured in a standard 4 h 51Cr-release assay, as described previously (Kammer et al., 1999; Moradpour et al., 2001; Gremion et al., 2004). In brief, target cells were labelled with Na2(51Cr)O4 (Amersham Biosciences) for 1 h and washed four times. They were then transferred to 96-well tissue-culture plates (2·5x103 target cells per well), mixed with effector cells at ratios indicated in the figures and incubated for 4 h to test for specific cytolytic activity. The fraction of lysed target cells was calculated as: (experimental release–spontaneous release)/(maximum release–spontaneous release). Maximum release was determined by lysis of target cells with 1 M HCl. Spontaneous release was <25 % of maximum release in all assays.

Vaccination of HLA-A2.1 transgenic mice.
HDD mice, transgenic for HLA-A2.1 MHC class I and deficient for both H-2Db and murine {beta}2-microglobulin (Ureta-Vidal et al., 1999), were kept at the animal-care facilities of the Department of Medicine, University of Bern, Switzerland, and experiments were conducted according to the international guidelines for animal experimentation. As described elsewhere in more detail (Engler et al., 2004), mice were immunized with liposomal formulations containing peptides for the NS31073–1081 epitope CINGVCWTV and the T-helper peptide TPPATRPPNAPIL, derived from aa 128–140 of the hepatitis B virus (HBV) nucleocapsid (Firat et al., 1999), with or without the CpG oligonucleotide 5'-TCCATGACGTTCCTGATGCT-3' (Whitmore et al., 2001). Mice received three injections at 2-week intervals. Two weeks after the last injection, spleen cells were restimulated with the NS3 peptide ex vivo and CD8+ T-cell responses were measured in 51Cr-release assays, using T1/NS3-4A cells as target cells.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Generation of stable cell clones
The human HLA-A2-positive APC line 174xCEM.T1, also termed T1, was chosen as the founder cell line for stable transfections. T1 cells were stably transfected with expression vectors pEFNS3-4A, pEFHCV(H)con and pEFEGFP to obtain cell lines T1/NS3-4A, T1/HCVcon and T1/EFEGFP, expressing the entire HCV polyprotein, the NS3–4A complex and EGFP, respectively (Fig. 1). The pEFEGFP construct was generated to optimize transfection and cloning procedures, the final protocol of which is presented in this paper. Initially, 600 µg G418 ml–1 was used for selection of stably transfected cells. Several independent clones expressing readily detectable levels of NS3–4A and EGFP were obtained, of which T1/NS3-4A-2F3.B and T1/EFEGFP-1G2.1 were kept in continuous culture for further analyses. Only a few cell clones were obtained for the pEFHCV(H)con expression construct and expression of the HCV polyprotein was generally very low. Clone T1/HCVcon-41.C, showing the highest HCV polyprotein-expression level, was kept for further analyses. Two more rounds of stable-transfection procedures were performed to generate further T1/HCVcon cell clones of independent origin. For selection and cloning of these cells, the G418 concentration was increased to 2000 µg ml–1. This allowed the isolation of several clones, of which the two independent cell lines T1/HCVcon-D1C2H5.C and T1/HCVcon-P1B6C12.B were kept in culture. During further analyses, this allowed verification of results in a total of three independent T1/HCVcon cell lines. All cell lines have been maintained in continuous culture for more than 1 year with stable characteristics.



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Fig. 1. Expression constructs used to generate T1/NS3-4A, T1/HCVcon and T1/EGFP cell lines. T1-derived cell lines were generated by stable transfection of the depicted constructs pEFNS3-4A, pEFHCV(H)con and pEFEGFP, encoding the NS3–4A complex, the entire HCV polyprotein and EGFP, respectively. All constructs were driven by the elongation factor-1{alpha} (EF 1{alpha}) promoter. Above, a schematic diagram of the HCV genome is shown. The single-stranded RNA genome of positive polarity consists of a long ORF of approximately 9000 nt between the 5' and 3' non-coding regions (NCRs). The ORF encodes a 3000 aa polyprotein precursor that is processed by the cellular signal peptidase ({lozenge}), as well as by the viral NS2–3 and NS3 proteases (indicated by arrows).

