Abstract
Differences in cytotoxic T lymphocyte activity in hepatitis C virus infection may account for the outcome of interferon monotherapy. To investigate this hypothesis, we analysed the response of peripheral CD8+ T cells that recognized epitopes presented by HLA-A*2402. We synthesized HLA/β2-microglobulin/peptide complexes using two epitopes. Production of interferon-γ by CD8+ T cells in response to plastic-bound monomeric HLA/peptide complex was observed frequently in sustained virus responders (SVR) (n = 13) against all the peptides, NS31296–1304 (the percentage of responding patients, 61·5%) and core 129–137 (53·8%), while no interferon-γ production was observed in non-responders (NR) (n = 13) for any of the peptides. Tetramer-staining showed the presence of CD8+ T cells specific for all the peptides except NS31296–1304 in two SVR at the end of interferon monotherapy, although hardly any such cells were found in four NR. Specific killing was observed against peptides NS31296–1304 (3/4) and core 129–137 (1/4) in sustained responders but none in non-responders. These results suggest that the responses of cytotoxic T lymphocytes (CTLs) were induced during interferon therapy in these patients and that interferon-γ production by CD8+ T lymphocytes against HCV NS31296–1304 and core 129–137 are well maintained in patients with SVR compared with those with NR. These findings emphasize the importance of the CD8+ T cell response in controlling HCV infection.
Keywords: cytotoxic, hepatitis C, HLA-A24, interferon-γ, T lymphocytes
Introduction
Hepatitis C is recognized as a major cause of chronic liver disease and hepatocellular carcinoma in Japan [1]. The majority of patients with acute infection with hepatitis C virus (HCV) develop chronic hepatitis [2]. Although the detailed mechanisms by which HCV is eliminated from patients are currently unknown, it has been suggested that T cell response plays a role in HCV elimination [3].
In patients with self-limited infection, the activity of cytotoxic T lymphocytes (CTLs) has been shown to recognize multiple epitopes [4,5]. However, loss of a virus-specific CD4+ T cell response [6] or CD8+ CTLs leads to persistent infection. In patients with chronic hepatitis C, the activity of CTLs has been demonstrated in the peripheral blood of patients with low-level viraemia [7], and responses of cellular immunity to HCV antigens have been shown in sustained viral responders to interferon (IFN) therapy [8,9]. These observations suggest a possible role of CTLs in the inhibition of HCV replication in chronic hepatitis C. Spontaneous eradication of HCV is estimated as infrequent in patients with chronic hepatitis C [10], and restoration of CTL activity against HCV antigen with therapy may be a rational approach for the elimination of the virus. To investigate this hypothesis, we evaluate the activity of CTLs in patients treated with IFN monotherapy.
To date, surveys of CTL activity in hepatitis C have been focused on the HLA-A*0201 allele, while the most common HLA-A allele in the Japanese population is A*2402 [11,12]. In this study we found that the activity of CTLs in peripheral blood and two recognized epitopes of HCV protein presented by HLA-A*2402 of five candidate epitopes that carry HLA-A*2402 peptide-binding motif bind well to the HLA-A*2402 molecule. Two epitopes were recognized by CTLs in patients in whom HCV had been eliminated and also stimulated CD8+ T cells to produce IFN-γ. These CTLs may contribute to the eradication of HCV by IFN monotherapy.
Methods
Subjects
Heparinized blood was obtained from 26 HLA-A24 positive patients with chronic hepatitis C (Table 1). As control subjects, four HLA-A24 negative patients with chronic hepatitis C and nine HLA-A24 positive healthy volunteers were also included. Chronic hepatitis C was diagnosed when there were elevated levels of serum alanine aminotransferase (ALT) for more than 6 months, and when anti-HCV antibody was detected (Third Generation Test, Ortho Diagnostics, Chicago, IL, USA) in the serum. Thirteen patients manifested a sustained virus response to IFN monotherapy, while the remaining 13 patients were non-responders (Table 1). Production of IFN-γ was tested at 6–36 months after the end of IFN therapy. Peripheral blood was also obtained from nine uninfected healthy volunteers who were negative for anti-HCV antibody. Blood samples were taken from individuals after written consent was obtained. Ethical permission was obtained from the Ethical Committee at Tohoku University School of Medicine. A liver biopsy showing active necroinflammatory changes in the liver was obtained from all patients.
