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International Immunology Advance Access published online on November 15, 2007
International Immunology, doi:10.1093/intimm/dxm121
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Priming and stimulation of hepatitis C virus-specific CD4+ and CD8+ T cells against HCV antigens NS4, NS5a or NS5b from HCV-naive individuals: implications for prophylactic vaccine

Wen Li1,*, Deepa K. Krishnadas1,*, Rakesh Kumar2, D. Lorne J. Tyrrell3 and Babita Agrawal1

1 Department of Surgery
2 Department of Laboratory Medicine and Pathology and
3 Department of Medical Microbiology and Immunology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada

Correspondence to: Correspondence to: B. Agrawal; E-mail: bagrawal{at}ualberta.ca


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Hepatitis C virus (HCV) is a devastating human pathogen, yet there is no vaccine available for this virus. From studies with acute or chronic HCV-infected humans and chimpanzees, T-cell responses against HCV-derived conserved non-structural antigens have been correlated with viral clearance. In this study, recombinant adenoviral vectors containing HCV-derived NS4, NS5a or NS5b genes were employed to endogenously express the HCV antigens in human dendritic cells (DCs). The DCs expressing these HCV antigens exhibited normal phenotype and function. Intriguingly, we found that the DCs expressing HCV NS4, NS5a or NS5b antigens were able to significantly stimulate autologous T cells obtained from uninfected healthy individuals. These T cells produced various cytokines and proliferated in an HCV antigen-dependent manner. Evidence of both CD4+ and CD8+ T-cell responses generated in vitro against HCV NS4, NS5a or NS5b were obtained. HCV NS4 was much less stimulatory for CD4+ and CD8+ T cells than NS5. Further, in secondary assays, the CD4+ T cells primed in vitro exhibited HCV antigen-specific proliferative responses against recombinant protein antigens. In summary, we provide conclusive evidence of in vitro stimulation of CD4+ and CD8+ T cells from HCV-naive individuals against HCV antigens NS4, NS5a and NS5b. The studies with naive T cells represent early events in the induction of cellular immune responses, which most likely govern the outcome of HCV infection. These studies have significant implications in designing vaccines for HCV infection in both prophylactic and therapeutic settings.

Keywords: dendritic cells, hepatitis C virus, immune response, phenotype, T lymphocyte activation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Hepatitis C virus (HCV) is a hepatotropic, enveloped, single-stranded RNA virus. The HCV genome consists of a single open reading frame, encoding a polyprotein of 3011 amino acids (aa). The translated polyprotein is processed by cellular and viral proteases to generate 10 proteins: structural proteins (Core, E1, E2 and P7) and non-structural proteins NS2 (cysteine protease), NS3 (serine protease and helicase), NS4 (cofactor for serine protease), NS5a (phosphoprotein) and NS5b (RNA-dependent RNA polymerase) (1).

Infection with HCV in most individuals (70–85%) results in chronic persistent infection with long-term consequences of severe liver disease and mortality. In contrast, 15–30% of the infected individuals get acute infections, followed with viral clearance. Initial infection with HCV often leads to asymptomatic acute infection, which is not diagnosed in most instances. Both host and viral factors may contribute to clearance or progression to persistent infection from acute infections.

Substantial evidence has accumulated to suggest the role of adaptive host immune responses in viral clearance in HCV infection. Control of acute primary viral infection is often associated with emergence and expansion of CD4+ and CD8+ T-cell responses to specific HCV antigens (2). Failure to generate an effective cellular immune response in the acute phase of infection is likely responsible for the high rate of chronicity. The kinetics of viremia and onset of adaptive CD4+ and CD8+ T-cell responses have been studied in depth in chimpanzees as well as humans infected acutely, transiently or persistently with HCV (3–6). It has been observed that onset of cellular immunity often lags behind the onset of viremia by a few weeks. The strong, multispecific and sustained adaptive immune responses lead to virus clearance, whereas weak or strong but waning immune responses result in transient clearance or persistent infection. Patients with chronic HCV infection fail to initiate, maintain and/or sustain a strong TH1 response that is targeted against several immunodominant proteins (5). From studies with humans and chimpanzees, CD4+ T-cell responses and IFN-{gamma} produced by CD4+ T cells have been suggested to be important in viral clearance (7, 8). Although the HCV genome is highly variable with hundreds of serotypes and six genotypes, several structural and non-structural proteins are conserved among genotypes and subtypes (9). Interestingly, a vigorous multispecific CD4+ T-cell response against some of these conserved protein epitopes have been suggested to be correlated to viral clearance (10). Strong T-cell proliferative responses against HCV Core, NS3, NS4 and NS5 have been found to be associated with self-limited infection (4, 11–14). For the T helper responses, several immunodominant epitopes have been recognized. The identified immunodominant epitopes are highly conserved among the known HCV isolates (12, 14). Many of these epitopes from conserved non-structural proteins of HCV are also permissive in that they can be presented in the context of several HLA haplotypes, suggesting their wide immunogenicity and potential as broadly applicable vaccine candidates (13).

It has been suggested that multispecific CTL responses against non-structural proteins may control HCV replication to some extent (15). Recently, CTL responses from humans and chimpanzees that cleared HCV infection have been analyzed (16, 17). A strong, multispecific and persistent T-cell response was associated with viral clearance in chimpanzees and humans acutely infected with HCV, and also with HCV seronegative humans exposed to HCV (16, 17). The CD8+ CTL response may lead to viral clearance through a direct lysis of infected cells or through cytokine-mediated inhibition of virus replication. An inverse correlation has also been obtained between levels of HCV-specific CTL activity and viral loads in humans (18), suggesting that HCV can be controlled to some extent by CTLs. These observations have also been supported by studies in chimpanzees (19). In contrast, persisting CTL responses are also detectable in some chronic HCV-infected patients, whereas virus-specific CD4+ T-cell responses are generally weak or absent (10, 12, 14). The relative contribution of CD4+ and CD8+ T cells to viral clearance in acutely HCV-infected patients is not fully defined. It appears that HCV persistence is predicted by a failure to generate or sustain CD4+ T-cell responses. This outcome was supported by persistence induced with anti-CD4 antibody treatment of immune chimpanzees, suggesting that HCV antigen-specific T cell help is a central event required for anti-viral immunity (8, 11).