 
HLA typing of T1 cells revealed the phenotype HLA-A2, A31(19); B60(40), B51(5); DR7, DRx. Analyses of HLA-A2 expression levels by FACS using fluorescein isothiocyanate-labelled HLA-A2-specific mAb BB7.2 confirmed high expression levels, comparable to those of the commonly used, EBV-transformed, human B-lymphoblastoid cell line JY (data not shown). To characterize our cell lines briefly with respect to MHC class II-dependent stimulation of CD4+ T cells, T1-derived cells were pulsed with TT and used as APCs to stimulate a TT-specific CD4+ T-cell line. In this context, T1-derived cells, as well as autologous APC lines, which were used as a positive control, were able to stimulate TT-specific CD4+ T cells (data not shown). No increased target-cell proliferation was observed when the MHC class II-negative mouse cell line L1210 was pulsed with TT as a negative control. We therefore concluded that T1 cells also express functional MHC class II molecules, which are able to stimulate CD4+ T cells.

HCV polyprotein processing in T1/NS3-4A and T1/HCVcon cells
Cell lines T1/NS3-4A-2F3.B, T1/HCVcon-41.C, T1/HCVcon-D1C2H5.C and T1/HCVcon-P1B6C12.B were analysed for polyprotein processing by Western blotting using mAbs against a panel of HCV proteins. As shown in Fig. 2, NS3 and NS4A proteins of the expected molecular masses of 70 and 6–7 kDa were detected in T1/NS3-4A and T1/HCVcon cells, respectively. Of note, detection of NS3 by mAb 1878 revealed additional bands below the 70 kDa band, which we interpreted as NS3-specific degradation products. The pattern of these bands differed when NS3 was expressed in the context of the entire HCV polyprotein or as the NS3–4A complex alone. Similarly, we previously observed different degradation-product patterns when various forms of NS3 were expressed in osteosarcoma cell lines (Wölk et al., 2000).



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Fig. 2. HCV polyprotein processing in T1/NS3-4A and T1/HCVcon cells. Cell lysates were separated by SDS-PAGE and analysed by immunoblotting using mAbs 1878 against NS3 (diluted 1 : 100), 8N against NS4A (1 : 500), A11 against E2 (1 : 100), 11H against NS5A (1 : 50) and 5B-3B1 against NS5B (1 : 5), as indicated. UHCVcon-57.3 cells cultured for 36 h in the absence of tetracycline to induce HCV protein expression were used as a positive control. Equal amounts of total cellular proteins were loaded for T1-derived cell lines, whereas UHCVcon lysates were used at a three- to fivefold dilution to adjust for lower HCV protein-expression levels of T1/NS3-4A and T1/HCVcon cells. Molecular mass markers are indicated on the left.

 
In all T1/HCVcon cell clones, E2, NS5A and NS5B were also detected with the expected molecular masses. Taken together, these results indicate that the NS3 serine protease is fully functional in T1/NS3-4A and T1/HCVcon cell lines. Moreover, detection of E2 of the correct size suggests that the cellular proteolytic machinery involved in processing of the HCV polyprotein precursor is also functional in T1/HCVcon cells.

HCV proteins show a cytoplasmic staining pattern in T1/NS3-4A and T1/HCVcon cells
Indirect immunofluorescence microscopy was performed to further characterize the HCV protein expression in T1-derived cell clones (Fig. 3). In T1/NS3-4A cells, both NS3 and NS4A were found in a cytoplasmic staining pattern. Whilst the cytoplasm of lymphocyte-derived cells typically appears as a small fringe around a dominant nucleus and is of limited value for detailed subcellular-localization studies, the overall distribution of NS3 and NS4A resembled our previous findings in tetracycline-regulated osteosarcoma cell lines, in which both proteins were found to co-localize with the endoplasmic reticulum when expressed together (Wölk et al., 2000). By contrast, in T1/HCVcon cells, NS3, NS4A, NS5A and NS5B were detected in a dot-like, cytoplasmic staining pattern (Fig. 3). This is in good agreement with the localization of HCV proteins expressed in the context of the polyprotein in specific and circumscript membrane alterations, designated membranous webs (Egger et al., 2002; Gosert et al., 2003). These membranous webs represent virus-replication complexes in HuH-7 cells harbouring HCV replicons (Gosert et al., 2003).



