A repetitive sequence of Epstein–Barr virus nuclear antigen 6 comprises overlapping T cell epitopes which induce HLA-DR-restricted CD4+ T lymphocytes

Éva Rajnavölgyi1,2, Noémi Nagy1, Britt Thuresson3, Zsuzsa Dosztányi4, Ágnes Simon4, István Simon4, Robert W. Karr5, Ingemar Ernberg1, Eva Klein1 and Kerstin I. Falk1

1 Microbiology and Tumorbiology Center, Karolinska Institute, 17 177 Stockholm, Sweden
2 Department of Immunology, L. Eötvös University, Jávorka S. 14, 2131 Göd, Hungary
3 Blood Centre University Hospital, 22 185 Lund, Sweden
4 Institute of Enzymology, 1518 Budapest, Hungary
5 Department of Immunology, G. D. Searle & Co. Ltd, St Louis, MO 63131-3850, USA

Correspondence to: É. Rajnavölgyi


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Most human adults carry the Epstein–Barr virus (EBV) and develop immunological memory against the structural and the virus-encoded cellular proteins. The EBV nuclear antigen 6 (EBNA6) elicits cytotoxic T cell responses and it also maintains a persistent antibody response. The majority of sera from EBV-seropositive individuals reacts with a synthetic peptide, p63, comprising 21 amino acids of a repetitive region of EBNA6. CD4+ T lymphocytes, with specificity for p63, could be recalled from the T cell repertoire of EBV carriers that expressed certain HLA-DR allotypes which were identified as good binders of p63 by an in vitro flow cytometric assay. Analysis of the HLA-DR/p63 interaction by molecular mechanics calculations indicated the presence of multiple overlapping epitopes which were predicted to bind in a HLA-DRB1 allo- and subtype-specific manner. Specific activation of p63-selected long-term CD4+ T cell cultures resulted in a proliferative response, in the production of IL-2 and in the secretion of high levels of tumor necrosis factor as measured by bioassays. Proliferation and cytokine production of p63-specific T cells could be induced by p63-loaded HLA-DR-matched antigen-presenting cells and by B cells co-expressing relevant HLA-DR molecules and EBNA6. Our results show that peptides of an EBNA6 repeat region induce CD4+ T cells which can react with EBNA6-carrying cells in many individuals. We suggest that these Th cells may be important in conditioning dendritic cells for initiation potent virus-specific immune responses, provide help for EBV-specific B cells, drive IgG isotype switch and support the sustained effector function of memory cytotoxic T lymphocytes.

Keywords: human CD4+ T lymphocyte, HLA-DR–peptide interaction, virus-specific immunity


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Almost every individual in the human population carries the Epstein–Barr virus (EBV), classified as a lymphotropic {gamma}-herpesvirus which upon primary infection can, but does not always, causes infectious mononucleosis (IM) (1). EBV has a strong tropism for B-lymphocytes resulting in latent infection with growth transforming capacity in vitro and with oncogenic potential in vivo (reviewed in 2,3). In vitro experiments and accumulated clinical experience established that the healthy virus carrier state can be ascribed to the function of the immune system which ensures that the immortalizing capacity of the virus is not manifested (reviewed in 2–4). The EBV-specific immune response includes the production of antibody and the expansion of T lymphocytes specific for both viral structural and virally encoded cellular antigens (59). Due to their killing potential MHC class I-restricted CD8+ cytolytic T lymphocytes (CTL) received special attention, and the targets and parameters of the EBV-specific response have been intensively studied (7,8). Using recently developed technologies for the detection of virus-specific CTL revealed that EBV-specific CD8+ T cells appear at high frequency after primary infection and a heterogeneous CTL pool, directed against both lytic cycle and latent EBV antigens, remains detectable for a long period of time (10,11).

EBV-immortalized lymphoblastoid B cell lines (LCL) express nine virally encoded proteins, six of them are localized in the nucleus hence the name of EBV nuclear antigen (EBNA) 1, 2, 3, 4, 5 and 6, and three latent membrane proteins (LMP) 1, 2A and 2B are associated with the plasma membrane (3,4). LCL expressing this set of viral antigen function as highly efficient professional antigen-presenting cells (APC) and can prime or challenge T cells in virus carriers or in vitro. An increased incidence of B cell lymphomas, which express a similar pattern of EBV latent antigen as LCL, occurs in immunocompromised individuals (12).

The continuous presence of viral antigen is not essential for developing CTL memory (13) but it can result in the reactivation of memory CTL that is required for conferring protection (14). The long-term expansion and sustained effector activity of activated T lymphocytes, however, has been attributed to CD4+ T cells as demonstrated in a murine {gamma}-herpes virus model which resembles the latent EBV infection of humans (15) and also in other persistent viral infections (reviewed in 16).

The knowledge concerning the fine specificity and function of CD4+ Th cells recognizing EBNA during acute or persistent EBV infection is rather limited (17,18). The goal of this study was to identify Th epitopes in EBNA6 and to characterize the functional properties of the specific Th cells. One of the characteristic features of the EBV genome is its high content of repetitive sequences within some of the protein coding regions (19). EBNA1, which is expressed in all EBV genome carrying cells (20) and can be detected in all EBV-related malignancies (4), contains a long Gly–Ala repeat which interferes with the ubiquitine-proteasome-mediated degradation of EBNA1, and consequently has an inhibitory activity on MHC class I-restricted presentation and CTL recognition (21). A poly-Pro region is found in EBNA2 (22) and two repetitive regions, unique for EBNA6, have also been identified (23). Defined repeats of the EBNA are dominant targets of antibody recognition (1,19,24). After primary infection the kinetics of the IgG-type antibody response against EBNA is sequential in the following order: EBNA2, 1 and 6 (25). The titer of antibody recognizing synthetic peptides comprising certain repetitive sequences of EBNA1 and EBNA6 increases after the onset of IM, and remains at an individually variable but permanent level while the EBNA2-specific titer declines (5).