In addition to examining the specific adaptive immune response in acute-clearing or persistent infection, it has been shown that recovering humans and chimpanzees are protected against re-exposure to the virus in the majority of cases, even against highly divergent viral strains. This protection is, however, not at the level of getting an acute infection but rather at the level of prevention of progression to chronic infection upon re-exposure (19–21). To date, studies illustrating the role of adaptive immunity in viral clearance in HCV infection support the notion that an effective prophylactic and/or therapeutic vaccine for HCV is an approachable target. In order to design and investigate promising vaccine candidates, the potential of healthy uninfected human donors' T cells to respond against HCV antigens has to be understood. In our previous studies, we have shown that HCV-derived NS3 and Core-specific T cells can be primed in vitro from HCV-naive healthy uninfected donors (22).

Dendritic cells (DCs) have been shown to be the most potent antigen-presenting cells (APCs) in the immune system, expressing high levels of MHC molecules, co-stimulatory molecules and adhesion molecules, to efficiently stimulate T cells (23). DCs can acquire antigens both endogenously and exogenously, process them and present them in the context of MHC class I and/or MHC class II molecules on the surface. In addition, DCs are capable of cross-priming, allowing the DCs to present exogenous antigens in context of both MHC class I and class II molecules (24). DCs have also been shown to successfully prime naive T cells in vitro against several known tumor antigens (25, 26). DCs pulsed with synthetic peptides have been used (25); however, in humans, this approach is limited to a relatively small number of previously identified peptides in the context of specific MHC class I or class II molecules. Use of whole recombinant protein antigens may be limiting because they may not be efficiently taken up and processed or presented by the APCs (27). In contrast, expression of viral or tumor antigens in DCs eliminates these limitations and leads to efficient processing and presentation of various peptides in the context of both MHC class I and class II molecules (28, 29), displaying a complete repertoire of presentable peptides to the T cells.

In the present study, we have examined whether DCs obtained from healthy uninfected donors' PBMCs, upon infection with recombinant adenoviral vectors containing HCV NS4, NS5a or NS5b genes, express these proteins in the cells and have normal phenotype and functions. In addition, we have examined whether DCs expressing HCV NS4, NS5a or NS5b antigens are able to prime/stimulate autologous CD4+ and CD8+ T-cell responses from normal healthy HCV-naive individuals in vitro. We herein present the first conclusive evidence of in vitro priming of both CD4+ and CD8+ T-cell responses against HCV antigens NS4, NS5a or NS5b. This priming was determined by T-cell proliferation, cytokine production, phenotype analysis and secondary T-cell proliferative responses of in vitro primed T cells against relevant recombinant proteins antigens. These studies are highly significant as they substantially advance our understanding of the host's adaptive immune responses against various non-structural antigens of HCV and pave the way for designing vaccines for HCV.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 Funding
 References
 
Cell line and culture
Monolayer of 293A cell line (QBiogene Inc., CA, USA), an adenovirus-transformed human embryonic cell line which provides phenotypic complementation of the E1 genes, was used for recombinant adenovirus plaque assays, amplification and virus titration (22, 30).

Plasmid construction
The NS4 (amino acids 1658–1972), NS5a (aa 1973–2420) and NS5b (aa 2421–3011) genes of HCV-1 strain (genotype 1a) were PCR amplified from the full-length clones of HCV (Supplementary Figure, available at International Immunology Online). The PCR products were cloned into the commercial pCR 2.1 vector (Invitrogen, Carlsbad, CA, USA) and the purified cDNA fragments were cloned into AdenoVator Transfer vector (pAdenoVator-CMV5-IRES-GFP, QBiogene) generating CMV5/GFP/NS4, CMV5/GFP/NS5a and CMV5/GFP/NS5b.

Recombinant adenovirus vectors
Recombinant adenoviruses were propagated, purified and stored as per the standard method provided in the manual (QBiogene) and as reported previously by us (22, 30). The recombinant adenoviral vectors were stored in aliquots at –80°C. Viral particles of Ad5/CMV-LacZ (with no gene insert) were provided by QBiogene and used as a control adenoviral vector (denoted as CV throughout the manuscript).

Preparation and infection of human peripheral blood monocyte-derived DCs
Peripheral blood samples were obtained from donors 30–60 years of age of both sexes after informed consent. Use of human blood samples was approved by institutional Health Research Ethics Board at the University of Alberta, Canada. DCs were generated from Human PBMCs (22, 30). In flow cytometry experiments, the co-expression of HLA-DR with CD11c was determined in the DC populations obtained from the 5-day cultures of adherent cells in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4 and observed that ~95% of the cells obtained from these cultures were double positive for CD11c and HLA-DR, providing the evidence of generation of DC population in the cultures.

Infection with adenovirus
DCs harvested on days 5–7 of the culture with GM-CSF and IL-4 were infected with recombinant defective adenoviruses expressing HCV NS4, NS5a or NS5b or control LacZ gene at a multiplicity of infection (MOI) of 100. In the experiments where LPS stimulation was performed, the LPS (Sigma–Aldrich, Oakville, Ontario, Canada) was added at 100 ng ml–1 24 h post-infection and allowed to further incubate for 4–24 h to mature the DCs.

RNA isolation, cDNA synthesis and reverse transcription
Total RNA was prepared (Roche Diagnostics, Mannheim, Germany), according to the instructions of the manufacturer from 2 x 106 DCs or 1 x 106 to 2 x 106 T cells, followed by cDNA synthesis from 0.5–1 µg of total RNA (30).

Indirect immunofluorescence
DCs (3 x 105 to 5 x 105) were grown on slides, infected with Ad/NS4, Ad/NS5a or Ad/NS5b for 24 h, followed by incubation with or without LPS for 24 h. The slides were washed two times with PBS and fixed in 3.5% PFA solution in PBS for 15 min at room temperature. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were washed once in PBS and blocked with 2% BSA (Sigma–Aldrich) in PBS. A 1:100 dilution of a primary mAb to HCV NS4 (Anogen, Mississauga, Ontario, Canada), NS5a (Research diagnostics Inc., Flanders, NJ, USA) or NS5b (undiluted) (gift from Chris Richardson, Halifax, NS, Canada) was prepared in PBS containing 1% BSA and incubated with the fixed cells for 1 h at room temperature to detect NS4, NS5a or NS5b protein expression. The cells were then incubated with secondary antibody anti-HLA-DR-FITC and 4',6-diamidino-2-phenylindole (DAPI) as described previously (30).

Antibody staining and flow cytometry analysis
DCs harvested 48 h after infection with adenoviral vectors and with or without LPS stimulation were used to assess the cell-surface phenotype by flow cytometry. The conjugated mAbs used include DEC-205 (FITC), HLA-DR (FITC or PE), CD11c (PE), CD80 (PE), CD86 (PE), CD40 (PE) and DC-SIGN (APC) from BD Biosciences PharMingen, San Diego, CA, USA and W6/32–FITC (IgG1, Sigma–Aldrich) as reported previously (31). The cells were gated on the basis of side and forward scatter to select DCs. More than 95% of the cells were positive for CD11c confirming the DC phenotype of the preparation (data not shown).