Table 1. Characteristics of patients with chronic hepatitis C and healthy volunteers.
| Characteristics | SVR | NR | Healthy | |
|---|---|---|---|---|
| Number of subjects | 13 | 13 | 9 | |
| Age (years) | 51 (36–67) | 49 (44–66) | n.s. | 35 (28–55) |
| Gender (M/F) | 7/6 | 7/6 | n.s. | 2/7 |
| Months after IFN | 18 (6–36) | 24 (6–36) | n.s. | – |
| AST (IU/l) | 19 (14–41) | 39 (16–107) | P < 0·005 | – |
| ALT (IU/l) | 21 (10–87) | 49 (15–191) | P < 0·005 | – |
| HCV genotype 1b : 2a : 2b* | 7:5:1 | 7:3:3 | n.s. | – |
| HCV RNA (kcopy/ml)* | 58 (<50–470) | 88 (<50–850) | P < 0·05 | – |
Numbers indicate median and ranges are shown in parenthesis. Hepatitis C virus (HCV) genotypes and viral load are obtained before the therapy. SVR: sustained virus responders; NR: non-responders; IFN: interferon.
Patients received 24 weeks of IFN-α treatment (IFN-α2b or natural IFN-α). All patients were followed for 24 weeks after completion of the treatment to determine whether there was a sustained viral response.
Tissue typing
Subjects were first screened for HLA class I phenotype using a conventional microlymphocytotoxicity test, and then the HLA genotype was determined by amplification mutation system (ARMS)-polymerase chain reaction (PCR) using sequence-specific oligonucleotide primers [13].
Synthetic peptides
Five 9-mer peptides (HCV E2719–727: EYVLLLFLL, HCV NS31296–1304: TYSTYGKFL, HCV NS52871–2879: CYSIEPLDL, HCV core 85–93: LYGNEGLGW, HCV core 129–137: GFADLMGYI, HCV core 173–181: SFSIFLLAL) were synthesized commercially by Research Genetics (Huntsville, AL, USA). They had the HLA-A*2402-binding motif of Y or F at position 2 and L, I or W at position 9 [14]. The purity and integrity of the synthesized peptides were confirmed by HPLC and mass spectrometry profiles.
CD8+ T cell separation
Peripheral CD8+ T cells were prepared by positive selection using the MACS system (Miltenyi Biotech, Bergisch-Gladbach, Germany). In brief, peripheral blood mononuclear cells were mixed with CD8 microbeads (Miltenyi Biotech; 20 µl/107 cells) in incubation buffer [phosphate buffered saline (PBS)/0·5% bovine serum albumin (BSA)/2 m M ethylene diamine tetra-acetic acid (EDTA); 80 µl/107 cells] for 15 min at 4°C. After washing, step cells were resuspended in the incubation buffer (107 cells/1 ml buffer), and positive selection was performed using LS + columns and a MidiMACS magnet according to the manufacturer's instructions. The purity of the resulting CD8+ T cell population was more than 95% as determined by flow cytometry for the surface markers CD3, CD8, CD4 and CD56.
Preparation of monomeric and tetrameric HLA/β2-microglobulin/peptide complex
Monomeric complexes were synthesized as described previously [15]. Briefly, the transmembrane and cytosolic region of HLA-A*2402 was substituted for a 15 amino acid substrate peptide (BSP) of BirA dependent biotinylation enzyme. The HLA-A*2402 BSP fusion protein and β2-microglobulin (β2m) were expressed in Escherichia coli and were folded in vitro with the specific antigen peptide. The correctly refolded HLA/β2m/peptide complex was purified by gel filtration chromatography and biotinylated on a single lysine within the BSP with the BirA enzyme (Avidity, LLC, Denver, CO, USA). The biotinylated HLA/β2m/peptide complexes were stored at −20°C and used subsequently to coat flat-bottomed 96-well microtitre plates. Tetramers were prepared by mixing the biotinylated HLA/β2m/peptide complex with streptavidin—phycoerythrin conjugate (Molecular Probes, Eugene, OR, USA) in a 1 : 4 molar ratio. The resulting tetramer was purified further by gel filtration chromatography and concentrated around 1 mg/ml.
IFN-γ production by CD8+ cells exposed to monomeric HLA/peptide complex
Monomeric HLA/peptide complexes were added to each test well at 1 µg/30 µl of PBS and allowed to bind to the plastic surface for 1 h at 25°C. Freshly isolated CD8+-enriched cells (5 × 106 cells/well) were then incubated in RPMI-1640 supplemented with l-glutamine and 10% pooled human AB serum (CosmoBio, Tokyo, Japan) in duplicate wells for 24 or 48 h at 37°C. Supernatant was harvested and the concentration of IFN-γ was measured using an enzyme-linked immunosorbent assay (ELISA) kit (human IFN-γ ELISA, Endogen, MA, USA), where the lower limit of detection was 7·8 pg/ml. Production of IFN-γ was confirmed by culturing purified CD8+ T cells at 105 cells/well with 211.LCL and peptides (5 µg/ml) for 24 h in triplicate and then visualized by enzyme-linked immunospot (ELISPOT) assay (ELISpot human IFN-γ, R&D Systems, MN, USA). Spots in each well were counted using an ELISpot reader system (Autoimmun Diagnostica Gmbh, Straßberg, Germany) and spots/104 CD8+ T cells were calculated by subtracting the number of spots in control wells (wells without peptide or wells without CD8+ T cells).
