Center for Vaccinology, Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium1
Division of Clinical Virology, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden2
Author for correspondence: Geert Leroux-Roels. Fax +32 9 2406311. e-mail geert.lerouxroels{at}rug.ac.be
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Abstract |
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Introduction |
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HLA class II molecules expressed on the cell surface membrane of antigen-presenting cells (APCs) play a key role in the regulation of humoral and cellular immune responses (Germain & Margulies, 1993 ; Germain, 1994
). Self-limited, acute HBV infections are associated with vigorous HLA class II-restricted CD4+ T cell responses towards the hepatitis B core antigen (HBcAg), whereas weak or no responses are observed in patients with chronic hepatitis (Ferrari et al., 1991
; Jung et al., 1992
, 1995
; Lohr et al., 1995
; Marinos et al., 1995
; Penna et al., 1996
, 1997
). These observations suggest that HBcAg-specific CD4+ T cell responses play an important role in the recovery from disease and control of the virus. The HLA system is highly polymorphic and the HLA phenotype of an individual largely determines the response to various antigens and outcome of disease. HLA DR13 has been associated with resistance to several infectious diseases, such as hepatitis C, AIDs and malaria (Hill et al., 1991
; Kuzushita et al., 1996
, 1998
; Chen et al., 1997
). Although earlier studies on the association between major histocompatibility class polymorphism and HBV infection led to conflicting results (Forzani et al., 1984
; Black et al., 1986
; Almarri & Batchelor, 1994
; Zavaglia et al., 1996
), three studies in larger cohorts of patients demonstrated independently that HLA DR13 is involved in the elimination of HBV. A first study reported that in a large study population in Gambia, the HLA allele DRB*1302 was associated with protection against persistent HBV infection in both children and adults (Thursz et al., 1995
). This observation has been confirmed in Caucasians (Hohler et al., 1997
) and in an Asian population (Ahn et al., 2000
). Recently, it was demonstrated that the beneficial effect of the HLA DR13 alleles on the outcome of HBV infection could be explained by a vigorous HBcAg-specific CD4+ T cell response (Diepolder et al., 1998
). This effect could be due to more proficient antigen presentation by the HLA DR13 molecules themselves or a linked polymorphism in a neighbouring immunoregulatory gene. To explore further the relationship between HLA DR13 and HBcAg-specific CD4+ T cell responses in patients with acute, self-limited HBV infection, we generated HLA DR13-restricted CD4+ T cell clones and cell lines from the HLA DR13-positive subjects who recovered spontaneously from acute HBV infection. Surprisingly, most T cells generated from these donors recognized a single epitope within HBcAg, aa 147156.
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Methods |
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Preparation of peripheral blood leukocytes (PBLs).
PBLs were isolated from fresh heparinized blood by FicollHypaque (Nycomed) density gradient centrifugation. PBLs were washed extensively and used immediately for the generation of HBcAg-specific T cell lines or frozen in liquid nitrogen for later use.
Reagents.
HBcAg is a 21 kDa protein that assembles to form a nucleocapsid particle. Recombinant HBcAg (rHBcAg, subtype ayw) expressed in Escherichia coli was purchased from DiaSorin. A total of 17 partially overlapping 20-mer peptides spanning the complete sequence of HBcAg (subtype ayw) were used in these studies (Table 1). A series of N-and C-terminally truncated peptides of HBc P15 and a panel of alanine-substituted peptides spanning HBc P15 were also employed. All peptides were synthesized with a multiple peptide synthesizer using standard Fmoc chemistry (Syro, MultiSynTech).
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Generation of HBcAg-specific T cell clones and cell lines.
PBLs (3x106 cells per well) from three AHB subjects were stimulated separately with rHBcAg (0·5 µg/ml) in 24-well plates in RPMI 1640 supplemented with 25 mM HEPES, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine (all from Life Technologies), 5x10-5 M 2-mercaptoethanol (Sigma) and 10% heat-inactivated human AB+ serum (hereafter called complete medium). After 5 days, fresh complete medium containing recombinant human interleukin-2 (IL-2, 1050 U/ml, Eurocetus) was added to the cultures. T cell clones from subject AHB1 were generated by culturing the cells at limiting dilutions (0·3 cells per well: there is one chance in three that each well be seeded with a cell) in 96-well, flat-bottomed plates in the presence of 0·1 µg/ml rHBcAg, 10 U/ml human rIL-2 and gamma-irradiated (2·5 Gy), autologous PBLs (3x104 PBLs per well). Every 2 weeks, the cultures were restimulated with rHBcAg and irradiated, autologous PBLs (2·5 Gy) as APCs. After 8 weeks, the growing T cell clones were tested for HBcAg specificity; HBcAg-specific clones were subsequently analysed for their fine specificity by confronting them with a series of overlapping synthetic HBc peptides. The phenotype of HBcAg-specific T cell clones was analysed by flow cytometry (FACScan, Becton Dickinson).
Because the observations made with the clones generated from subject AHB1 needed to be confirmed, HBcAg-specific T cell lines from two additional subjects who recovered from AHB (AHB2 and AHB3) were generated by culturing the cells at a density of 10 cells per well in the presence of rHBcAg (0·2 µg/ml), rIL-2 (25 U/ml) and irradiated (3·5 Gy), autologous or DR13-matched, allogenic PBLs as APCs.
Proliferation and restriction assay.