Purified CD8+ T cells were added to the infected DCs (with or without LPS stimulation) and stained to identify their effector phenotype: anti-CD8 (QR) (Sigma), anti-CD11b (APC–Cy7) (e-Bioscience) and anti-Granzyme B (GrB) (PE) (Caltag Laboratories, Burlingame, CA, USA). Cell suspensions containing 4 x 105 CD8+ T cells were stained with anti-CD8 and anti-CD11b. For intracellular staining for GrB, cells were first pre-incubated with 100 µl of cold permeabilization buffer (2% FBS + 0.3% saponin + 5% normal human serum in PBS) for 5 min at 4°C, and then anti-GrB was added. After incubation for 30 min at 4°C, cells were washed with FACS wash intracellular (2% FBS + 0.1% saponin in PBS) and read the cells using FACS Scan, FACS-Canto and CellQuest software (Becton Dickinson, Mountain View, CA, USA). In all staining experiments, corresponding isotype-matched control mAbs were used to establish background fluorescence. The marker was set to exclude >97% of isotype control antibody-stained cells.

Cell purification
CD4+ and CD8+ T-cell populations were purified by magnetic cell sorting magnetic affinity column separation (MACS) using magnetic beads (Miltenyi Biotec) according to the manufacturer's instructions. In brief, ~2 x 108 non-adherent cells were re-suspended in 1.6 ml MACS buffer (0.5% BSA + 2mM EDTA in PBS, pH 7.2) and incubated with 400 µl CD4+ or CD8+ microbeads for 15 min at 4°C. The cells were washed and re-suspended in 1 ml of MACS buffer and applied onto the LS column. The unlabeled cells were collected which passed through and washed the column with MACS buffer. The cells that bind to the column were flushed out with 5 ml buffer and contained purified CD4+ or CD8+ T cells. These purified populations were found to be at >97% purity by flow cytometry.

Real-time PCR for cytokines
Cytokine gene expression was quantified by real-time PCR on the LightCycler® (Roche Diagnostics Inc.) according to the manufacturer instructions. Primers used in this study have been reported previously (22, 30).

Recombinant HCV proteins
All recombinant HCV proteins, control superoxide dismutase (SOD) protein, SDS lysate and Escherichia coli lysate were kindly provided by M. Houghton, Chiron Corporation (Emeryville, CA, USA). The HCV proteins were genotype 1a and had >99% homology with H77 sequence. These proteins were NS4 (SOD 5-1-1, aa 1694-1735), NS4 (c100-3 aa 1569-1931), NS4 (SOD-c25, aa 2-120 and 1192-1935), NS5 (NS5a and NS5b) (SOD-NS5, aa2054-2995), control antigen yeast extract, E. coli extract, rhSOD and rhSOD-SDS.

T-cell proliferation assay
Proliferative responses of T cells were measured in triplicate cultures in flat-bottom 96-well microtiter plates (Corning Costar , New York, USA). For allogeneic T-cell response, 2 x 105 allogeneic non-adherent cells (as obtained during removal of adherent cells) were co-cultured with different concentrations of infected or non-infected DCs (103–104) in 200 µl of AIM-V medium (GIBCO) at 37°C for 5 days as described previously (30).

For antigen-specific T-cell response, 2 x 105 autologous T cells were cultured with different concentrations of infected or non-infected DCs (103 to 1 x 104) in 200 µl of AIM-V medium (GIBCO) at 37°C for 5 days. Non-adherent cells isolated after removing adherent cells were used as T cells. Non-adherent cells comprised of >80% CD3+ T cells (data not shown). The assay included negative (no antigen) and positive (PHA, 1 µg ml–1) controls. In experiments using purified CD4+ and CD8+ T cells, the non-adherent cells were used to purify CD4+ and CD8+ T cells using MACS (MS spectrometry or LS columns). The purified CD4+ or CD8+ T cells were then co-cultured with autologous DCs expressing various HCV antigens at the stated cell concentration for 4 days. T-cell proliferation was measured by [3H]-thymidine ([3H]-TdR) incorporation assay. In order to determine the cytokines secreted in the supernatant of cultured T cells, supernatant was collected before adding [3H]-TdR and were used to perform ELISA using commercial ELISA kits (Biosource International Inc., Camarillo, CA, USA).

To determine the secondary T-cell responses against recombinant protein antigens, replica-plating assays were performed (22). Initially, ~25 to 48 wells of a 96-well plates were plated with NS4, NS5a or NS5b or control adenoviral infected DCs (104 per well) together with 2 x 105 autologous T cells (or purified CD4+ T cells) in total 200 µl AIM-V media for 6 days. On the sixth day, each well was split into three equal wells on three different 96-well plates. On the first plate, no antigen was added and on the second plate, the relevant recombinant proteins (NS4 protein for NS4 group and NS5 protein for NS5a or NS5b group) were added at 10 µg ml–1. On the third plate, irrelevant antigen (NS4 for NS5 group and NS5 for NS4 group), control antigens, SOD, SDS extract, E. coli extract, etc. were added in 5–12 replicates. Each well was supplemented with irradiated autologous PBMCs (1 x 105 per well) and cultured for another 5 days. At the end of 5 days, 0.5 µCi per well [3H]-TdR was added, followed by harvesting the cells on the day 6 and counting the levels of [3H]-TdR incorporation into the cellular DNA.

Cytokine secretion
Detection of secreted cytokines was carried out using IL-10, IFN-{gamma}, IL-6, IL-2, transforming growth factor-ß (TGF-ß) and tumor necrosis factor-{alpha} (TNF-{alpha}) sandwich ELISA kits purchased from Biosource International Inc. The assay was performed as outlined in the instructions. A dilution of 1:5 to 1:20 was used for the samples and the standards ranged from 15.6 to 2000 pg ml–1.

Statistical analyses
Statistical analyses were done by Tukey's test, using SPSS 11.5 software.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 Funding
 References
 
Endogenous expression of HCV-derived NS4, NS5a or NS5b proteins in human DCs
The recombinant adenovirus vectors containing HCV-derived NS4, NS5a, NS5b or control (Ad/LacZ) were incubated at 50–200 MOI with human monocyte-derived immature DCs (iDCs) for 24 h followed by stimulation with 100 ng ml–1 LPS to mature the DCs. In our previous studies, we found that an MOI of 100 provides the best efficiency of gene expression through adenoviral transfer (30), and therefore, 100 MOI was used in all the experiments described below. The DCs infected with recombinant adenoviral vectors had mRNA for respective HCV antigens (NS4, NS5a or NS5b) (data not shown).

