1 Division of Gastroenterology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-8575, Japan
2 Research & Development Department, Mitsubishi Kagaku Bio-Clinical Laboratories Inc., 3-30-1 Shimura, Itabashi-ku, Tokyo 174-8555, Japan
3 Division of Gastroenterology, Omiya Medical Center, Jichi Medical School, 1-847 Amanuma-cho, Omiya-ku, Saitama 330-8503, Japan
4 Second Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan
Correspondence
Michio Imawari
imawari{at}med.showa-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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HLA-A24 is one of the most common HLA-A antigens in Asians (Chandanayingyong, 1986), occurring in more than 60 % of Japanese (Date et al., 1996
). To study the immunopathogenesis of HCV infection in Japanese and other Asians and to develop HCV-specific CTL vaccines for these populations, identification of HCV-specific CTL epitopes with recognition restricted by HLA-A24 is therefore important. An HLA-A24 allele in Japanese people is almost exclusively HLA-A*2402 (Date et al., 1996
). Yet few HLA-A*2402-restricted, HCV-specific CTL epitopes have been reported, in contrast to more numerous reports concerning HLA-A2.1-restricted, HCV-specific CTL epitopes (Ward et al., 2002
), representing the HLA most common in Caucasians.
A recently reported enzyme-linked immunospot (ELISpot) assay provides a rapid, inexpensive and efficient way to define a given HLA class I molecule-restricted, novel virus-specific CD8+ T cell epitope and to characterize the breadth of CTL responses (Altfeld et al., 2000). To identify HLA-A*2402-restricted, HCV-specific CD8+ T cell epitopes, we synthesized 87 peptides derived from the total protein content of HCV and carrying HLA-A*2402-binding motifs (Ibe et al., 1996
) and assessed the ability of the peptides to stimulate CD8+ T cells by counting interferon (IFN)-
-releasing cells in HLA-A*2402-positive patients with acute or chronic hepatitis C using the ELISpot assay. Using the epitopes identified, we then studied the effects of treatment with IFN-
on frequencies of HCV-specific CD8+ T cells in two HLA-A*2402-positive patients with unresolved acute hepatitis C.
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METHODS |
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ELISpot assay.
Analysis of anti-peptide immune responses of peripheral blood CD8+ T cells was performed using an IFN--based ELISpot assay kit (Mabtech). Briefly, 105 CD8+ T cells, 104 monocytes as antigen-presenting cells and a peptide mixture or individual peptides at 10 µg ml1 each were placed in duplicate in 96-well plates with a PVDF membrane at the bottom (MAIP S45; Millipore). Well bottoms were coated with anti-IFN-
monoclonal antibody (mAb). Cells were cultured for 40 h at 37 °C in a humidified 5 % CO2 atmosphere. No peptide was added to the negative control wells. After culture, IFN-
spot-forming cells (SFCs) were visualized as described previously (Lalvani et al., 1997
). Responses were considered significant when a minimum of five SFCs were present per well, representing at least twice the number of SFCs in negative control wells. In preliminary studies, monocytes did not present IFN-
SFCs in response to stimulation with HCV peptides.
To enrich peptide-specific CD8+ T cells that might recognize a known HLA-A*2402-restricted epitope, 2x106 PBMC pulsed with 10 µg peptide 10311039 ml1 (Kurokohchi et al., 2001) were cultured in a 24-well flat-bottom plate for 9 days in RPMI 1640 medium supplemented with 10 % human serum AB blood type and 50 U recombinant human interleukin (rhIL)-2 ml1 added on day 2. The cells were harvested after 9 days of culture and CD8+ T cells were isolated for an ELISpot assay.
Generation of HCV-specific CTLs.
HCV-specific CTLs were generated as described previously (Hiroishi et al., 2002). Briefly, PBMC were suspended at a cell density of 106 cells ml1 in RPMI 1640 medium supplemented with 10 % human AB serum and a single peptide was added on day 0. Cells were incubated at 37 °C in a humidified 5 % CO2 atmosphere. On day 2, rhIL-2 was added at a final concentration of 20 U ml1. On day 7, the culture was restimulated with the single peptide and irradiated autologous PBMC. Cytotoxic activity of peptide-induced effector cells was assessed on days 1416.
CTL assay.