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Fig. 3. Subcellular localization of HCV proteins in T1/NS3-4A and T1/HCVcon cells. Cell lines were processed for CLSM as described in Methods, using hybridoma supernatants of mAbs 1B6 against NS3, 8N against NS4A, 11H against NS5A (each diluted 1 : 10 in blocking buffer) and 5B-12B7 against NS5B (diluted 1 : 2). Images were recorded by CLSM using a Zeiss LSM 510 Meta microscope. To depict all fluorescent signals (green) within cells, stacks of 0·50 µm thick layers comprising entire cell volumes were recorded with identical settings for each antibody. Image layers were then projected in the z direction by using Zeiss LSM software and laid on top of a single differential interference contrast image plane (grey) so that cell contours were also shown. NS3 and NS4A were detectable in T1/NS3-4A and T1/HCVcon cells, but not in parental T1 cells. NS5A and NS5B could only be detected in T1/HCVcon cells. Viral proteins showed a predominantly dot-like staining pattern in T1/HCVcon cells, whereas they exhibited a cytoplasmic reticular pattern in T1/NS3-4A cells.

 
Core-specific mRNA can be detected in T1/HCVcon cells
For unknown reasons, we failed to detect HCV core protein by immunoblotting or immunofluorescence microscopy. To exclude truncation of the chromosomally integrated HCV cDNA, we analysed HCV-specific mRNA by RT-PCR using NS3- and core-specific primer pairs (Fig. 4). Core-specific mRNA was detected in T1/HCVcon-41.C, T1/HCVcon-D1C2H5.C and T1/HCVcon-P1B6C12.B cell clones only, whereas NS3-specific mRNA was also detectable in T1/NS3-4A-2F3.B cells. No PCR-amplification products were found in T1 cells at any time, nor in any sample when reverse transcriptase was omitted. We therefore concluded that all T1/HCVcon clones expressed mRNA specific for the core region, but that the low levels of HCV proteins in our system impeded detection of core and E1 proteins by Western blot or immunofluorescence analyses.



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Fig. 4. Core-specific mRNA can be detected in T1/HCVcon cells, but not in T1/NS3-4A or T1 cells. Total cellular RNA isolated from the cell clones indicated above was analysed by RT-PCR with (+) or without (–) reverse transcriptase (RT), using specific primers for the NS3 protease (upper panel)- or core (lower panel)-coding regions. PCR products were separated by agarose-gel electrophoresis and stained with ethidium bromide. Plasmid pBRTM/HCV(H)con was used as a positive control and water as a negative control. An internal control (IC) mRNA with the corresponding primer pair was provided by the manufacturer of the RT as a control. Sizes of the expected PCR products for the core or partial NS3 region were 602 and 632 bp, respectively. Size markers are indicated on the left.

 
T1/NS3-4A and T1/HCVcon cells function as targets for HCV-specific CTLs
To evaluate the presentation of endogenously processed HCV epitopes, T1/NS3-4A and T1/HCVcon cells were used as targets in 51Cr-release assays with human CTL lines specific for the core131–140 or the NS31073–1081 epitope. External loading of parental T1 cells with HCV core131–140 or NS31073–1081 peptide showed specific cytolytic activity of these cells (data not shown). As shown in Fig. 5, T1/N3-4A and T1/HCVcon clones were lysed readily by CTL lines specific for the NS31073–1081 epitope. Parental T1 cells used as negative controls were not recognized by these CTL lines. Interestingly, T1/HCVcon cells showed lower 51Cr release than T1/NS3-4A cells. This might reflect lower NS3 expression levels in these cells, but might also indicate differences in antigen processing and presentation. In addition, all T1/HCVcon cell clones were also lysed by the core-specific CTL line, whereas T1/NS3-4A and T1 cells were not lysed. When parental T1 cells and T1/HCVcon cell clones were, in addition, loaded externally with HCV core131–140 peptide, all target cells showed specific lysis values of up to 90 % (data not shown). We concluded, therefore, that T1/NS3-4A and T1/HCVcon cell lines present naturally processed, HCV-specific MHC class I epitopes, which are recognized by HCV-specific CTLs. Notably, all T1/HCVcon cell clones were lysed by core-specific CTLs, indicating that core protein is expressed, but is below the detection limit of our immunoblot and immunofluorescence assays.



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Fig. 5. T1/NS3-4A and T1/HCVcon function as target cells for HCV-specific human CTL lines. T1/NS3-4A and T1/HCVcon cell clones were used as target cells in 4 h 51Cr-release assays with CTLs specific for the NS31073–1081 or the core131–140 epitopes as effector cells. The percentage of specific lysis is shown. {blacksquare}, HCV protein-expressing target cells; {circ}, non-transfected T1 cells used as negative controls. The specificity of CTLs is shown on the left. E : T ratio, effector- to target-cell ratio. T1/NS3-4A cells were only recognized by NS3-specific CTLs, whereas T1/HCVcon cells were also lysed by core-specific CTLs.