We determined the percentage of DCs expressing HCV NS4, NS5a or NS5b in our experiments by multicolor immunofluorescence staining. As shown in the Fig. 1, among both mature and iDC populations the DAPI staining corresponded almost 100% to HLA-DR staining and overlapped completely with anti-NS4, anti-NS5a or anti-NS5b staining, suggesting that ~100% of DCs were infected with the recombinant adenovirus vectors expressing HCV NS4, NS5a or NS5b. The Green fluorescent protein (GFP) expression was significantly dimmer than HLA-DR-FITC staining (data not shown). DCs infected with control adenovirus vector were used as controls. These did not stain with anti-NS4, anti-NS5a or anti-NS5b antibodies, but did show staining with anti-HLA-DR and DAPI (data not shown).


Figure 1
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  		Fig. 1. Immunofluorescence staining of DCs expressing NS4, NS5a and NS5b antigens. Single staining with DAPI, HLA-DR-FITC and anti-NS4, NS5a or NS5b antibodies and overlap of the three colors are shown. The DAPI stains the nucleus, HLA-DR is used as a marker for DCs and anti-NS4, anti-NS5a and anti-NS5b are used to locate the HCV antigen expression.

 
Cytokine induction in human DCs expressing HCV-derived NS4, NS5a or NS5b proteins
DCs are sentinels of the immune system and respond to external stimuli, antigens, viruses and bacteria by inducing cytokine expression to stimulate or anergize naive T cells against specific antigens. The induction of cytokines TNF-{alpha}, IFN-{gamma}, IL-12p40, IL-10, IL-2 and IL-6 was determined by real-time reverse transcription (RT)–PCR assays from both iDCs and mature DCs (mDCs) expressing HCV NS4, NS5a or NS5b proteins (Fig. 2).


Figure 2
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  		Fig. 2. Real-time RT–PCR analyses of induction of cytokine mRNAs in DCs expressing HCV NS4, NS5a or NS5b antigens. The data represent three repeated experiments with different donors.

 
In the iDCs, the expression of HCV antigens did not induce IFN-{gamma}, IL-6, IL-12 or IL-10 compared with control vector-infected DCs. In contrast, expression of HCV NS4, but not NS5a or NS5b led to a significant increase in IL-2 mRNA expression in iDCs. HCV NS4, NS5a and NS5b expression induced TNF-{alpha} in iDCs. Also IL-10 induction was reduced upon expressing HCV NS5a and NS5b, but not NS4 in these cells. Upon maturation with LPS for 4 h, the overall levels of TNF-{alpha}, IL-12p40 and IL-6 were significantly higher compared with iDCs. In mDCs expressing NS4, TNF-{alpha} and IL-6 were not induced above background levels (control vector). In the mDCs expressing NS5b, TNF-{alpha}, IFN-{gamma}, IL-12p40, IL-2 and IL-6 were increased significantly compared with either the control vector or NS4 or NS5a (Fig. 2). IL-10 mRNA was not increased significantly in mDCs expressing NS5b compared with DCs expressing control vector (Fig. 2), in contrast to DCs expressing NS4 and NS5a. Therefore, the expression of HCV-derived proteins NS4, NS5a or NS5b in DCs led to differential induction of various cytokines, which could affect downstream events and the ability of DCs to stimulate naive antigen-specific T cells.

Phenotype analysis of iDCs and mDCs expressing HCV-derived NS4, NS5a or NS5b proteins
We determined the expression of various lineage, maturation and co-stimulation markers expressed by DCs to investigate whether the expression of HCV antigens NS4, NS5a or NS5b leads to modulation in DC phenotype which could affect T-cell stimulation (Fig. 3). Among the iDCs, CD11c was expressed on >96% of uninfected DCs indicating the myeloid DC lineage of these cells. Upon infection with control vector, NS4, NS5a or NS5b recombinant adenoviral vectors, CD11c was not altered (data not shown). Other activation markers for DCs such as CD86, CD40 and HLA-DR were significantly up-regulated on DCs matured with LPS. However, in both the iDCs or mDCs, the expression of these markers was not altered upon infection with CV or the recombinant adenoviral vectors containing HCV NS4, NS5a or NS5b genes. The expression of HLA class I molecules, as detected by a pan anti-class I antibody (HLA-A,B,C) was also not altered upon expression of NS4, NS5a or NS5b in both iDCs and mDCs. DC-specific C-type lectin, DC-SIGN and the macrophage mannose receptor homolog, DEC-205 (32, 33), were also examined in iDCs and mDCs expressing HCV NS4, NS5a or NS5b antigens or control vector. Expression of both of these DC markers was not altered when various HCV antigens were expressed. The culture incubation time for the phenotype analysis was the same as the immunofluorescence experiments. Therefore, at the time of phenotype analysis, the HCV proteins were being expressed by almost all the DCs.


Figure 3
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  		Fig. 3. Phenotypic analyses and comparison of uninfected DCs, control vector-infected DCs, rAd-NS4-infected DCs, rAd-NS5a-infected DCs and rAd-NS5b-infected mDCs. The data represent three repeated experiments.

 
Stimulation of allogeneic T cells by iDCs and mDCs expressing HCV-derived NS4, NS5a or NS5b proteins
Allogeneic T-cell stimulation by limiting numbers of DCs is used as a hallmark of DCs' function as professional APCs. The ability of iDCs and mDCs expressing HCV NS4, NS5a or NS5b to stimulate allogeneic T-cell proliferation was determined. Varying DC:T ratios (1:200, 1:100, 1:40 and 1:20; i.e. 1000, 2000, 5000 and 10 000 DCs per well) were used in these studies. iDCs expressing NS4, NS5a, NS5b or control vectors stimulated allogeneic T cells to proliferate less than the mDCs (data not shown). The expression of NS4, NS5a or NS5b in DCs did not have a significant effect on allo-T-cell stimulation by iDCs or mDCs. Therefore, the DCs expressing HCV NS4, NS5a or NS5b proteins have a normal capability to stimulate allogeneic T-cell proliferation.