The immunodominance of B cell epitopes located in the EBNA repeats prompted us to investigate the role of a repeat region in the activation of MHC class II-restricted CD4+ Th lymphocytes. The approach was based on previous results showing that Th cell epitopes of certain proteins are often localized close to or within B cell epitopes (26), and the induction of a humoral response to internal viral proteins requires co-localization of B and Th cell epitopes in the same protein (27). We have chosen the synthetic peptide p63, which comprises a segment of the EBNA6 region encoded by the nx39bp tandem repeat, for further studies on the role of EBNA repeats in immune recognition. In earlier studies our group identified this EBNA6 region as a dominant antibody epitope recognized by ~80% of sera defined as EBV- seropositive by the EBNA-specific antibody test (5). Here we demonstrate that the sequence of p63 comprises multiple overlapping regions which function as promiscuous epitopes recognized by Th cells in the context of certain HLA-DR allotypes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antigens and peptides
The soluble gp340 protein, produced in a baculovirus expression system, was a generous gift of A. J. Morgan (University of Bristol, Bristol, UK). The p63 peptide, comprising the ProAlaProGlnAlaProTyrGlnGlyTyrGlnGluProProAlaProGlnAlaProTyr sequence of the EBNA6 protein (corresponding to position 100665–100724 in the B95-8 genome) was synthesized as described previously (5). The human influenza A virus (H1N1) hemagglutinin (HA) peptides HA317-329 (H1 and H3 subtypes) and HA306-341 (H1 subtype), used as controls for the T cell activation assays, were described previously (28).

HLA typing
HLA typing was performed by the serological lymphocytotoxicity method (29). Subtype determination was performed by genomic typing using the PCR-SSP technique (30). The typing kits were purchased from Dynal (Oslo, Norway).

Cell lines
B95-8-transformed LCL were established from peripheral blood mononuclear cells (PBMC). The mouse mAb L243 (ATCC HB55) and LB.3.1 (31,32), both specific for the human HLA-DR {alpha} chain, the LG-2 (homozygous for HLA-DRA*0101; DRB1*0101; HLA-DR1) and the Priess (homozygous for HLA-DRA*0101; DRB1*0401; HLA-DR4Dw4) LCL were the generous gift of Zoltán Nagy (La Roche, New York). The MHC class II-negative BLS-1 (33) and Sweig (34) (homozygous for HLA-DRA*0101; DRB1*1101; HLA-DR5Dw11) LCL were kindly provided by Alexander Diehl (MTC, Karolinska Institute, Stockholm, Sweden).

The EBV-negative Burkitt's lymphoma (BL) cell line BL41 and its EBV-converted subline, BL41/95 (35), express HLA-DR1 and HLA-DR6Dw6. Bjab and DG75 BL and their EBNA6 transfected sublines (36,25) were typed as HLA-DRA*0101; DRB1*1201 (HLA-DR5Dw12/HLA-DR6Dw13) and HLA-DRA*0101; DRB1*0404 (HLA-DR4Dw14/HLA-DR6) respectively. The Burkitt's lymphoma Raji cells express EBNA1–5 but not EBNA6. The cell lines were cultured in RPMI supplemented with antibiotics and 10% FCS. The murine L cells transfected with human HLA-DRA*0101 in combination with HLA-DRB1*0101 (L57.23, HLA-DR1), HLA-DRB1*0401 (L243.6), HLA-DRB1*0404 (L300.7) or HLA-DRB1*0402 (L164.II) were cultured in IMDM supplemented with 10% FCS and 5x10–5 M 2-mercaptoethanol. The IL-2-dependent CTLL-2 cells were cultured as described previously (36). The WEHI-164 clone 13, sensitive for tumor necrosis factor (TNF)-mediated apoptosis, was used as described (37).

Antibody assays
The detection of human serum antibody against the EBV nuclear antigen EBNA1–5 or to EBNA6 was performed by the anti-complement immunofluorescence (ACIF) test using Raji cells and the EBNA6 transfected DG75 BL cells respectively (25). The level of serum antibody specific for the soluble gp340 protein and for the p63 peptide was measured by ELISA and detected by human IgG isotype-specific mouse mAb (Southern Biotechnology Associates, Birmingham, AL). Antibody binding was detected by biotinylated anti-mouse antibody and horseradish peroxidase-labeled streptavidin (Amersham, Arlington Heights, IL).

Measurement of peptide-specific T cell activation
PBMC of healthy individuals were isolated by Hypaque (Pharmacia, Uppsala, Sweden) separation and cell suspensions were distributed in U-bottomed 96-well plates at 1x106/ml concentration (200 µl) in serum-free medium (AIMV; Gibco, Frederick, MD) in the presence of 10 µg/ml peptide. Seven days later the cultures were transferred to flat-bottomed plates and fed with 10 ng/ml IL-2 (a generous gift of Ajinomoto, Kyoto, Japan) and/or with 10 ng/ml IL-4 (Peprotech EC, London, UK). On day 14 aliquots of cells were reactivated with 10 µg/ml peptide in the presence of irradiated (30 Gy) autologous PBMC (1x105 cells/well) or irradiated (70 Gy) autologous LCL (1x104 cells/well) used as APC. Cultures, set up in the absence of peptide, were used as negative controls. T cell proliferation was monitored by DNA synthesis measured by [3H]thymidine incorporation in the last 16 h of the 3 day cultures. The c.p.m. values measured in the positive and negative cultures were used to calculate the stimulation index (SI). Cultures characterized by an SI > 3.0 were considered as positive. The proliferation of T cell lines was measured by a similar method using various numbers of irradiated LCL or other APC. The proliferative potential of irradiated APC was monitored in control wells of all experiments.