The cytotoxic activity of peptide-induced effector cells was assessed using a standard 4 h sodium chromate (51Cr) release assay. Briefly, EpsteinBarr virus-transformed B-lymphoblastoid cell lines (B-LCL) were labelled with 100 µCi (3·7 MBq) 51Cr. The 51Cr-labelled B-LCL were suspended in RPMI 1640 medium supplemented with 10 % fetal calf serum and incubated overnight at 37 °C with a synthetic peptide or infected with recombinant vaccinia virus (rVV) that endogenously expressed HCV antigens (K. Funatsuki & H. Ishiko, unpublished results). An m.o.i. of 5 was used for an 18 h incubation with rVV. Then, after incubating the effector cells with the target cells for 4 h at 37 °C in a humidified 5 % CO2 atmosphere, supernatants were collected and radioactivity was measured with a gamma counter.
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RESULTS |
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The magnitude of IFN- SFC responses to single-peptide stimulation ranged from 4 to 139 SFC per 105 CD8+ T cells and summed frequencies of the SFCs in patients 2 and 4 were 146 and 193 SFC per 105 CD8+ T cells, respectively. Of the 11 peptides, two were 9-mers, seven were 10-mers and two were 11-mers. These epitopes were distributed throughout the entire HCV protein; one epitope was localized in each of the E1 and E2 regions, two in the NS2 region, three in the NS3 region, two in the NS4 region and three in the NS5 region. None of the 11 epitopes had been reported previously.
The individual peptides previously reported as HLA-A*2402-restricted, HCV-specific CTL epitopes did not elicit an ex vivo IFN- SFC response in cells from any patient studied. The assay was repeated after CD8+ T cells were expanded by stimulating CD8+ T cells from patients 1 and 4 with the known HLA-A*2402-restricted, HCV-specific CTL epitope peptide 10311039 (Kurokohchi et al., 2001
) for 9 days in the presence of rhIL-2 to enrich CD8+ T cells with specificity for the peptide. Although stimulation of the CD8+ T cells with the epitope peptides identified in the present study enriched peptide-specific CD8+ T cells, peptide 10311039-specific CD8+ T cells could not be enriched to attain a detectable level (Fig. 1
).
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To define the HLA molecules that restricted recognition of peptides by CD8+ T cells other than peptide 22802288, we attempted to induce CTL lines by stimulating PBMC from patients with the individual peptides. We could generate CTLs specific for peptides 910919, 947956 and 12431252 from PBMC of patient 4 and CTLs specific for peptide 14431452 from PBMC of patient 6. The peptide-induced CTLs lysed not only peptide-pulsed autologous B-LCL but also B-LCL that had been infected with rVV, resulting in HCV protein expression including the peptide sequence in infected cells (data not shown). HLA restriction of peptide recognition by CTLs was studied using a panel of autologous and allogeneic B-LCL with known HLA haplotypes as target cells. CTLs induced by peptide 910919 or 12431252 selectively lysed B-LCL expressing HLA-Cw3 (HLA-Cw*0303 or HLA-Cw*0304 or both) that had been pulsed with the individual peptides (Fig. 2a and b), indicating that recognition of both HCV NS2 peptide 910919 and NS3 peptide 12431252 was restricted by HLA-Cw*0303 and HLA-Cw*0304 molecules. However, since the NS3 peptide 12431252-specific CTL lysis restricted by HLA-Cw*0303 and HLA-Cw*0304 was less than the total lysis for the peptide, the recognition of the peptide also might be restricted by HLA-B*4002 (Fig. 2b
). CTL induced by peptide 947956 selectively lysed peptide-pulsed B-LCL expressing HLA-B61 (HLA-B*4002 or HLA-B*4006; Fig. 2c
) indicating that recognition of HCV NS2 peptide 947956 was restricted by HLA-B*4002 and HLA-B*4006 molecules. CTL induced by peptide 14431452 selectively lysed peptide-pulsed B-LCL expressing HLA-A*0206 (Fig. 2d
), indicating that the recognition of the peptide 14431452 was restricted by an HLA-A*0206 molecule, although the possibility that the CTLs also could recognize the targets in an HLA-A*0207 molecule-restricted manner cannot be ruled out from the data in Fig. 2(d)
. CTLs induced by peptide 14431452 did not lyse peptide-pulsed B-LCL expressing HLA-A*0201 (data not shown).
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Recognition of truncated and overlapping HCV peptides by CD8+ T cells
To define further the epitopes within the peptides that elicited an IFN- SFC response, truncated and overlapping peptides were synthesized and assayed for their ability to elicit a response from CD8+ T cells obtained from patients 4, 6 and 7 (Table 4
).