Uninfected healthy donors' T cells proliferate upon stimulation with autologous DCs endogenously expressing HCV-derived NS4, NS5a or NS5b antigens
In order to examine if naive T cells from uninfected donors can be stimulated in vitro to proliferate against HCV-derived NS4, NS5a or NS5b antigens, the iDCs or mature (with 100 ng ml–1 LPS) DCs expressing NS4, NS5a or NS5b antigens were cultured with autologous T cells for 5 days at various DC:T cell ratios (1:200 to 1:20). As controls, uninfected DCs or control-vector infected DCs were used as antigen-negative DCs (Fig. 4). T-cell proliferation was determined as a measure of T-cell stimulation. The data from three representative donors are shown individually in Fig. 5. These experiments were done with T cells obtained from nine individual donors and out of these, HCV antigen-dependent T-cell responses were obtained from eight donors. After 5 days of culture, in vitro NS5a- or NS5b-specific proliferation seemed to be significantly higher in all the donors when compared with negative control groups. Upon stimulation with mDCs expressing NS5a or NS5b, proliferation was higher than upon stimulation with iDCs expressing HCV NS5a or NS5b. The proliferation against NS5a or NS5b was significantly higher than against control vector-infected DCs (P < 0.05 at DC:T cell ratios of 1:100, 1:40 and 1:20, in all three donors). The antigen-specific proliferative response against NS4 appeared to be much lower than that against NS5a or NS5b antigens. In most instances, NS4-specific proliferative response was not apparent when iDCs were used to stimulate T cells (data not shown). Upon using mDCs expressing NS4, T-cell proliferation was still not observed above background (at all DC:T ratios) (P > 0.1)).


Figure 4
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  		Fig. 4. Primary stimulation of naive autologous T cells by mDCs expressing HCV antigens NS4, NS5a or NS5b. The data are shown with three individual donors' T cells and represent eight repeated experiments from individual donors.

 

Figure 5
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  		Fig. 5. Primary proliferative response of naive autologous CD4+ and CD8+ T cells upon stimulation by DCs expressing HCV antigens NS4, NS5a or NS5b. The top panel shows stimulation of CD4+ T cells and the bottom panel shows stimulation of CD8+ T cells with iDCs and mDCs expressing HCV antigens. The data shown are representative of three repeated experiments with individual donors.

 
The experiments described in Fig. 4 were done with bulk non-adherent cells (comprised of ~85% T cells). In order to determine specifically whether CD4+ or CD8+ T cells are proliferating in response HCV antigens, these T cells were purified from non-adherent cells using magnetic columns (>97% purity) and cultured for 5–6 days with autologous immature or mature autologous DCs expressing NS4, NS5a or NS5b antigens (Fig. 5). In these experiments, uninfected or control vector-infected DCs were used as controls. DCs uninfected or infected with various adenoviral vectors and cultured by themselves, or purified CD4+ and CD8+ T cells cultured alone, provided background counts per minute (CPMs) (<500 CPM). Upon stimulating purified CD4+ and CD8+ T cells by autologous iDCs or mDCs expressing HCV antigens NS4, NS5a or NS5b, significantly higher antigen-specific proliferation was observed (Fig. 5). With purified CD4+ T cells, the response to NS4, NS5a and NS5b was in the order of NS5a > NS5b > NS4 with both iDCs and mDCs. With purified CD8+ T cells, no significant antigen-specific T-cell proliferation was observed with NS4-expressing DCs, but with both NS5a and NS5b significant antigen-dependent proliferation was observed at higher DC:T cell ratios (1:20). The proliferation was significantly higher with CD4+ T cells than with CD8+ T cells. When comparing the antigen-dependent proliferation via stimulation indices (SI = CPM in the presence of antigen/CPM in the absence of antigen or control vector-infected DCs), it was observed that even in the primary responses, SI of 4–34 was observed with CD4+ T cells and SI of 1.5–5.5 were obtained with CD8+ T cells stimulated with NS5a or NS5b at various DC:T cell ratios. With NS4, however, with CD4+ T cells, SI of 3.6–12 were obtained and SI of <1.5 was obtained with CD8+ T cells.

DCs expressing HCV-derived NS4, NS5a or NS5b antigens stimulate various cytokines in autologous CD4+ and CD8+ T cells from HCV-naive individuals
The mDCs expressing NS4, NS5a, NS5b, control vector or uninfected were used in limiting numbers to stimulate autologous T cells (non-adherent cells) from HCV-naive individuals. After 24 h co-culture, the cells were harvested, CD4+ and CD8+ T cells were purified by magnetic columns and then mRNA was isolated, followed by RT to cDNA which was then used to run real-time PCR to quantitate the message for various cytokines such as IFN-{gamma}, TNF-{alpha}, IL-2, IL-6 and IL-10 (Fig. 6).


Figure 6
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  		Fig. 6. Primary stimulation of naive autologous CD4+ or CD8+ T cells by mDCs expressing HCV antigens NS4, NS5a or NS5b leads to TH1 cytokine mRNA expression in antigen-dependent manner. The data are representative of two repeated experiments.

 
CD4+ and CD8+ T cells demonstrated a distinct pattern of cytokine mRNA induction in response to stimulation by autologous DCs expressing NS4, NS5a and NS5b antigens. From the CD4+ T cells, IFN-{gamma}, TNF-{alpha} and IL-6 were shown to be induced in response to all three antigens relative to control vector, although there were quantitative differences in response to different antigens (Fig. 6). Induction of IL-2 mRNA in CD4+ T cells was only observed in NS4-stimulated CD4+ T cells but not in NS5a- or NS5b-stimulated CD4+ T cells. IL-10 mRNA was not induced in CD4+ T cells stimulated with any of the three HCV antigens.

Interestingly, NS4 did not induce IFN-{gamma}, IL-2 or IL-6 above background in CD8+ T cells, whereas NS5a and NS5b stimulated significantly high levels of mRNA for both IFN-{gamma} and IL-2. TNF-{alpha} mRNA was induced in response to all three HCV antigens above background. IL-10 was not induced significantly above background in CD8+ T cells stimulated with DCs expressing any of the three non-structural HCV antigens examined (Fig. 6).

We also measured cytokines secreted in the supernatants of CD4+ and CD8+ T lymphocytes incubated for 4 days with DCs expressing HCV antigens NS4, NS5a and NS5b (Fig. 7A and B). High levels of antigen-dependent IFN-{gamma} and TNF-{alpha} were observed from CD4+ T cells stimulated by NS4, NS5a or NS5b with both iDCs and mDCs, relative to control DCs. IL-2 was only observed in the CD4+ T cells stimulated with iDCs but not mDCs. Also, IL-2 was not induced much above the background DCs expressing control vector. IL-6 was only detected in CD4+ T cells stimulated by mDCs in an antigen-dependent manner, with background levels being negligible.