Establishment and maintenance of CD4+ T cell lines
Microcultures were repulsed with 10 µg/ml p63 peptide presented on irradiated (70 Gy) autologous LCL (104 cells/well) in AIMV medium supplemented with 1 ng/ml IL-2. T cell proliferation was supported by feeding the cultures every second day with AIMV medium containing gradually increasing amounts (2, 4 and 8 ng/ml) of IL-2. After four rounds of re-stimulation the cultures were reactivated biweekly by 2 µg/ml phytohemagglutinin together with 1x105 cells/well irradiated (30 Gy) PBMC in combination with increasing amounts of lymphokines. Antigen-specific activation of T cells was measured 10–14 days after the last challenge.

Peptide binding assay and flow cytometry
N-terminal biotinylation of the p63 peptide and the binding assay was performed as described earlier (38). Cells (2x106) were incubated for 5 h at 37°C with 20 and 10 µmol biotinylated peptide. Inhibition of the peptide binding by the LB.3.1 mAb, specific for the peptide binding site of HLA-DR molecules (31,32) was performed under the same conditions in the presence of 2 µl mAb-containing supernatant. Fluorescence intensity was measured by a Becton Dickinson (San Jose, CA) FACStar and CellQuest software was used to collect data and for analysis. Viable cells were gated on the basis of forward and side scatter. Data are documented as percent increase of fluorescence (arbitrary units) calculated from the median values measured for the peptide-preincubated and control samples incubated without the biotinylated peptide. The HLA-DR expression of APC was monitored by the LB.3.1 and L243 (31,32) mouse mAb detected by biotinylated anti-mouse antibody (Southern Biotechnology Associates) and streptavidin–phycoerythrin (PE) (Sigma, St Louis, MO). Phenotypic characterization of the T cell lines was studied after four peptide-specific activation using the following antibodies: (FITC) RPE-labeled anti-CD4 (MT310) and anti-CD8 (DK25), both purchased from Dako (LOCATION???, Denmark) and FITC-labeled anti-CD3 (Leu-4) (Becton Dickinson, Glostrup, Denmark).

Monitoring T cell activation by lymphokine production
T cells (1–2x104) were cultured in 96-well U-bottom tissue culture plates (Nunc, LOCATION??) in complete RPMI in the presence of graded concentrations of peptide and 1–2x104 autologous, irradiated LCL (70 Gy) or graded numbers of irradiated (70 Gy) or viable BL cells positive or negative for EBNA6. Culture supernatants (25–75 µl) were removed at 24 h of culture and the amount of secreted IL-2 was measured by the proliferation of CTLL-2 detector cells (28). The TNF content of the supernatants was titrated by the detection of its killing activity for WEHI-164 clone 13 cells (37). The background activity of the various irradiated APC was measured in control wells, and these percent killing values were used as background and were subtracted from the percent values measured in the other cultures. The amount of IL-4 and IFN-{gamma} in the cell culture supernatants was measured by ELISA using the antibody pairs purchased from PharMingen (San Diego, CA) and R&D Systems (Minneapolis, MN) respectively.

Computer graphics and energy minimization
Computer graphics and energy minimization were carried out using the INSIGHT II software package, containing DISCOVER (Biosym Technology, San Diego, CA) on a Silicon Graphics Indigo Workstation. Coordinates of HLA-DR1, complexed with an influenza virus (H3N2) HA peptide (HA306-318) (39), were taken from the Brookhaven Database. Co-ordinates of the HLA-DRA*0101; DRB1*0402 molecule were created from the X-ray data of HLA-DRA*0101; DRB1*0401 complexed with a peptide of human collagen type II, kindly provided by A. Dessen (40), after replacing four residues in the protein: ß86 Gly -> Val in pocket 1, which reduces pocket size, ß71 Lys -> Glu, which alters the overall charge of pockets 4 and 7, ß70 Gln -> Asp in pocket 4, which modifies the size and shape of this pocket, and ß67 Leu -> Ile in pocket 7, which has an effect on pocket shape. The bound peptides were replaced by dodecapeptides corresponding to different alignments in the p63 peptide sequence. Three parameters were chosen to characterize the HLA-DR-p63 interaction: (i) the total number of H-bonds created between amino acids of the peptide and of the HLA-DR binding groove, (ii) the area of the anchoring residues still accessible by solvent after occupying pockets 1 and 4, and (iii) the number of contacts between the amino acids of the peptide with residues ß70 and ß71 of pocket 4. As a positive control the same parameters were given for the original complex of HA306-318 and HLA-DR1. Hydrogen atoms were added to the heavy atoms and energy minimization was performed using CVFF force field. The cut-off distance of 15 Å was used for unbound interactions, and energy minimization with steepest descent and conjugate gradient algorithms went on until the maximal derivative of the energy function was <0.1 kcal/mol/Å. The number of H-bonds was calculated by considering donor and acceptor atoms closer than 2.5 Å and within an angle of 120–180° degrees. The solvent-accessible area of peptidic amino acids, which is indicative of the occupation of pockets 1 and 4, was calculated by the MSRoll program, using a probe radius of 1.4 Å (41).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The EBV-specific antibody response
The sera of 10 EBV carriers but not the serum of one seronegative individual (MA) of known HLA-DR haplotypes exhibited reactivity with EBNA detected by the ACIF method (Table 1Go). Some of these sera reacted with the EBNA6- derived p63 peptide, showing substantial quantitative variations (Fig. 1BGo). These antibodies were of the IgG1 and IgG3 isotypes, low levels of IgG2 or IgG4 were detected (Fig. 2A and DGo). The sera had variable levels of antibody specific for the lytic antigen gp340 viral membrane protein (Fig. 1AGo) confined also to the complement-fixing IgG subclasses with dominance of IgG1. The isotype distribution of the antibody raised against a lytic and a latent EBV antigen was similar, but the magnitude of the two responses did not correlate (Fig. 1Go).