 
T1/NS3-4A and T1/HCVcon cells allow the study of HCV-specific CD8+ T-cell responses generated in human HLA-A2.1 transgenic mice
Current HCV vaccination strategies are aimed at the induction of HCV-specific T-cell responses (Forns et al., 2002). In this context, HLA-A2.1 transgenic mice represent an attractive animal model for the preclinical evaluation of vaccine candidates (Ureta-Vidal et al., 1999), but the limited tropism of HCV prohibits its use to challenge mouse immunity. We therefore addressed whether our T1-derived cells could function as a readout system to assess HCV-specific CD8+ T-cell responses of HLA-A2.1 transgenic mice that were immunized with various liposome formulations. As shown in Fig. 6, T1/NS3-4A cells were lysed efficiently by CD8+ T lymphocytes derived from these mice 14 days after vaccination. Addition of CpG to the liposome formulation enhanced CD8+ T-cell responses. Interestingly, when we used peptide-pulsed JY cells as targets in similar experiments, we were not able to reveal the effect of CpG in liposome formulations (Engler et al., 2004). Thus, our T1-derived cell lines can be used as a sensitive and well-defined readout system to evaluate the quality of CD8+ T-cell responses induced by vaccine candidates in the HLA-A2.1 transgenic-mouse model system.



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Fig. 6. CTLs from HLA-A2.1 transgenic mice efficiently recognize and lyse T1/NS3-4A cells. HLA-A2.1 transgenic mice were immunized with liposomes containing HCV CTL epitope NS31073–1081 and an HBV T-helper peptide with or without the oligonucleotide CpG, as described in Methods. Specific lysis of T1/NS3-4A-2F3.B cells used as targets in 51Cr-release assays is illustrated, with mouse-derived CD8+ T cells as effector cells. Four of the animals were immunized with liposome formulations (data shown as solid symbols) and, as a control, one animal was immunized with the HCV NS31073–1081 peptide in saline only ({circ}). The left diagram depicts data from two mice ({blacksquare},{blacktriangleup}) that were immunized with liposomes containing the HCV NS3 and HBV T-helper peptide. On the right, two mice ({blacklozenge},*) were injected with liposomal formulations that additionally contained CpG. E : T ratio, effector- to target-cell ratio.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have presented new lymphoblastoid cell lines, termed T1/NS3-4A and T1/HCVcon, that express the HCV NS3–4A complex or the entire HCV polyprotein, respectively, in a continuous fashion. The cellular and viral proteolytic machineries involved in HCV polyprotein processing were found to be functional in these cell lines, as shown by Western blot analyses (Fig. 2). Moreover, subcellular localization of viral proteins was in agreement with previous findings in other expression systems (Moradpour et al., 1998; Wölk et al., 2000; Egger et al., 2002), as shown by indirect immunofluorescence staining and CLSM (Fig. 3). Of note, we could not detect core protein in T1/HCVcon cells (data not shown). However, core-specific mRNA was detected readily by RT-PCR in all T1/HCVcon cell clones (Fig. 4). Moreover, all T1/HCVcon clones were lysed specifically by core-specific CTL lines. We therefore concluded that core expression in these cell lines occurs at levels that might frustrate detection by conventional protein-detection methods, but still lead to a significant response of core-specific CTLs. However, we cannot rule out the possibility that core and E1 proteins may be degraded or modified post-translationally in a cell-specific manner, which would also impair detection in our Western blot and immunofluorescence assays. In general, compared with transient- and inducible-expression systems, viral protein expression was low in our cells and probably resembles infection levels in vivo more closely.

Importantly, our cell lines are based on professional APCs. They have high HLA-A2 expression levels and function well as target cells for HCV-specific T lymphocytes (Fig. 5). Moreover, these cell lines are also able to activate CD4+ T cells, as found for the TT antigen (data not shown). This indicates functional MHC class II expression and may be an essential feature for future applications. T1/NS3-4A and T1/HCVcon cells complement each other. T1/NS3-4A cells not only serve as well-characterized internal-control APCs, but will also allow identification of putative alterations in HCV antigen processing in the context of the entire HCV polyprotein in future studies. Our novel cell lines have been maintained in continuous culture for more than 1 year without notable changes in their characteristics. Thus, they make up highly reproducible and well-characterized APC lines, which efficiently present endogenously processed, HCV-specific MHC class I ligands and are a novel and valuable tool to study HCV-specific CD8+ T-lymphocyte function.