Figure 7
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  		Fig. 7. Primary stimulation of naive autologous CD4+ or CD8+ T cells by DCs expressing HCV antigens NS4, NS5a or NS5b leads to TH1 cytokine production in an antigen-dependent manner. Panel (A) represents cytokine production from CD4+ T cells and panel (B) represents cytokine production from CD8+ T cells.

 
Both IFN-{gamma} and TNF-{alpha} were not detected in the supernatants from the purified CD8+ T cells stimulated with iDCs or mDCs expressing HCV antigens NS4, NS5a and NS5b (Fig. 7B). Interestingly, IL-2 was present in the supernatants of control CD8+ T cells but was not increased in the supernatants of CD8+ T cells stimulated with DCs expressing HCV antigens. IL-6 secretion also did not appear in an antigen-dependent pattern in CD8+ T cells except those stimulated with DCs expressing NS5a. These showed above background values compared from mDCs with vector alone. IL-10 was not detectable in any of the CD8+ T cell cultures stimulated in vitro with DCs expressing various HCV antigens.

Activated effector cytotoxic CD8+ T cells are stimulated in response to autologous DCs expressing HCV-derived antigens
In order to determine whether DCs expressing HCV NS4, NS5a or NS5b antigens are leading to CD8+ T-cell activation, resulting in armed effector CTL, intracellular GrB expression was determined in CD8+ T cells after stimulation with autologous DCs expressing HCV NS4, NS5a and NS5b antigens (Table 1). Purified CD8+ T cells stimulated with DCs expressing various HCV antigens or control vector were cultured for 5 days, followed with four-color staining for CD8, CD11b, CD28 and intracellular GrB. Compared with control DCs, CD8+ T cells stimulated by DCs expressing HCV antigens showed a 2-, 5- and 3-fold increases in the CD28–GrB+CD8+CD11b+ T cells, in response to HCV NS4, NS5a and NS5b, respectively. This suggests that DCs expressing these HCV antigens are able to activate armed effector cytotoxic T cells.


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  		Table 1. Effector phenotype of CD8+ T cells stimulated by DCs expressing HCV antigens NS4, NS5a or NS5b

 
T cells primed in vitro by autologous DCs expressing HCV NS4, NS5a or NS5b antigens proliferate in an antigen-specific manner in secondary cultures
The T cells primed in vitro in limiting dilution cultures by autologous DCs expressing HCV NS4, NS5a or NS5b were re-stimulated with autologous irradiated PBMCs and the respective NS4, NS5a or NS5b recombinant protein antigens. These experiments were done by replica plating (22), so we could examine the NS4-, NS5a-or NS5b-specific responses as compared with no antigen or irrelevant antigens. In addition, recombinant NS5 protein was used as a negative specificity control antigen in NS4-stimulated cultures and vice versa. As shown in Fig. 8(A), the second re-stimulation showed NS4-, NS5a- or NS5b-specific responses in individual wells, which was statistically significant for both NS5a and NS5b antigens (P < 0.05). However, for NS4 antigen, there was no antigen-specific proliferative response (Fig. 8A). In fact, upon adding NS4 proteins to the cultures, the CPMs were significantly reduced compared with controls with no antigen. As controls, we also stimulated T cells with uninfected or control vector-treated DCs for 6 days followed by re-stimulation with no antigen, NS4, NS5 or control antigens (data not shown). In both of these control cultures, the proliferation in response to HCV NS4, NS5a or NS5b recombinant proteins was lower than the no-antigen control wells, and the average CPMs of all the wells was also lower than the no antigen or control groups. As controls, we also stimulated naive T cells in primary cultures with DCs loaded with recombinant protein antigens and did not observe antigen-specific proliferation (data not shown).


Figure 8
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  		Fig. 8. (A) NS4-, NS5a- or NS5b-specific T-cell proliferation in secondary cultures. The bottom three panels show average CPMs from 25 wells of the top three graphs. The data are representative of two repeated experiments from two different donors. (B) Purified CD4+ T-cell proliferation in secondary cultures with NS4, NS5a or NS5b recombinant protein antigens. The antigen-specific proliferation was obtained by subtracting the background CPMs (no antigen) from the experimental CPMs of the replica well. The bottom three panels show average CPMs (standard error is shown as error bar) from 48 wells of the top three graphs. The data are representative of two repeated experiments from two different donors.

 
In addition to determining antigen-specific T-cell responses against recombinant protein antigens in whole T cell culture as shown in Fig. 8(A), CD4+ T cells were purified and stimulated with autologous DCs expressing NS4, NS5a, NS5b or control vector, followed by a replica-plating experiment to determine antigen-specific proliferation of CD4+ T cells (Fig. 8B). Recombinant protein antigen-specific proliferation was evident in the purified CD4+ T cells, in the secondary cultures with all three HCV antigens tested. This contrasted with the unpurified T-cell response, where NS4 protein showed inhibition.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 Funding
 References
 
Strong CD4+ and CD8+ T-cell responses specific to HCV have been found to be associated with viral clearance. In contrast, weak T cell immunity has been correlated with progression to persistent chronic infection. Understanding human T-cell responses against various HCV antigens is therefore of utmost importance in understanding host protective immune responses to HCV and for design of immunotherapeutic approaches including vaccine candidates. The non-structural proteins of HCV are conserved proteins, and peptides derived from them have been shown previously to be targets of CD4+ and CD8+ T-cell recognition (13). In addition, broad, vigorous and multi-epitope-specific T cells have been shown to be important in controlling primary HCV infection (2). Therefore, understanding human T-cell responses against each of the HCV antigens is fundamental to understanding overall immunity against HCV. In the current study, we have focused on HCV-derived non-structural antigens NS4, NS5a and NS5b.

First, we determined whether human DCs endogenously expressing HCV antigens NS4, NS5a and NS5b are functionally and/or phenotypically normal and can efficiently stimulate T cells, so as to use them as antigen expressing autologous APCs for in vitro priming of naive T cells. Using recombinant adenoviral vectors, 100% of the iDCs and mDCs were infected and expressed HCV NS4, NS5a or NS5b proteins, as indicated by immunofluorescence (Fig. 1). These initial experiments not only verified our ability to efficiently express HCV NS4, NS5a and NS5b proteins in human DCs but also suggested that the expression of these HCV proteins does not lead to cell death or toxicity to cells.