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Table 1. The HLA-DR haplotype, the presence of EBNA-specific serum antibody and the proliferative T cell response to secondary in vitro p63 stimulation of the selected donors
 


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Fig. 1. The EBV-specific antibody titers measured in the serum of the tested donors. The titer of gp340 protein- (A) and the p63 peptide- (B) specific IgG1 and IgG3 antibody was measured by indirect ELISA as described in Methods. Data are given as optical densities (OD405) measured at 100- (A) or 10- (B) fold serum dilutions. Mean values of duplicates of a typical experiment out of three are given.

 



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Fig. 2. The antibody and T cell response of donors GK and KM to p63 and HA peptides. Sera were titrated at various dilutions for reactivity with the p63 peptide and bound IgG1 ({blacklozenge}), IgG2 ({blacksquare}), IgG3 ({blacktriangleup}) and IgG4 (•) isotypes were detected (A and D). PBMC of donors KM (B and C) and GK (E and F) were cultured with the peptides for 2 weeks, and after secondary re-stimulation with the same peptide the selected cells were tested for peptide-specific proliferation as described in Methods. DNA synthesis was detected by the [3H]thymidine incorporation assay in the presence of autologous LCL (empty columns) and peptide-loaded LCL (black columns). The data demonstrate the proliferative response detected in individual cultures containing PBMC of two donors as examples for the frequency of responding cultures and for the relative responsiveness of the EBNA6-derived p63 (C and F) and of the influenza-derived HA306-341 (B) or HA317-329 (E) peptides. The insert in (A) shows the effect of p63 absorption on peptide-specific antibody levels detected by anti-IgG1 secondary antibody.

 
A survey of the 10 selected donors (Table 1Go) and 76 HLA-typed seropositive individuals (data not shown) showed 70% positive reactions with the p63 peptide with no obvious correlation with the HLA haplotype. The sera of BK, KM, EK and D266 donors (Table 1Go) with high p63-specific antibody titers were absorbed with solid-phase bound p63 peptide, and the reactivity of sera with p63 and with EBNA6-transfected DG75 cells was tested. The insert of Fig. 2Go(A) shows that the serum of KM has substantially lost p63 reactivity and EBNA6-specific antibodies were undetectable in the ACIF test (Table 1Go). Similar results were obtained with BK, D266 and EK donors (Table 1Go), indicating that the serum antibody recognized the same epitope in EBNA6 and in the p63 peptide. This finding confirmed previous results and suggested that the recognition of the repetitive region in the EBNA6 protein dominated the antibody response (5).

The reactivity of blood T lymphocytes with the p63 peptide
PBMC of one EBV-seronegative and nine EBV-seropositive donors with known HLA-DR haplotypes (Table 1Go) were tested for reactivity with the p63 peptide. T cell activation was estimated after secondary exposure of the in vitro cultured cells to the p63 peptide by the number of microcultures in which DNA synthesis was induced and by the magnitude of the response expressed as SI. Irradiated autologous LCL or PBMC were used as APC. The results given in Table 1Go showed that the responding individuals BK, ST, D3, KM and D266 carried the HLA-DR1 or the HLA-DRB1*0404 or HLA-DRB1*0401 alleles. The cells of donors GK and RS, both carrying HLA-DR4,B1*0402, did not respond. The response of KM T cells to the HA306-341 influenza peptide demonstrated that the magnitude of the p63-specific response was comparable to the immunodominant HLA-DR1-restricted HA306-318 epitope (Fig. 2B and CGo). The T cell response of GK to the influenza virus HA peptide (HA317-329 H1) ruled out the possibility that the negative results obtained with these T cells were due to technical factors or impaired reactivity (Fig. 2E–FGo). Thus p63-specific T cells could be recalled from the repertoire of EBV-seropositive individuals and the differential responsiveness of the HLA-typed individuals suggested that the peptide was recognized in a HLA-DR-restricted manner. However, the contribution of HLA-DP or HLA-DQ molecules in p63 presentation could not be excluded. The occurrence of high p63-specific antibody levels in individuals without detectable p63-specific Th cell responses (in donors D1, RS and EK, Table 1Go) indicated that Th cells, recognizing epitopes of EBNA6 outside of the repetitive region in the context of other HLA-class II allotypes, may also provide cognate help for p63-specific B cells.