Tetramer staining of peptide-specific CD8+ T cells
Peripheral blood mononuclear cells were stained with anti-CD8-FITC [FITC-conjugated mouse anti-human CD8 monoclonal antibody (mAb), Becton-Dickinson, Mountain View, CA, USA], anti-CD38-APC (APC-conjugated mouse antihuman CD38 mAb, Becton-Dickinson), Viaprobe (Becton-Dickinson) and PE-conjugated HLA class I/NS3 1296–1294, HLA class I/core 129–137 tetramers for 30 min at 4°C in the dark in 100 µl of FACS buffer (PBS containing 2% BSA and 0·02% sodium azide). PE-conjugated mouse IgG antibodies were used as isotype control. Cells were then washed twice with FACS buffer; sample data were acquired and analysed on a BD Biosciences FACSCalibur instrument and then analysed using CellQuest software (BD Biosciences, Mountain View, CA, USA). Cells were first gated on a lymphocyte gate and then on CD8-positive cells. Results were expressed as percentages of tetramer-binding cells in the CD8 positive population. Results were considered as positive for tetramer-binding cells when the level was above 0·05% in CD8+ cells.
Generation of peptide-specific CD8+ T cell lines
CD8+ cells were cultured in RPMI-1640 medium containing 10% pooled human AB serum using a 96-well flat-bottomed tissue culture plate precoated with one of the HLA A*2402/β2 microglobulin/peptide complexes. After 2 days of culture, they were supplemented with 10 U/ml of recombinant human interleukin 2 (rhIL-2, Shionogi Pharmaceutical Inc., Osaka, Japan). Thereafter, RPMI-1640 containing 10% pooled human AB serum with 10 U/ml rhIL-2 was added to each well at 3–4-day intervals. Peptide-specific CD8+ T cells were used as effector cells for cytotoxicity assay after 14–28 days of culture.
51Cr release cytotoxicity assays
An HLA-A*2402-positive EBV-transformed B lymphocyte line (211.LCL) was provided by the Cell Resource Center for Biomedical Research, Tohoku University Institute of Development, Ageing and Cancer, and HLA-A*24-negative T2 cell line was purchased from ATCC (Manassas, VA, USA). The cells were incubated overnight with synthetic peptides at 5 µg/ml. After labelling with 100 µCi of 51Cr (Amersham Bioscience Co., Tokyo, Japan) for 45 min, the cells were used as target cells. Cytotoxic activity was measured by the standard 5-h split-well 51Cr release assay using a U-bottomed 96-well plate with 5000 target cells/well. Peptide-specific CD8+ T cells were added to the 96-well plate at E/T ratios of 20 : 1. To confirm HLA-A24 specificity, a T2 cell line that expresses HLA-A2 but lacks A24 expression was also used as a target cell in some experiments. Supernatants (100 µl) were harvested, and the amount of 51Cr released was measured using a gamma counter. Spontaneous 51Cr release was determined in wells containing target cells alone, and maximum release was determined in wells of target cells lysed with 10% Triton-X 100 (Sigma Chemicals, St Louis, MO, USA). Spontaneous release was < 20% of maximum release in all experiments. The percentage of specific lysis was calculated by the following formula: % specific lysis = [(cpm test wells — cpm control spontaneous release wells)/(cpm maximum release wells — cpm control spontaneous release wells)] × 100. Results were considered as positive for peptide-specific killing when killing of peptide-labelled targets was 15% above that of control targets.