DCs initiate T-cell responses mainly by three mechanisms, production of cytokines, provision of co-stimulatory molecules and stimulation of T cells. In our studies, we examined all these three functions of DCs expressing HCV NS4, NS5a or NS5b proteins. Our results demonstrated that HCV-derived antigens NS4, NS5a or NS5b are able to induce pro-inflammatory cytokines from human iDCs as well as mDCs. This initial induction of cytokines by the DCs would be very important in priming and initiating T-cell responses against these antigens. The mRNA copy number of IL-12p40 was the highest in control iDCs suggesting that the expression of HCV proteins in iDCs possibly leads to down-regulation of induction of TH1 responses by DCs. Alternatively, a difference in kinetics of the mRNA induction could be responsible for this observation. In contrast, IL-10 was reduced in NS5a and NS5b expressing iDCs suggesting that these antigens induce the differentiation of tolerogenic iDCs to stimulatory DCs. In the DC population, there were some contaminating lymphocytes (<5%) which may also contribute to the measured cytokine mRNAs. However, in all these experiments, there was a clear distinction in the induction of cytokine expression by the CV which was significantly lower than that of the HCV antigens, suggesting an HCV antigens' dependent induction of inflammatory cytokines. The exact mechanism of how these HCV antigens induce the expression of these cytokines from DCs remains to be determined.

Upon examining the phenotype of mDCs expressing HCV NS4, NS5a or NS5b antigens, we did not observe significant differences in expression of CD11c or CD80 (data not shown), CD86, CD40, DEC-205, DC-SIGN, HLA class I and class II (Fig. 3). Taken together, these results suggest that expression of these HCV-derived antigens in DCs does not alter them phenotypically, change their ability to provide co-stimulation to T cells or negatively affect their antigen-presenting capability. Upon stimulation with LPS, all these markers were up-regulated to approximately the same extent in DCs whether or not they were expressing HCV antigens. However, it is possible that a stimulus such as dsRNA or poly I:C-mediated maturation of DCs may be affected by these HCV antigens, as the virus may have evolved to evade these signaling pathways. In agreement with the results obtained from the DC phenotype analysis and cytokine induction, the allogeneic T-cell stimulation by DCs expressing NS4, NS5a or NS5b antigens was not reduced compared with the control DCs whether infected or not (data not shown).

The results obtained with human DCs expressing HCV NS4, NS5a or NS5b antigens thus suggested that these DCs are normal in phenotype and fully functional and could potentially prime and stimulate naive T cells against these HCV antigens.

Next, we used the DCs expressing HCV antigens to stimulate autologous T cells in vitro. As an initial measure of T-cell activation, unpurified mixed T cells were stimulated in vitro with autologous DCs expressing HCV antigens NS4, NS5a or NS5b (Fig. 4). In each of the donors tested, there was antigen-dependent proliferation, compared with control vector or uninfected DCs. DCs are known to present both endogenous and exogenous antigens (34). However, stimulation with CV-infected DCs was much lower than the ones expressing NS5a or NS5b antigens, suggesting that adenoviral antigens may be much less stimulatory than HCV antigens NS5a and NS5b, but not NS4 because NS4-specific proliferation was not much higher than control vector. It is also possible that due to frequent exposure to adenovirus in humans, induction of T-cell tolerance occurs against adenoviral antigens.

In our initial T-cell stimulation experiments (Fig. 4), bulk, non-adherent cells were used as T cells. In order to determine whether CD4+ and CD8+ T cells are proliferating in responses to autologous DCs expressing HCV antigens NS4, NS5a or NS5b, we purified CD4+ or CD8+ T cells from non-adherent cells using MACS. The purified T cells were then stimulated followed by examination of proliferation (Fig. 5). CD4+ T cells exhibited significantly more proliferation than CD8+ T cells. With all three antigens, using iDCs or mDCs, there was a highly significant antigen-dependent proliferation in CD4+ T cells compared with those stimulated by control vector-infected DCs. In the CD8+ T-cell proliferation, interestingly, there was significant proliferation against NS5a and NS5b when higher numbers of DCs were used (10 000 cells per well), whereas there was no proliferation in CD8+ T cells in response to NS4 (Fig. 5). It is possible that stimulation of CD8+ T cells against NS4 requires TH help in order to be optimum, or it is possible that NS4 is not able to stimulate and/or expand CD8+ T cells by itself.

After obtaining convincing evidence of T-cell proliferation with CD4+ and CD8+ T cells, we used real-time RT–PCR to examine the mRNA induction of various cytokines (IFN-{gamma}, TNF-{alpha}, IL-2, IL-6 and IL-10) in these primary stimulated CD4+ and CD8+ T cells (Fig. 6). These experiments were done in a way that cytokine induction in CD4+ or CD8+ T cells could be determined independent of induction of cytokines in DCs.

Induction of IFN-{gamma} mRNA in both CD4+ and CD8+ T cells correlated well with relative proliferation of these cells against each of the HCV antigens NS4, NS5a or NS5b. From the CD8+ T cells stimulated with DCs expressing NS4, IFN-{gamma} was not induced above background control, consistent with proliferation data. This is an interesting observation. In agreement with IFN-{gamma} data, IL-6 was also not induced in NS4-stimulated CD8+ T cells, whereas NS5a- and NS5b-stimulated CD8+ T cells had high levels of IL-6 mRNA. With CD4+ T cells, however, all three of the HCV antigens induced IL-6 mRNA in an antigen-dependent manner. Induction of TNF-{alpha} was notably different for NS4, in that TNF-{alpha} mRNA was induced in both CD4+ and CD8+ T cells upon stimulation with DC expressing NS4, NS5a or NS5b. IL-2 mRNA induction presented a different pattern. NS4 induced IL-2 mRNA in CD4+ T cells, but not CD8+ T cells. IL-2 mRNA induction was reduced in NS5a- or NS5b-stimulated CD4+ T cells as compared with control vector. It is possible that kinetics of IL-2 mRNA induction was different with more stimulatory NS5a and NS5b antigens. Alternatively, it is possible that after antigen priming, the naive T cells which mainly produced IL-2 differentiate into antigen-specific effector cells producing cytokines such as IFN-{gamma}. IL-2 mRNA was increased in CD8+ T cells in response to HCV NS5a or NS5b. With all three HCV antigens, IL-10 expression was not induced in CD4+ or CD8+ T cells in an antigen-dependent manner.