Binding of the biotinylated p63 peptide to various HLA-DRB1-defined allotypes
Two types of APC, LCL homozygous for defined HLA-DRB1 alleles and murine L cells transfected with the HLA-DRA and with various HLA-DRB1 genes, were used to test the binding of the p63 peptide to HLA-DR molecules expressed on the surface of viable cells relevant for T cell recognition. In order to validate the use of various LCL for estimating the peptide binding capacity, their expression of HLA-DR molecules was tested by reactivity with two HLA-DR specific mAb. The results showed that all the tested cells expressed similar levels of HLA-DR molecules (Table 2Go). The results of peptide binding to the LCL surface HLA-DR are given in Fig. 3Go(A). LCL expressing HLA-DRB1*0101 (LG-2), -DRB1*0401 (Priess) or -DRB1*1101 (Sweig) molecules bound the biotinylated p63 peptide in a dose-dependent manner. Specificity was verified by the lack of binding to the MHC class II-negative BLS-1 cells and by inhibition with the LB.3.1 mAb which reacts with the peptide binding site of the HLA-DR molecules. KM LCL, which expresses HLA-DR1 molecules (Table 1Go), also bound the p63 peptide and the interaction could be inhibited by the LB.3.1 mAb. Since this blocking LB3.1 mAb is toxic for LCL in long-term cultures (31), it was used only in the short-term binding assays.


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Table 2. The expression of HLA-DR molecules on different LCL and transfected murine L cells
 


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Fig. 3. Binding of the p63 peptide to various HLA-DR molecules. The binding of biotinylated p63 peptide to the homozygous LCL LG-2 (HLA-DRB1*0101), Priess (HLA-DRB1*0401), Sweig (HLA-DRB1*1101) (A) or to HLA-DR transfectants L57.23 (HLA-DRB1*0101), L300.7 (HLA-DRB1*0404), L243.6 (HLA-DRB1*0401) and L164.II (HLA-DRB1*0402) (B) was detected at 20 (dark columns) or 10 (open columns) µmol p63 peptide concentrations. Cells (2x106) were washed with serum-free medium and incubated with the peptide for 5 h at 37°C in the presence of 0.1% BSA. After removal of unbound peptides cell surface bound p63 was detected with PE-labeled streptavidin. In the inhibition experiments the binding site-specific LB.3 mAb was present throughout the culture period (shaded columns). To detect non-HLA-DR-related unspecific binding the same assay was performed with the HLA-DR-negative human BLS-1 (A) and the murine A9 (B) cells. Mean values of the increase of mean fluorescence ± SD were calculated from five to eight independent experiments and are given in arbitrary units determined as described in Methods.

 
Based on the reactivity with the HLA-DR-specific mAb, the expression of HLA-DR molecules was similar on the various transfected L cells but it was lower than on the LCL (Table 2Go). Cells transfected with the genes encoding for the human HLA-DRA in combination with the HLA-DRB1*0101 (L57.23), -DRB1*0401 (L243.6) or -DRB1*0404 (L300.7), but not with the -DRB1*0402 (L164.II) allele bound the biotinylated p63 peptide in a dose-dependent manner. The specificity imposed by the presence of the HLA-DR expression was revealed by the lack of binding to the A/9 HLA-DR-negative cells which was the related subline of the recipient of the transfected genes. In addition, peptide binding to the transfectants was inhibited by the LB.3.1 mAb (Fig. 3BGo).

Thus the p63 peptide bound promiscuously, but in a subtype-specific manner to the related HLA-DR1, -DR11(5) and certain -DR4 peptide binding grooves. The specificity pattern provided a reasonable explanation for the differences in the p63-specific T cell responses seen in the panel of the lymphocyte donors (Table 1Go).

The structural background of p63 binding to different HLA-DR allotypes
To predict core regions, which may interact with the peptide binding grooves of HLA-DR1, -DR11(5) and certain -DR4 subtypes, dodecapeptides comprising overlapping sequences within p63 were placed to the binding groove of HLA-DR molecules created on the basis of known three dimensional structures of HLA-DR–peptide complexes as described in Methods. Interaction of peptides, covering different alignments of p63, with HLA-DR1 and HLA-DRB1*0402 molecules was characterized by selected parameters calculated after energy minimization of the complexes. The total number of H-bonds, which link the docking peptide to HLA-DR residues, reflects overall fitting of the peptide backbone. The water-accessible area of amino acids contacting pockets 1 and 4 is indicative of acceptance of the corresponding amino acid side chains by the pocket. The number of atomic contacts between peptide amino acids and ß70 and ß71 of pocket 4 also indicates the vicinity of peptide side chains to amino acids of the pocket.

The p63 sequence offers multiple possible alignments for HLA-DR binding as listed in Table 3Go. In good correlation with the results of the binding assay (Fig. 3Go), various dodecapeptides were predicted to interact with HLA-DR1 but none of these alignments fulfilled the requirements of efficient interaction with the HLA-DRB1*0402 binding site (Table 3Go). Glu at position 12 is completely buried in pocket 4 of HLA-DR1 (Table 3Go, row 2) but fails to interact with the same pocket formed by the HLA-DRB1*0402 ß chain harboring a critical Lys -> Glu substitution at position ß71 which modifies its charge characteristics. Alignments which place Tyr into pocket 1 are not accepted by HLA-DRB1*0402 since the ß86 Gly -> Val substitution apparently excludes Tyr from this pocket (Table 3Go, row 1,3) and pocket 4 cannot be occupied by Glu (Table 3Go, row 2). Pocket 4 of HLA-DR1 can accommodate Gln. The alignments, which place this amino acid to position 4, allow less contacts with the crucial ß70 and ß71 residues in HLA-DR4,B1*0402 than in HLA-DR1, although two out of the four possible alignments result in comparable solvent-accessible areas in both proteins (Table 3Go, rows 4–7).