Results
Cytotoxic activity of major histocompatibilty complex (MHC)/peptide-stimulated CD8+ T cells
Short-term CD8+ T cell line was obtained in six cases [four sustained virus responders (SVR) patients and two non-responder (NR) patients] by culturing in MHC/peptide monomeric complex-bound wells 28-day culture with adding IL-2 every 3 days. These short-term T cell lines were tested for peptide specific lysis against 211.LCL target cells pulsed with the same peptides. Specific cytotoxicity against peptide-pulsed targets was observed only with NS31296–1304 and core 129–137 (Tables 2 and 3, Fig. 1a), although cytotoxicity against targets pulsed with E2719–727, NS52871–2879, core 85–93 or core 173–181 was not observed (data not shown). Killing was not observed by these CD8+ T cells when HLA-A24-lacking T2 cells were used as target cells, and therefore recognition of these two peptides was restricted by the HLA-A24 (Fig. 1b). Cytotoxicity against NS31296–1304 was observed in three of the four SVR patients and that against core 129–137 was in two of the three SVR patients (Fig. 1c). In addition, ex vivo ELISPOT assays with freshly isolated CD8+ T cells from an HLA-A24 positive patient with SVR, an HLA-A24 negative patient with SVR and an HLA-A24 positive healthy volunteer showed that response to these peptides was restricted by HLA-A24 (Fig. 2). CD8+ T cells from two NR patients, without detectable production of IFN-γ, showed no cytotoxicity against targets labelled with peptides. Amino acid sequences of these two peptides were well conserved among HCV genotypes (Table 2).
Table 2. List of hepatitis C virus (HCV) peptides with HLA A*2402-binding motif.
| Comparison by genotype(%)2 | ||||||||
|---|---|---|---|---|---|---|---|---|
| Protein | aa position | aa sequence1 | 1a | 1b | 2a | 2b | 3a | |
| NS3 | 1296–1304 | TYSTYGKFL | 100 | 100 | 99 | 93 | 100 | |
| Core | 129–137 | GFADLMGYI | 100 | 99 | 98 | 100 | 100 | |
Amino acid positions were deduced from a prototype strain HC-J4 (genotype 1b, accession number AF054250) [18].
Homology of amino acid sequences was calculated using prototype hepatitis C virus (HCV) sequences of genotype 1a (n = 4), genotype 1b (n = 33), genotype 2a (n = 18), genotype 2b (n = 3) and genotype 3a (n = 3).
Table 3. Peptide-specific killing by CD8+ T cells stimulated with monomeric HLA A*2402/β2 microglobulin/peptide complex.
| Specific killing (%) against peptide-labelled target | ||
|---|---|---|
| Outcome of IFN therapy | NS31296–1304 | Core 129–137 |
| SVR | 0·0 | n.d. |
| SVR | 25·7 | 12·8 |
| SVR | 19·3 | 27·5 |
| SVR | 17·2 | 4·0 |
| NR | −2·3 | −0·8 |
| NR | 6·5 | 0·0 |
One cytotoxic assay was performed using growing T cells obtained from six of 26 tested cases [four sustained virus responders (SVRs) and two non-responders (NRs)]. From healthy subjects growing T cells were not recovered. Killing activity was tested using HLA A*2402-positive B cell line (211.LCL) labelled with hepatitis C virus (HCV)-derived peptide by 51Cr-release assay. Specific killing was calculated as specific killing in peptide-labelled targets minus specific killing in control targets, and more than 15% was considered positive. IFN: interferon; n.d.: not done.
Fig. 1.
Recognition of NS31296–1304 and core 129–137 peptides by cytotoxic T lymphocyte (CTL) lines. Cytolysis of a CD8+ T cell line stimulated with each peptide was evaluated in a standard 51Cr release assay against a target cell (211.LCL) pulsed with NS31296–1304 and core 129–137 peptides at a concentration of 5 µg/ml. Cytotoxic assay was performed at E : T ratios of 20 : 1, 5 : 1 and 1 : 1 (a). HLA restriction was confirmed by 51Cr release assay against the 211.LCL (HLA-A24 positive) and T2 cell lines (HLA-A24 negative). Both cell lines were pulsed with either peptide (5 µg/ml) and used as target cells (b). T cell lines were prepared in four sustained virus responders (SVR) and two non-responders (NR) (c, NS31296–1304; white column, core 129–137; black column).
Fig. 2.
HLA restrictions of interferon (IFN)-γ by CD8+ T cells in response to NS31296–1304 and core 129–137. Freshly purified CD8+ T cells from a sustained virus responder (SVR) (HLA-A24 positive) were incubated with NS31296–1304 and core 129–137 (5 µg/ml) on anti-IFN-γ-bound 96 wells in triplicate. CD8+ T cells from a sustained virus responder (SVR) (HLA-A24 negative) and a healthy volunteer (HLA-A24 positive) were incubated in the same way. Spots were visualized and counted. Finally, the number of spots specific for each peptide was calculated by subtracting the number of spots in wells without peptide added. Stimulation with phytohaemagglutinin (PHA) was used as a positive control.