Proliferation assays of T cell clones or lines were performed by incubating 2x104 T cells per well for 4 days in the presence of different antigens (rHBcAg, TT and HBc peptides) and irradiated, autologous PBLs as APCs (5x104 PBLs per well). All cultures were performed at least in duplicate and in triplicate when sufficient cells were available. At 18 h before harvesting the cultures, 0·5 µCi (1·85 MBq) [3H]thymidine (TdR) was added and the radioactivity incorporated by the cells was determined by -counting.
In peptide stimulation assays performed to determine the specificity of T cell clones, autologous EpsteinBarr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) that were treated previously with mitomycin C (50 µg/ml) were used as APCs (5x104 cells per well). LCLs were used as APCs rather than fresh PBMCs because the former adequately presented peptides and because we wanted to limit the number and volume of phlebotomies to a strict minimum.
In order to determine the restriction of T cell recognition, blocking assays were performed in which T cell proliferation was measured in the presence or absence of mouse anti-human HLA class I, class II (DR, DP and DQ) and isotypic control antibodies (1 µg/ml). In confirmatory assays, T cell proliferation was measured following antigen presentation by HLA-typed allogeneic PBLs displaying different haplotypes.
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Results |
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Discussion |
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Our analysis of the T cell recognition of HBcAg by three HLA DR13-positive subjects who recovered from an acute HBV infection shows that all three subjects display a significant response to recombinant HBcAg and that each individual recognizes at least four different HBcAg-derived peptides. This confirms the observations of Diepolder et al. (1998) who analysed eight DR13-positive AHB patients. The frequency of peptide recognition by our patients displayed the following hierarchy: P1 and P15 (aa 120 and 141160) were recognized by three of three subjects; P2, P5 and P12 (aa 1130, 4160 and 111131) were recognized by two of three subjects and P4, P6, P10, P13 and P14 (aa 3150,5170, 91110, 121140 and 131150) were recognized by one of three subjects. This differs somewhat from the frequencies reported by other investigators (Ferrari et al., 1991
; Jung et al., 1995
; Penna et al., 1997
; Diepolder et al., 1998
) who found peptide spanning aa 5069 to be the most immunodominant one (it was recognized by seven of eight subjects) and further disclosed the following hierarchy of recognition: of eight subjects, peptide 6185 was recognized by six, peptide 81105 was recognized by five, peptide 141165 was recognized by four, peptide 101125 was recognized by three, peptides 125, 2145, 121145 and 161183 were recognized by two and peptide 4165 was recognized by one. The difference in the hierarchy observed may be due to differences in the patient populations examined, differences in the qualities of the peptides employed and differences in the culture methods used. However, when the peptide recognition pattern of DR13-positive and -negative subjects was compared, it was observed that the three immunodominant peptides were the same in both groups of patients and that only one peptide, namely peptide 141165, was preferentially recognized by HLA DR13-positive individuals (P<0·01). The investigators were, however, unable to demonstrate that this peptide (aa 141165) was presented to the T cells by the DR13 molecule. Our study provides ample evidence that this is indeed the case. Using blocking experiments with mAbs directed against DR and lymphoproliferation assays wherein HBcAg or peptide 141165 were presented to P15-specific T lymphocytes by haploidentical PBLs, we demonstrated that six clones and one T cell line specific for P15 recognized this peptide in the context of the HLA DR13 molecule.
To exclude the occurrence of some inexplicable or accidental phenomenon causing all T cell clones generated from subject AHB1 to recognize the same peptide P15 (aa 141160), we started a new cloning procedure with cells from subject AHB1 and, in addition, we produced HBcAg-specific T cell lines from PBLs of two other AHB patients (AHB2 and AHB3). Each experiment led to the production of P15-specific T lymphocytes, making the possibility that results were experimental artefacts very unlikely and suggesting that P15-specific T cells are consistently induced during an acute HBV infection in DR13-positive patients.
In an attempt to understand the molecular basis of the preferential presentation of P15 by the HLA DR13 molecule, we analysed the recognition characteristics of four T cell clones using a series of N- and C-terminally truncated and single alanine-substituted peptides spanning HBcAg aa 141160. All four T cell clones recognized the minimal epitope HBcAg 147TVVRRRGRSP156. Our analysis further revealed the critical role of certain (V148, V149 and R151), and the less important role of other (R150, R151, G153 and P156), amino acids, but the information gathered did not allow us to define which amino acids might be involved in the binding to the DR13 molecule and which are recognized by the T cell receptor. Importantly, this analysis showed the polyclonal nature of the response to this dominant epitope within HBcAg.
In the past, therapeutic immunizations with HBV envelope proteins have produced disappointing results (Couillin et al., 1999 ; Pol et al., 2001
). Recent studies have shown that transfer of immunity to HBV can be achieved by bone marrow transplantation from immune donors (Ilan et al., 1993
; Shouval et al., 1993
; Shouval & Ilan, 1995
; Lau et al., 1997
). Recently it was demonstrated that this resolution of HBV infection was associated with transfer of HBcAg-specific T cells present in the marrow graft (Lau et al., 2002
). Interestingly, in this study, freshly isolated PBMCs from four immune donors responded to HBcAg P1, P2, P5, P6, P7, P8, P15 and P16 (Lau et al., 2002
). Similar responses were observed with freshly isolated PBMCs from recipients. Moreover, the responding T cells found in the recipients were shown to be of donor origin. These results, together with previous observations (Diepolder et al., 1998
) and our results, strongly indicate that recognition of the T cell epitope at aa 141160 can be correlated with virus clearance in HLA DR13 subjects.
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Acknowledgments |
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References |
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Received 5 April 2002;
accepted 22 July 2002.
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