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Since both peptides 790798 and 910919 had an HLA-A*2402-binding amino acid residue at the position next to the C terminus, peptides with truncation of the HLA-A*2402-binding amino acid at the C terminus were synthesized and assayed for antigenicity. Neither of the truncated peptides retained antigenicity, indicating that C-terminal amino acids of peptides 790798 and 910919 were essential for antigenicity. The effect of truncation of the N-terminal amino acids was not studied. No studies of effects of truncation were performed for the other eight peptides.
HCV-specific CD8+ T cell epitopes that were identified and their HLA restriction are shown in Table 5.
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DISCUSSION |
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In the present study, we sought to identify HCV-specific, CD8+ T cell epitopes, with recognition restricted by HLA-A*2402, the most frequent HLA class I allele in Japanese and other Asians (Chandanayingyong, 1986). We screened the epitopes by an ELISpot assay based on IFN-
release by CD8+ T cells obtained from HLA-A*2402-positive patients with acute or chronic hepatitis C in response to peptide stimulation. Eighty-seven peptides were synthesized based on HLA-A*2402-binding motifs and the amino acid sequence of type 1b HCV. We could identify 10 HCV-specific CTL epitopes that induced IFN-
release by CD8+ T cells from a total of five of seven HLA-A*2402-positive patients with acute or relatively early chronic hepatitis C but not in any of four patients with persisting chronic hepatitis. The findings indicate that the response of HCV-specific CTLs to the panel of peptides is very low in patients with prolonged HCV infection. Consistent with this interpretation, an HLA-B*3501-restricted CTL epitope peptide that induced strong HCV-specific CTL responses in peripheral blood cells in the acute phase of HCV infection reportedly failed to induce CTL responses in seven of seven patients with chronic hepatitis C (Ibe et al., 1998
). Frequencies of HLA-B*3501-restricted, HCV-specific CTL also have been reported to be very low in the peripheral blood of patients with chronic hepatitis C, although CTLs were detectable among the PBMC by flow cytometric analysis using HLA-B*3501 tetramers (Sobao et al., 2001
). In still other reports, frequencies (Lechner et al., 2000
; Rehermann et al., 1996
; He et al., 1999
) and IFN-
-production potential (Gruener et al., 2001
; Wedemeyer et al., 2002
) of antiviral CTLs were low in patients with chronic HCV infection.
Unexpectedly, only one of the ten CTL epitopes identified in HLA-A*2402-positive patients was found to be definitely HLA-A*2402-restricted. In addition, the frequency of CD8+ T cells that responded to stimulation with the epitope was far less than for other CTL epitopes in this patient. The HLA class I molecules that restricted recognition of the other five epitopes by CD8+ T cells were thought to be HLA-Cw*0303 and HLA-Cw*0304 for two epitopes, HLA-B*4002 and HLA-B*4006 for one, HLA-A*0206 for one and probably HLA-Cw*0801 for another. The HLA class I molecules that restricted recognition of the remaining four epitopes by CD8+ T cells have not yet been defined. One more CD8+ T cell epitope was identified using the peptides with HLA-A*2402-binding motifs in one of two HLA-A*2402-negative patients with acute hepatitis C, although the HLA class I molecule restricting recognition of the epitope has not been determined. In two of the six patients with positive CD8+ T cell responses the target epitopes were multiple and only one of the 11 peptides was targeted in more than one patient. These findings indicate that a universally immunogenic HLA-A*2402-restricted, HCV-specific CD8+ T cell epitope may not exist; epitopes with recognition by CD8+ T cells restricted by HLA molecules other than HLA-A*2402 presumably were contained in the synthetic peptides with HLA-A*2402-binding motifs. CTL responses to HCV infection are heterogeneous, as concluded by Lauer et al. (2002).