IFN-γ production of CD8+ T cell against HLA A*2402/β2 microglobulin/peptide complexes
Peripheral CD8+ cells from 26 patients with chronic hepatitis C were stimulated for 48 h with plastic-bound HLA A*2402/β2 microglobulin/peptide complexes. In sustained virus responders, IFN-γ production in response to each of the peptides was 8/13 for NS31296–1304 [38·0 ± 13·3 pg/ml, mean ± standard error of the mean (s.e.m.)] and 7/13 for core 129–137 (33·0 ± 12·1 pg/ml). However, no IFN-γ production was observed in non-responders (n = 13) for either of the peptides (Fig. 3). Peripheral blood CD8+ cells from nine HLA A*2402-positive healthy subjects did not respond to these peptides, although one of them responded to core 129–137. In addition, four HLA-A24 negative patients with chronic hepatitis C were tested in the same way and there was no production of IFN-γ against these peptides. The difference in production of IFN-γ was significant between the SVR patients and NR patients for both NS31296–1304 (P < 0·001) and core 129–137 (P < 0·05) (Mann–Whitney U-test, Fig. 4).
Fig. 3.
Production of interferon (IFN)-γ by CD8+ T cells stimulated with monomeric HLA A*2402/β2 microglobulin/peptide complex. Production of IFN-γ was measured by enzyme-linked immunosorbent assay (ELISA) (pg/ml) in supernatant after 48 h of incubation of freshly isolated CD8+ T cells in duplicate with plastic bound monomeric HLA-A*2402/peptide complexes. We repeated the experiment twice in selected samples, including both positive cases and negative cases for production of IFN-γ, and interexperimental deviations were less than 10%.
Fig. 4.
Comparison of interferon (IFN)-γ production in response to NS31296–1304 and core 129–137 among patient groups. Concentrations of IFN-γ in the supernatants were measured after 48 h of incubation of freshly isolated CD8+ T cells in triplicate with plastic bound monomeric HLA-A*2402/peptide complexes by enzyme-linked immunosorbent assay (ELISA) (pg/ml). Each dot represents the individual case (filled circle, NS31296–1304 and open circle, core 129–137) and detection limit was indicated by a dotted line.
IFN-α therapy and changes in response of CD8+ T cells
IFN-γ production and tetramer staining were evaluated (Fig. 5a, b). Tetramers of peptides NS31296–1304 and core 129–137 were less than 0·05% of CD8 population in control subjects (four HLA-A24− patients with chronic hepatitis C and four HLA-A24+ normal subjects). In cases with SVR, production of IFN-γ in response to peptides NS31296–1304 or core 129–137 was observed only at the end of the therapy, and tetramer-positive CD8+ T cells against these two peptides increased (Table 4). Two patients with NR had tetramer positive cells against NS31296–1304 before therapy and at the end of therapy, while tetramers against core 129–137 were negative in all three cases (Table 4). In one of NR cases, IFN-γ production was observed in response to NS31296–1304. The results of ELISPOT assay using the same samples obtained at the end of the therapy in these five patients indicated that there were IFN-γ-producing CD8+ T cells in samples which were positive for tetramer-staining and production of IFN-γ in response to monomeric peptide/HLA complex stimulation. In addition, the results of ELISPOT assay also suggested the lower limit of the IFN-γ production in stimulation with monomeric peptide/HLA complex was frequency of specific CD8+ T cell at between 11·11 and 18·52/104 CD8+ T cells.
Fig. 5.
Peripheral cytotoxic T lymphocyte (CTL) numbers specific for NS31296–1304 and core 129–137 at the end of interferon (IFN)-α monotherapy. Tetramer/NS31296–1304 (a) and tetramer/core 129–137 (b) mark the specific CD8+ T cell lines, which were expressed mainly as surface CD38. Specific CD8+ T cells were assumed to be present in peripheral blood when the tetramer-positive percentage was more than 0·05% (the cut-off value deduced from the results of control subjects).