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Table 3. The predicted fitting of different p63 sequences to the HLA-DR1 and HLA-DR4,B1*0402 peptide binding sites
 
The same analysis of the HLA-DR11(5), HLA-DRB1*0401 and HLA-DRB1*0404 binding sites revealed that multiple alignments could fulfil the requirements of efficient binding to these HLA-DR molecules (data not shown). These predictions are in line with the results of the p63 binding assay (Fig. 3Go) and with the p63-specific T cell reactivity of EBV-seropositive individuals (Table 1Go).

Specificity and functional characteristics of p63-specific T cells
Several responder cultures of D2, KM, BK and ST were further expanded, and their CD4/CD8 phenotype and the pattern of cytokine secretion was monitored. No CD8+ T lymphocytes were detected in the peptide-reactive cultures while stimulation with high numbers of autologous LCL induced CD8+ T cells as well. Similar to the LCL-stimulated T cells, the p63-specific T cell cultures (>15) produced IL-2 and TNF, and in some cultures IFN-{gamma} but not IL-4 was detected. Homogeneous populations of CD3+CD4+CD8 T lymphocytes of two long-term lines, derived from donor KM, i.e. KMB5 and KMD6, were analyzed for functional activity. KMB5 T cells entered DNA synthesis (Fig. 4AGo) and secreted IL-2 (Fig 4BGo) when exposed to the p63 peptide presented on murine L cells which carried HLA-DR1, HLA-DRB1*0401 or HLA-DRB10404 allotypes but not by the L cells expressing HLA-DR4,B1*0402 molecules (Fig. 4Go). Since IL-2 is consumed upon T cell proliferation, in some cases high T cell proliferation was accompanied by reduced level of free IL-2 (Fig. 4BGo). According to these results the KMB5 T cells recognized and responded to the p63 peptide if presented by the autologous HLA-DR1 and by the related HLA-DR4 molecules.



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Fig. 4. Activation of KMB5 T cells by the p63 peptide presented on murine L cells carrying different HLA-DR allotypes. Peptide-specific proliferation (A) and IL-2 production (B) were tested by co-culturing 5x103 KMB5 T cells with 1x105 irradiated HLA-DR transfectants without (control) or in the presence of 50, 10 or 2 µg/ml p63 peptide. Supernatants for IL-2 detection were harvested after 24 h of culture, DNA synthesis was measured after 48 h. Proliferation of KMB5 cells (A) or that of the CTLL-2 indicator cells (B) was given as c.p.m. values obtained from duplicates in the [3H]thymidine incorporation assay and corrected by the c.p.m. values measured in the corresponding control cultures (lacking the p63 peptide). The HLA-DR-negative A9 murine fibrosarcoma cell was used as a negative control for the HLA-DR-mediated response induced by the transfectants. A typical experiment out of three is given.

 
KMB5 T cells were stimulated by the p63 peptide presented on autologous LCL but not on the HLA class II-negative BLS-1 cells (Fig. 5BGo). They were activated in a dose-dependent manner also by high numbers of autologous LCL irrespective of p63 peptide loading (Fig. 5AGo). Since irradiated LCL had no proliferative capacity in the absence of T cells, these results suggested that KM LCL not only presented the exogenously added p63 peptide but also expressed HLA-DR1–peptide complexes which were generated from endogenous EBNA6 and recognized by the KMB5 T cells. T cell proliferation was accompanied by the secretion of IL-2 (Fig. 4BGo) and TNF (Fig. 5CGo).



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Fig. 5. Activation of KMB5 T cells by autologous LCL. KM LCL (1x106) were preincubated without or with 20 µg/ml p63 peptide for 1 h at 37°C and added at graded numbers to 1x103 T cells (A and C). Proliferation of 1x103 T cells, cultured in the presence of graded doses of p63 peptide and 1x104 autologous KM LCL or BLS-1 cells (B) was measured on day 3 of culture. TNF secretion (C) was detected in cell culture supernatants taken after 24 h. Percentage of apoptotic indicator cells incubated with the cell culture supernatant was given as mean values of duplicates of a typical experiment out of three are documented.

 
In a further step we addressed the question whether EBNA6 expressed in B cells would generate peptides recognized by the apparently p63-specific KMB5 T cells. We used the EBNA6 transfected sublines of the EBV-negative DG75 and Bjab BL lines which expressed HLA-DR4,B1*0404 and HLA-DR11(5) molecules respectively. The EBV-negative parental cells could present the exogenously added p63 peptide to KMB5 T cells in a dose-dependent manner (data not shown). In mixed cultures of KMB5 T cells and irradiated EBNA6-carrying cells DNA synthesis was detected indicative for T cell activation (Fig. 6A and BGo). In addition IL-2 (data not shown) and TNF (Fig. 6C and DGo) secretion was also detected. The BL41/95 cells, which carried the EBV genome as a result of in vitro infection by EBV, but lacked the EBNA6 protein, did not activate the KMB5 T cells, although they expressed HLA-DR1 molecules (Fig. 6CGo). The results obtained with the EBNA6-positive cells suggested that EBNA6-derived peptides encompassing the repetitive sequence of the EBNA6 protein could be generated from intracellular sources, loaded to HLA-DR molecules and presented on the cell surface. The fact that the addition of the p63 peptide to KMB5 T cells, without any additional APC, did not result in T cell activation (data not shown) confirmed that T cells by themselves were not able to present the EBNA6-derived peptides.