We analysed sensitivity and specificity of previously reported HLA-A*2402-restricted, HCV-specific CTL epitopes (Kurokohchi et al., 2001; Ito et al., 2001
) in HLA-A*2402-positive patients with acute and relatively recently acquired chronic hepatitis C. These two epitope peptides did not induce IFN-
SFC responses, suggesting that their immunogenicity might be low compared with other epitopes. However, HCV NS3 peptide 10311039 identified by Kurokohchi et al. (2001)
has been reported to induce HCV-specific CTLs in three of four HLA-A*2402-positive patients with chronic hepatitis C. It has been reported that in vitro expansion of CD8+ T cells by stimulation with known HLA-A2-restricted CTL epitopes and culture in the presence of rhIL-2 revealed the existence of CD8+ T cells specific for the peptide, although IFN-
SFC responses ex vivo could not be induced (Lauer et al., 2002
). However, we could not confirm immunogenicity of the 10311039 epitope, even after stimulation and expansion with culture in the presence of rhIL-2. The limited number of patients in our study may have happened to lack CTLs responsive to stimulation with peptide 10311039; alternatively, the IFN-
-based ELISpot assay might detect a CD8+ T cell population that is functionally different from the CTLs identified by Kurokohchi et al. (2001)
. Consistent with this speculation, a stunned CD8+ T cell population has been reported to emerge in the acute phase of HCV infection, retaining potent HCV-specific cytotoxicity but only limited capacity for IFN-
production (Lechner et al., 2000
; Thimme et al., 2001
). CTLs responsive to the 10311039 peptide may belong to such a population.
Reliability of T cell epitope prediction based on HLA-binding motifs or algorithms (Rammensee et al., 1999) has been reported to be limited (Lauer et al., 2002
; Anthony et al., 2002
; Day et al., 2001
). Therefore, establishing that no immunodominant HLA-A*2402-restricted, HCV-specific CD8+ T cell epitope exists would require screening HLA-A*2402-restricted, HCV-specific CD8+ T cell epitopes by an IFN-
ELISpot assay using overlapping peptides that span the entire HCV protein; such a study, in progress in our laboratory, may identify new HLA-A*2402-restricted, HCV-specific CD8+ T cell epitopes without known HLA-A*2402-binding motifs. This study is intended to define the hierarchy of immunodominance of CTL epitopes in patients with HCV infection. Lauer et al. (2002)
demonstrated multiple unpredicted specificities of HCV-specific CD8+ T cell epitopes by an ELISpot assay using overlapping peptides that spanned the entire HCV protein, but none of the new epitopes that they found corresponded to those identified in the present study.
Using the CD8+ T cell epitopes currently identified, we sequentially monitored frequencies of CD8+ T cells secreting IFN- in response to stimulation with the epitope peptides during and after treatment of two patients with unresolved acute hepatitis C with IFN-
. Although effects of IFN-
therapy on HCV-specific CD8+ T cell responses have been reported from several laboratories (Löhr et al., 1999
; Vertuani et al., 2002
; Barnes et al., 2002
), results are conflicting. Löhr et al. (1999)
have reported that augmentation of HLA class I-restricted tumour necrosis factor (TNF)-
responses by IFN-
therapy contributes to a better treatment outcome in patients with chronic hepatitis C. The decline of serum HCV RNA during IFN-
therapy has been described as having two phases: a rapid early phase, thought to reflect direct inhibition of HCV replication by IFN-
; and a slower second phase, thought to be mediated by cellular immune responses, especially those of CTLs (Neumann et al., 1998
). Augmentation of TNF-
-releasing HCV-specific CD8+ T cell responses by IFN-
may beneficially affect the second phase of HCV RNA decline. However, in our present study, numbers of HCV epitope peptide-sensitized CD8+ T cells in peripheral blood declined in parallel with decreases and disappearance of serum HCV RNA and with ALT normalization. The reason why our results differed from those of Löhr et al. (1999)
is not known, but it could involve differences in degree of chronicity of disease, decrease rates of serum HCV RNA, doses of IFN-
or ethnicity of patients.
In conclusion, we have newly identified one definite HLA-A*2402-restricted, HCV-specific CD8+ T cell epitope and 10 probably non-HLA-A*2402-restricted epitopes by an IFN--based ELISpot assay using synthetic HCV peptides with HLA-A*2402-binding motifs. We could find HCV-specific CTL epitopes only in patients with acute or relatively early chronic hepatitis C. CD8+ T cell responses to HCV infection were heterogeneous. The findings indicate a need to identify as many HCV-specific CD8+ T cell epitopes as possible in large numbers of patients with acute or recently acquired chronic hepatitis C to understand better how immune responses eliminate HCV and contribute to pathogenesis. The ultimate aim is development of new strategies for enhancing immune responses for more effective control of HCV infection.
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ACKNOWLEDGEMENTS |
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Received 12 November 2003;
accepted 5 February 2004.
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