Table 4. Comparison of interferon-γ (IFN) production before and at the end of interferon-α monotherapy and tetramer-positive CD8+ T cells in five patients with chronic hepatitis C.
| Peptides | ||||
|---|---|---|---|---|
| Outcome of IFN therapy | Time | T cell response | NS31296–1304 | Core 129–137 |
| SVR | Before | IFN-γ1 | <7·8 | <7·8 |
| Tetramer positive2 | n.d. | n.d. | ||
| End | IFN-γ | 11·87 | 15·84 | |
| Tetramer positive | 0·36 | 0·53 | ||
| ELISPOT | 41·67 | 94·44 | ||
| SVR | Before | IFN-γ | <7·8 | <7·8 |
| Tetramer positive | <0·05 | <0·05 | ||
| End | IFN-γ | 10·78 | 10·35 | |
| Tetramer positive | 1·17 | 0·12 | ||
| ELISPOT | 108·89 | 71·11 | ||
| NR | Before | IFN-γ | <7·8 | <7·8 |
| Tetramer positive | 0·12 | <0·05 | ||
| End | IFN-γ | <7·8 | <7·8 | |
| Tetramer positive | 0·10 | <0·05 | ||
| ELISPOT | 13·89 | 11·11 | ||
| NR | Before | IFN-γ | <7·8 | <7·8 |
| Tetramer positive | n.d. | n.d. | ||
| End | IFN-γ | <7·8 | <7·8 | |
| Tetramer positive | <0·05 | <0·05 | ||
| ELISPOT | <1·0 | <1·0 | ||
| NR | Before | IFN-γ | <7·8 | <7·8 |
| Tetramer positive | 0·08 | <0·05 | ||
| End | IFN-γ | 14·92 | <7·8 | |
| Tetramer positive | 0·08 | <0·05 | ||
| ELISPOT | 18·52 | 7·41 | ||
After 48 h of incubation with plastic-bound monomeric HLA A*2402/peptide complexes, the supernatant was harvested. Concentration of IFN-γ was detemined by enzyme-linked immunosorbent assay (ELISA) (pg/ml).
Peptide-specfic CD8+ T cells were defined as percentages of tetramer-positive cells in the CD8+ T cell population. SVR, sustained virus response; NR, non-response; n.d., not done. ELISPOT: enzyme-linked immunospot.
Discussion
In individuals with recovered from acute self-limited HCV infection, the presence of cytotoxic CD8+ T cells against HCV antigens has been well reported [5,16]. It is suggested that CTLs display functional heterogeneity during viral infections [17–19] and a functional defect has been reported in chronic HCV infection [20,21]. The production of IFN-γ by CD8+ T lymphocytes in response to two HCV epitopes presented in the context of HLA-A*2402 was well preserved in some of patients with SVR when compared with those with NR and therefore may affect the course of HCV infection as well as killing of infected targets [22].
We selected two CTL epitopes, namely NS31296–1304 and core 129–137 of five candidate epitopes and identified the presence of CTLs in peripheral blood by tetramer-staining and also evaluated IFN-γ production by CD8+ lymphocytes after stimulation with monomeric HLA/peptide complexes. The results suggest that the function of CTLs which recognize NS31296–1304 and core 129–137 are characterized readily by the production of IFN-γex vivo. Detection of the functional CD8+ CTL response by cytotoxicity assay usually requires in vitro re-stimulation [23], and functional characterization is necessary on the ex vivo-detected tetramer-positive cells to evaluate their functional properties. Neither method is therefore suitable to evaluate the presence of functional CTLs ex vivo in the situation of low frequencies of peripheral CTLs such as in HCV infection [24]. Comparison with the results of the ELISPOT assay suggested the lower detection limit of IFN-γ production assay by stimulation with monomeric peptide/HLA complex as 0·11–0·18% in CD8+ T cells, indicating that the method is less sensitive than tetramer staining.
The response of CD8+ T cells to peptides NS31296–1304 and core 129–137 was maintained in peripheral blood after IFN monotherapy in patients with sustained viral responses. A patient with NR was positive in IFN-γ production in response to NS31296–1304 at the end of the therapy, although IFN-γ production in response to either of these epitopes was not detectable after the end of the therapy. ELISPOT assay also showed the presence of antigen-specific CD8+ T cells in this patient. Therefore, it is conceivable that response of CD8+ T cells to these antigens may not persist after the withdrawal of IFN therapy in patients with HCV recurrence, while it persists in SVR patients. The role of the response of IFN-γ production by CD8+ T cells in the elimination of HCV during IFN therapy was not determined in this study. It is possible that functional CTLs recovered following the clearance of HCV by treatment in patients with SVR. It has been shown that HCV-specific CTLs are of an immature phenotype in patients with chronic infection [25,26] and that HCV infection has a pervasive effect on CD8+ T cell population [27]. IFN monotherapy may induce the alteration in CD8+ T cells to produce IFN-γ in response to HCV antigens in SVR patients, although the effects of IFN-α therapy may vary from augmentation to suppression of specific CTL response according to the dose of IFN-α[28,29]. These previous reports from others and our observation of varying response of specific CD8+ T cells in SVR indicate the possibility of other factors co-operating in the induction and maintenance of specific CTL response in addition to IFN-α therapy. Further detailed study is needed to evaluate the immune regulatory mechanisms, including specific CD4 T+ cells and regulatory T cells, as long-term IL-10 therapy has been reported to produce the decrease in disease activity as measured by inflammatory activity within the liver and serum ALT levels while serum HCV viral levels were increased by alteration of immunological viral surveillance [30].