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Fig. 6. Activation of KMB5 T cells by EBNA6-carrying B cells. Proliferation (A and B) of 2x103 T cells was measured in the presence of 1x105/well (dark columns), 5x104/well (shaded columns) or 2.5x104/well (empty columns) irradiated DG75 (A) or Bjab (B) BL cells and with their EBNA6 transfected DG75-EBNA6 and Bjab-EBNA6 sublines. TNF production (C) of 2x103 T cells was measured in the presence of graded numbers of irradiated EBNA6-negative Bjab, BL41, BL41/95 BL cells or with the EBNA6-positive Bjab-EBNA6. TNF production was compared in supernatants of cultures set-up with irradiated or viable Bjab, Bjab-EBNA6 or Bjab-EBNA3 used as a vector control (D). The percentage of apoptotic WEHI-164 indicator cells, proportional to the amount of T cell-derived TNF, was calculated by correction with the background values detected in the supernatant of cultured B cells. Mean values of duplicates of a typical experiment out of three are documented.

 
In these experiments the EBNA6-derived peptides could have been generated either by an endogenous source or by exogenous EBNA6 released from cells as a consequence of irradiation-induced apoptosis. Monitoring the activation of T cells by TNF production it was possible to compare lymphokine production in response to both irradiated and viable APC. Using either KMB5 (Fig. 6DGo) or KMD6 (data not shown) T cells, the viable APC were better stimulators. Although it was difficult to exclude the release of nuclear proteins in these cultures, this finding might suggest that peptides, recognized by p63-specific T cells, could also be generated from the endogenous EBNA6 protein and presented by viable B cells.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We examined an EBNA6 repetitive region for its potential to function as a target of Th cell recognition by using the p63 synthetic peptide which comprises the sequence of one and a half repeats. Promiscuous binding of this peptide to certain HLA-DR molecules substantiated the pattern of p63 recognition by the T lymphocytes of HLA-typed individuals. Molecular analysis of the HLA-DR–peptide interaction predicted multiple overlapping epitopes within the repeat region. Selection of p63-specific Th cells from high responder virus carriers demonstrated the existence of such Th cells in the periphery and showed that this region of the EBNA6 protein is able to induce CD4+ Th lymphocytes in a HLA-DR-restricted manner. The functional assays revealed that the peptide-specific Th cells produce IL-2, TNF and some of them IFN-{gamma} upon stimulation. p63-specific T cells recognized EBNA6-positive B cells which indicated that the nuclear antigen was available for the MHC class II-related processing pathways and generated fragments which encompassed repeat sequences.

The p63 peptide induced a proliferative T cell response in five of nine individuals. The pattern of response suggested promiscuous p63 binding to various HLA molecules, among them HLA-DR allotypes. Binding of the p63 peptide to HLA-DR1, HLA-DRB1*0404, HLA-DRB1*0401 and HLA-DRB1*1101 but not to HLA-DRB1*0402 revealed that specificity is governed by allo- and subtype-related differences. Based on the known peptide binding motifs (4246) various alignments of the p63 sequence could be postulated to generate core regions fitting to the peptide binding grooves of HLA-DR1, -DR5 and -DR4 allotypes. The three-dimensional structure of HLA-DR1 and -DR4 molecules, complexed with various peptides, revealed that the interaction of amino acid side chains with pockets 1 and 4 could significantly enhance the binding efficiency maintained primarily by conserved H-bonds along the peptide backbone (47). Our results suggest that overlapping sets of peptides, encompassed in the repetitive sequence of EBNA6, could bind to HLA-DR1 and to the related HLA-DR allotypes in a subtype-specific manner providing a structural background for generating Th cell epitopes recognized in those individuals which carry the binder allotype(s).

Peptides with degenerate MHC class II binding properties are frequently recognized in viral infections and in cancer patients (46,48). Promiscuous binding of a sequence to various HLA-DR molecules is necessary but it is not sufficient to act as universal T cell epitope (49). The intrinsic structural properties of the protein antigen, which confer preferential release of certain fragments upon antigen processing and consequently the availability of peptides for MHC class II loading, are important factors in determining immunodominance for CD4+ T lymphocyte recognition. The putative processing motif XPXX, which was observed in natural peptides (42,44), is located at both termini of the nonamer core of the Gly9–Glu12 alignment (Table 3Go, row 2) which may result in a preferential elaboration of this peptide under in vivo conditions. The stimulation of p63-specific T cells by EBNA6-transfected cells provided strong evidence that fragments, encompassing the epitope(s) identified in the p63 peptide, were generated upon EBNA6 processing. Our preliminary results demonstrated that overlapping nonapeptides of the p63 peptide, which comprise various combinations of the predicted alignments, were efficient activators of KM T cells (N. Nagy et al., unpublished results).

Our antibody absorption experiment with the p63 peptide demonstrated that the sequence comprised in p63 was a dominant target of EBNA6-specific antibody and suggested that it was available for antibody recognition in EBNA6. Tandem repeats of such B cell epitopes allow polyvalent antibody binding which may support the selection of memory B cells with this specificity. Multiple copies of linear T cell epitopes can function as super activators by mediating appropriate clustering of peptide–MHC–TCR complexes, which is a major factor in T cell stimulation (50). Differences in the magnitude of the p63-specific antibody and Th cell responses may be the consequence of the variable copy numbers of EBNA6 repeats present in different virus isolates (51). The question why these repeats are maintained in the EBV genome still remains open.

