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
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ABSTRACT |
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These authors contributed equally to this work.
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.
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INTRODUCTION |
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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.
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METHODS |
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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
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 NS34A 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 core131140 epitope (ADLMGYIPLV, aa 131140 of the HCV H polyprotein) and the NS310731081 epitope (CINGVCWTV, aa 10731081) 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 ml1. 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 -counter. Autologous EpsteinBarr 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 releasespontaneous release)/(maximum releasespontaneous 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 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 NS310731081 epitope CINGVCWTV and the T-helper peptide TPPATRPPNAPIL, derived from aa 128140 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.
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RESULTS |
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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 67 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 NS34A 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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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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DISCUSSION |
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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 NS34A 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.
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ACKNOWLEDGEMENTS |
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Received 22 December 2004;
accepted 16 February 2005.
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