In summary, our data suggest that IFN-γ production by CD8+ T lymphocytes against HCV NS31296–1304 and core 129–137 are well maintained in patients with SVR compared with those with NR. The responses of CTLs are presumed to be induced during IFN therapy in these patients. These findings emphasize the importance of CD8+ T cell response in controlling HCV infection. In future studies the attempt to enhance the functional response of HCV-specific CTLs in patients with chronic HCV infection must be taken into account.
Acknowledgments
This study was supported by grant no. 12877084 from Ministry of Education, Culture, Sports, Science and Technology of Japan.
References
- 1.Iino S. Natural history of hepatitis B and C virus infections. Oncology. 2002;62(Suppl. 1):18–23. doi: 10.1159/000048271. [DOI] [PubMed] [Google Scholar]
- 2.Alter HJ, Seeff LB. Recovery, persistence, and sequelae in hepatitis C virus infection. a perspective on long-term outcome. Semin Liver Dis. 2000;20:17–35. doi: 10.1055/s-2000-9505. [DOI] [PubMed] [Google Scholar]
- 3.Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med. 2001;194:1395–406. doi: 10.1084/jem.194.10.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Lechner F, Wong DK, Dunbar PR, et al. Analysis of successful immune responses in persons infected with hepatitis C virus. J Exp Med. 2000;191:1499–512. doi: 10.1084/jem.191.9.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Cooper S, Erickson AL, Adams EJ, et al. Analysis of a successful immune response against hepatitis C virus. Immunity. 1999;10:439–49. doi: 10.1016/s1074-7613(00)80044-8. [DOI] [PubMed] [Google Scholar]
- 6.Gerlach JT, Diepolder HM, Jung MC, et al. Recurrence of hepatitis C virus after loss of virus-specific CD4(+) T cell response in acute hepatitis C. Gastroenterology. 1999;117:933–41. doi: 10.1016/s0016-5085(99)70353-7. [DOI] [PubMed] [Google Scholar]
- 7.Hiroishi K, Kita H, Kojima M, et al. Cytotoxic T lymphocyte response and viral load in hepatitis C virus infection. Hepatology. 1997;25:705–12. doi: 10.1002/hep.510250336. [DOI] [PubMed] [Google Scholar]
- 8.Lohr HF, Schmitz D, Arenz M, Weyer S, Gerken G, Meyer zum Buschenfelde KH. The viral clearance in interferon-treated chronic hepatitis c is associated with increased cytotoxic T cell frequencies. J Hepatol. 1999;31:407–15. doi: 10.1016/s0168-8278(99)80030-0. [DOI] [PubMed] [Google Scholar]
- 9.Vertuani S, Bazzaro M, Gualandi G, et al. Effect of interferon-alpha therapy on epitope-specific cytotoxic T lymphocyte responses in hepatitis C virus-infected individuals. Eur J Immunol. 2002;32:144–54. doi: 10.1002/1521-4141(200201)32:1<144::AID-IMMU144>3.0.CO;2-X. [DOI] [PubMed] [Google Scholar]
- 10.Mazzeo C, Azzaroli F, Giovanelli S, et al. Ten year incidence of HCV infection in northern Italy and frequency of spontaneous viral clearance. Gut. 2003;52:1030–4. doi: 10.1136/gut.52.7.1030. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Sobao Y, Sugi K, Tomiyama H, et al. Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A*2402, the most common HLA class I allele in East Asia. J Hepatol. 2001;34:922–9. doi: 10.1016/s0168-8278(01)00048-4. [DOI] [PubMed] [Google Scholar]
- 12.Kondo Y, Kobayashi K, Kobayashi T, et al. Distribution of the HLA class I allele in chronic hepatitis C and its association with serum ALT level in chronic hepatitis C. Tohoku J Exp Med. 2003;201:109–17. doi: 10.1620/tjem.201.109. [DOI] [PubMed] [Google Scholar]
- 13.Browning MJ, Krausa P, Rowan A, et al. Tissue typing the HLA-A locus from genomic DNA by sequence-specific PCR: comparison of HLA genotype and surface expression on colorectal tumor cell lines. Proc Natl Acad Sci USA. 1993;90:2842–5. doi: 10.1073/pnas.90.7.2842. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kondo A, Sidney J, Southwood S, et al. Prominent roles of secondary anchor residues in peptide binding to HLA-A24 human class I molecules. J Immunol. 1995;155:4307–12. [PubMed] [Google Scholar]