Stimulation of p63-specific Th lymphocytes in the presence of p63-loaded LCL or murine fibroblasts carrying the appropriate HLA-DR molecules showed that the activation of these T cells could be achieved with different APC irrespective of their co-stimulatory molecules. Reactivity of KMB5 T cells with both LCL and BL cells, co-expressing the appropriate HLA-DR molecules and EBNA6, also confirmed that the stimulation of these T cells did not depend on the phenotype or on the activation state of the presenting B cells. This result indicates that the in vitro selected p63-specific T cells have the characteristics of memory Th cells which require less stringent co-stimulation for antigen-induced responsiveness (52).

In latent EBV infection the continuous presence of certain viral antigens can support the maintenance of immunological memory although recent data revealed that T cell memory can develop in the absence of antigen (13). The EBNA6 protein is localized in the nucleus of latently infected B cells and as such it is not available for antibody recognition. The p63-specific antibody response, detected in the majority of virus carriers, implies that EBNA6 is released from the nucleus of the cells. In healthy individuals the EBNA6 protein may derive from EBV genome-carrying cells which are destroyed by effector mechanisms of the immune response. The viral genome is maintained in resting B cells which express EBNA1 only (20). If such cells are activated they express other EBV-encoded proteins among them EBNA6 and function as potent professional APC. Peptides, derived from these virally encoded antigen can be presented on the cell surface in the context of both MHC class I and class II molecules, and thus the APC becomes target of effector cells with cytotoxic activity (4,7,8,17). By this way destroyed activated B lymphocytes may provide a continuous source of nuclear antigen which maintain the antibody and Th responses at a constant level. EBNA6, released from damaged cells, can be taken up and processed by professional APC such as B cells with surface Ig specific for the protein and also by monocytes, macrophages or dendritic cells (DC) which engulf the released EBNA6 or the EBNA6-carrying apoptotic cells (53,54). These cells can then present the peptides derived from exogenous EBNA6 to CD4+ T cells. The possibility that loading of HLA-DR molecules could be achieved also by the endogenous pathway requires further investigations. Earlier data showed that certain viral proteins can efficiently be processed and presented for MHC class II-restricted Th cells via the endogenous pathway (55), and the involvement of both pathways in the presentation of an HLA-DQ-restricted EBNA-2 epitope was also reported (18). In a recent report, EBNA1-specific CD8+ T cell clones were shown to recognize peptides derived from exogenous EBNA1 in the context of MHC class I molecules (56) and the selection of MHC class II-restricted CD4+ T cells with cytotoxic activity, which recognized EBNA1-derived peptides generated by the endosomal processing pathway, was also reported (17). Thus it seems that the EBV-encoded proteins with nuclear localization can be handled by the classical as well as by certain minor processing pathways for presentation to MHC class I- and class II-restricted T cells.

Priming of Th cells merely by B lymphocytes favors the differentiation of Th2 effector cells and thus the tropism of EBV could bias the virus-specific immune response (57). The dominant IgG1/IgG3-type antibody-secreting cells and the cytokine profile of in vitro selected T cells, specific for a defined epitope of a latent EBV-encoded antigen, suggest polarization of the EBV-specific response to the Th1 phenotype, a tendency demonstrated in IM (58). The failure of the immune system to eliminate EBV-infected B cells in X-linked lymphoproliferative disease has now been attributed to the newly identified gene product (59) that modulates the co-stimulation of EBV-specific Th cells by the signaling lymphocyte activation molecule (SLAM), which is able to redirect Th2 responses to a Th1 or Th0 phenotype (60). Activation of EBV-specific Th1 cells can deliver proper signals for conditioning DC via the CD40–CD40 ligand interaction (61) and supports further polarization to a Th1 response.

The biological significance of the immune response to the EBNA6 repetitive region is not known. We propose that Th cells, directed against the EBNA repeats, may participate in the maintenance of a stable CTL memory repertoire (62). As it has been shown recently, CD4+ Th cells are not essential for the generation of CD8+ memory T cells but they are required for the survival and sustained effector activity of CTL. Th cells can confer these effects by the production of growth and trophic factors such as IL and chemokines which support the expansion, migration and the functional activity of antigen-specific CTL effectors (15,16). The lymphokine pattern of p63-specific T cells, i.e. secretion of IL-2 and TNF without a measurable amount of IL-4, suggest that in concert with other CTL-derived lymphokines they can potentiate cell survival and may also act on virus infected or tumour targets resulting either in their activation or damage (63). By these mechanisms these Th cells may modulate immunosurveillance and/or may play a role in the development or in controlling EBV-positive malignancies.


    Acknowledgments
 
This work was supported by the Swedish Cancer Society (Cancerfonden) and Concern Foundation/Cancer Research Institute. É. R. was supported as a visiting professor by the Swedish Medical Research Council and by the Hungarian FKFP grant no. 0186-1999.


    Abbreviations
 
ACIF anti-complement immunofluorescence
APC antigen-presenting cell
BL Burkitt's lymphoma
CTL cytotoxic T lymphocyte
DC dendritic cell
EBNA EBV nuclear antigen
EBV Epstein–Barr virus
HA hemagglutinin
IM infectious mononucleosis
LCL lymphoblastoid cell line
LMP latent membrane protein
PBMC peripheral blood mononuclear cells
PE phycoerythrin
SI stimulation index
TNF tumor necrosis factor
XLP X-linked lymphoproliferative disease

    Notes
 
Transmitting editor: I. Pecht

Received 30 May 1999, accepted 10 November 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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