We also measured cytokines secreted in the supernatant by CD4+ and CD8+ T cells stimulated by iDCs or mDCs expressing HCV antigens NS4, NS5a or NS5b (Fig. 7A and B). CD4+ T cells appeared to produce higher amounts of type 1 cytokines than CD8+ T cells, and NS5a induced the maximum cytokine production compared with NS4, NS5b or control vector. From the CD8+ T cells, IFN-{gamma} and TNF-{alpha} could not be detected from any sample because these cytokines were produced in very minute quantities, were not produced at all or were used up by the cultures. In conclusion, the determination of cytokine production by both the mRNA levels and by secreted proteins concentration, provided conclusive evidence of antigen-specific priming of both CD4+ and CD8+ T cells in the primary in vitro cultures. In a few instances (Figs 6 and 7), the mRNA copy number and cytokine protein levels in the supernatant did not correspond to each other. This could be due to differential kinetics of induction of cytokine mRNA in different groups, differential stability of mRNA and/or degradation or use of cytokine proteins by the cell cultures.

CD8+ T cells are effector cytotoxic T cells. In the preceding experiments (Figs 5, 6 and 7), we demonstrated evidence of antigen-specific CD8+ T-cell proliferation and cytokine induction, especially in T cells stimulated with NS5a and NS5b but not with NS4. We then determined whether the primary stimulation of CD8+ T cells led to stimulation of armed antigen-specific effector cytotoxic T cells. CD8+CD11b+ cells were gated for activated cytotoxic T cells and analyzed for CD28– and GrB+ to examine the population of armed effector cytotoxic T cells. There was a significant increase (2.4-, 4.7- and 3-fold increase, respectively) in CD28–GrB+CD8+CD11b+ T cells upon stimulation with DCs expressing NS4, NS5a or NS5b compared with the control vector (Table 1). Interestingly, CD8+ T cells stimulated by NS5a exhibited maximum increase in GrB+ cells. These results suggest that HCV antigen-dependent stimulation of armed effector CD8+ cytotoxic T cells occurred after in vitro priming. Granzymes have been shown to be essential molecules for effector cytotoxic T cells capable of controlling primary virus infection (35) and may play a role in protection against HCV infection.

In our next experiments, we were able to identify antigen-specific T cell proliferation in secondary cultures (Fig. 8A and B). For these experiments, the DCs expressing HCV NS4, NS5a or NS5b antigens were used in the priming cultures and the irradiated autologous PBMCs, along with recombinant proteins or control antigens, were used as APCs in the secondary cultures. Since it is expected that the frequency of antigen-specific T cells would be very low in HCV-naive individuals, we performed these experiments in replica-plating cultures (22). The proliferative responses of T cells in replica-plating experiments provided conclusive evidence of HCV antigen NS5a- and NS5b-specific T-cell proliferation in the primary in vitro cultures and also in vitro priming of T cells against HCV antigens NS5a and NS5b using autologus DCs expressing these HCV antigens. However, in the secondary cultures the NS4 protein antigen did not show any proliferative response, and in fact the proliferation was lower than the controls with no peptide or irrelevant antigens. For NS4 protein antigen, three different recombinant proteins were obtained and they all showed the same results. It is possible that NS4 protein is not efficiently taken up, processed and/or presented by PBMCs, or it is possible that NS4 protein inhibits specific APC or T-cell functions leading to inhibition in the background proliferation. However, upon using purified CD4+ T cells (Fig. 8B), significant antigen-specific proliferation was obtained suggesting that NS4 is able to be taken up, processed and presented by APCs to stimulate antigen-specific CD4+ T cells primed in the primary cultures. It is possible that in bulk, unpurified T-cell cultures, NS4 is inhibiting a specific cell type or inducing them to produce inhibitory factors influencing the overall proliferative response. We examined the production of TGF-ß and IL-10 in the NS4-stimulated cultures and did not observe an increase in their secretion in the unpurified T cells (data not shown). It is possible that the impaired response to NS4 protein in the unpurified T-cell population is related to lack of proliferative response of CD8+ T cells in the primary culture (Fig. 5). We are currently investigating the mechanism behind this observation.

In this paper, we describe human CD4+ and CD8+ T-cell responses from HCV-naive (uninfected) individuals specific to HCV antigens NS4, NS5a and NS5b. It could be argued that the observation of HCV antigen-specific T-cell stimulation from naive uninfected individuals is due to cross-reactivity with antigens from other pathogens as suggested previously (36–38). However, this does not appear to be the case because, in contrast to our studies, recombinant HCV antigen-specific proliferation has not been demonstrated (36–38). Our studies with the generation of T cells specific for HCV antigens from individuals naive to HCV represent early events in the induction of cellular immune responses against HCV which most likely play a decisive role in the outcome of infection. These studies have advantages over previous reports (4–8, 10) because they explore anti-HCV T-cell activity generated in healthy, immunocompetent individuals in response to HCV antigens presented by professional APCs. This study eliminates the use of T cells from HCV-infected people, which could be defective and/or modulated. In addition, by using T cells and DCs from healthy individuals, the potential of the T-cell repertoire unaffected by long-term presence of HCV in the body can be examined. These studies have huge potential for the investigation and development of prophylactic as well as immunotherapeutic vaccines for HCV infection. In addition, because the DCs expressing HCV antigens are able to prime the antigen-specific T cells in vitro, they will have the potential to be harnessed as cellular vaccines or as T-cell adoptive transfer therapy in vivo in both prophylactic and therapeutic settings. HCV is a worldwide health problem and therefore protective and/or immunotherapeutic vaccines need to be developed as soon as possible.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
Funding
 References
 
Supplementary Figure is available at International Immunology Online.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
References
 
Canadian Institutes of Health Research (EOP 79327); Alberta Heritage Foundation for Medical Research; Canadian Foundation for Innovation New Opportunities to B.A.; Canadian Vaccine Network to B.A. and D.L.J.T.


   Acknowledgements
 
Thanks to Jens Bukh for providing H77 HCV plasmid. The authors sincerely thank Mark Peppler for critically reviewing the manuscript. B.A. is a recipient of Senior Scholar Award from the Alberta Heritage Foundation for Medical Research.


   Abbreviations
 
APC, antigen-presenting cell
aa, amino acid
CPM, counts per minute
CV, control adenoviral vector
DAPI, 4',6-diamidino-2-phenylindole
DC, dendritic cell
GFP, Green fluorescent protein
GM-CSF, granulocyte macrophage colony-stimulating factor
GrB, Granzyme B
HCV, hepatitis C virus
[3H]-TdR, [3H]-thymidine
iDC, immature DC
LPS, lipopolysaccharide
MACS, magnetic affinity column separation
mDC, mature DC
MOI, multiplicity of infection
RT, reverse transcription
SI, stimulation index
SOD, superoxide dismutase
TGF-ß, transforming growth factor-ß
TNF-{alpha}, tumor necrosis factor-{alpha}

   Notes
 
* These authors contributed equally to this study. Back

Transmitting editor: A. Falus

Received 13 June 2007, accepted 17 October 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 

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