- 15.Altman JD, Moss PA, Goulder PJ, et al. Phenotypic analysis of antigen-specific T lymphocytes. Science. 1996;274:94–6. [PubMed] [Google Scholar]
- 16.Cucchiarini M, Kammer AR, Grabscheid B, et al. Vigorous peripheral blood cytotoxic T cell response during the acute phase of hepatitis C virus infection. Cell Immunol. 2000;203:111–23. doi: 10.1006/cimm.2000.1683. [DOI] [PubMed] [Google Scholar]
- 17.Moser JM, Altman JD, Lukacher AE. Antiviral CD8+ T cell responses in neonatal mice: susceptibility to polyoma virus-induced tumors is associated with lack of cytotoxic function by viral antigen-specific T cells. J Exp Med. 2001;193:595–606. doi: 10.1084/jem.193.5.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Welsh RL. Assessing CD8 T cell number and dysfunction in the presence of antigen. J Exp Med. 2001;193:F19–F22. doi: 10.1084/jem.193.5.f19. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Chisari FV. Cytotoxic T cells and viral hepatitis. J Clin Invest. 1997;99:1472–7. doi: 10.1172/JCI119308. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Gruener NH, Lechner F, Jung MC, et al. Sustained dysfunction of antiviral CD8+ T lymphocytes after infection with hepatitis C virus. J Virol. 2001;75:5550–8. doi: 10.1128/JVI.75.12.5550-5558.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Wedemeyer H, He XS, Nascimbeni M, et al. Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. J Immunol. 2002;169:3447–58. doi: 10.4049/jimmunol.169.6.3447. [DOI] [PubMed] [Google Scholar]
- 22.Lancaster T, Sanders E, Christie JM, Brooks C, Green S, Rosenberg WM. Quantitative and functional differences in CD8+ lymphocyte responses in resolved acute and chronic hepatitis C virus infection. J Viral Hepat. 2002;9:18–28. doi: 10.1046/j.1365-2893.2002.00330.x. [DOI] [PubMed] [Google Scholar]
- 23.Belyakov IM, Wang J, Koka R, et al. Activating CTL precursors to reveal CTL function without skewing the repertoire by in vitro expansion. Eur J Immunol. 2001;31:3557–66. doi: 10.1002/1521-4141(200112)31:12<3557::aid-immu3557>3.0.co;2-o. [DOI] [PubMed] [Google Scholar]
- 24.Lechner F, Gruener NH, Urbani S, et al. CD8+ T lymphocyte responses are induced during acute hepatitis C virus infection but are not sustained. Eur J Immunol. 2000;30:2479–87. doi: 10.1002/1521-4141(200009)30:9<2479::AID-IMMU2479>3.0.CO;2-B. [DOI] [PubMed] [Google Scholar]
- 25.Appay V, Dunbar PR, Callan M, et al. Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections. Nat Med. 2002;8:379–85. doi: 10.1038/nm0402-379. [DOI] [PubMed] [Google Scholar]
- 26.Barnes E, Harcourt G, Brown D, et al. The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection. Hepatology. 2002;36:743–54. doi: 10.1053/jhep.2002.35344. [DOI] [PubMed] [Google Scholar]
- 27.Lucas M, Vargas-Cuero AL, Lauer GM, et al. Pervasive influence of hepatitis C virus on the phenotype of antiviral CD8+ T cells. J Immunol. 2004;172:1744–53. doi: 10.4049/jimmunol.172.3.1744. [DOI] [PubMed] [Google Scholar]
- 28.Rehermann B, Chang KM, McHutchinson J, et al. Differential cytotoxic T-lymphocyte responsiveness to hepatitis B and C viruses in chronically infected patients. J Virol. 1996;70:7092–102. doi: 10.1128/jvi.70.10.7092-7102.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Gehring S, Gregory SH, Kuzushita N, Ands JR. Type 1 interferon augments DNA-based vaccination against hepatitis C virus core protein. J Med Virol. 2005;75:249–57. doi: 10.1002/jmv.20264. [DOI] [PubMed] [Google Scholar]
- 30.Nelson DR, Tu Z, Soldevila-Pico C, et al. Long-term interleukin 10 therapy in chronic hepatitis C patients has a proviral and anti-inflammatory effect. Hepatology. 2003;38:859–68. doi: 10.1053/jhep.2003.50427. [DOI] [PubMed] [Google Scholar]