So far, analysis of HCV-specific CD8+ T-cell responses has been dependent on professional APCs that were loaded exogenously with synthetic, HCV-specific peptides of known or predicted sequence. The study of CTL responses to naturally processed HCV antigens has been hampered due to the limited panel of cell-culture systems with stable HCV protein expression. The available expression systems are rarely based on professional APCs and thus express only low levels of MHC class I molecules. In this context, it is worth mentioning that we recently enhanced functional HLA-A2 expression levels in tetracycline-regulated osteosarcoma cells to levels found in professional APCs by stable transfection of an HLA-A2 expression construct into HCV protein-expressing founder cells (Gremion et al., 2004; unpublished data). However, these modified, inducible osteosarcoma cell lines grow in adherent monolayer cultures and CTL assays using these cells have been technically more challenging. In the future, it will be interesting to employ a similar approach to study HCV-specific antigen processing and presentation in hepatocytes to identify possible subtle differences in the currently available APC model systems.

To our knowledge, there has only been one report of professional APC lines that express HCV antigens stably so far. Chen et al. (1998) generated EBV-transformed B-LCLs, although these only expressed the structural region of the HCV-J genome. Therefore, our T1-derived cell clones represent the first professional APC lines stably expressing the complete HCV polyprotein and the NS3–4A complex. Importantly, our cells are HLA-A2-positive. This opens up a wide field of applications, as HLA-A2 is highly prevalent in most study populations and many specific tools are already available for this HLA type. This makes our novel cell lines an ideal readout system for ongoing and future studies, as shown for vaccination studies in the HLA-A2.1 transgenic-mouse model (Fig. 6). Indeed, studies using these cell lines have already allowed the exploration of novel vaccination strategies (Engler et al., 2004) and analysis of bystander killing by HCV-specific CTLs (Gremion et al., 2004). Importantly, T1-derived cells grow in suspension cultures and are easy to handle. Furthermore, T1 cell cultures can easily be expanded to a large scale, which opens up the possibility of using these cells for the isolation of MHC class I molecules and the isolation of naturally processed, HCV-specific MHC class I ligands (Schirle et al., 2000).

Interestingly, B-cell lymphoma cell lines infected persistently with HCV have recently been reported (Sung et al., 2003). So far, however, these cells are not widely available and their value as APCs remains to be determined. Lymphocytes isolated from infected patients have also been reported to harbour replicating HCV, but this is an ongoing matter of debate (Lerat et al., 1998; Sansonno et al., 1998a, b; Laskus et al., 2000; Boisvert et al., 2001; De Vita et al., 2002). In this context, it is worth noting that detection of HCV proteins in our T1-derived cell lines by indirect immunofluorescence was challenging, due to the low expression levels and relatively high non-specific background in parental T1 cells. Indeed, the low expression levels of T1/NS3-4A and T1/HCVcon cells might be similar to expression levels found in cells of infected patients, in which detection of HCV proteins has been difficult. Thus, the optimization steps that were needed to allow us reproducible detection of HCV proteins expressed at low levels in lymphocyte-derived cells may also be necessary for reliable detection of HCV proteins in infected lymphohaematopoietic cells of patients with hepatitis C. T1/NS3-4A and T1/HCVcon cells may serve as valuable controls for such efforts.

In conclusion, T1/NS3-4A and T1/HCVcon cells are unique, professional APCs that express HCV proteins continuously and allow the study of cellular HCV-specific immune responses. Ultimately, these cell lines may contribute to the evaluation of novel vaccine and immunotherapeutic strategies against HCV.


   ACKNOWLEDGEMENTS
 
The authors gratefully acknowledge Werner Pichler and Lynn Dustin for helpful discussions, Charles M. Rice for plasmid pBRTM/HCV1-3011con, Jean Dubuisson and Harry Greenberg for mAb A11, Jan Albert Hellings for mAbs 8N and 11H and François A. Lemonnier for HLA-A2.1 transgenic mice. This work was supported by grants from the European Commission, Brussels, Belgium (QLK2-CT1999-00356 and QLK2-CT2002-01329), the Bundesministerium für Bildung und Forschung, Bonn, Germany (01 KI 9951) and the Swiss National Science Foundation.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 22 December 2004; accepted 16 February 2005.



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