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
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Abstract |
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Keywords: human CD4+ T lymphocyte, HLA-DRpeptide interaction, virus-specific immunity
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Introduction |
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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 -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 GlyAla 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.
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Methods |
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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 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 EBNA15 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 5x105 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 EBNA15 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 1014 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 streptavidinphycoerythrin (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 (12x104) were cultured in 96-well U-bottom tissue culture plates (Nunc, LOCATION??) in complete RPMI in the presence of graded concentrations of peptide and 12x104 autologous, irradiated LCL (70 Gy) or graded numbers of irradiated (70 Gy) or viable BL cells positive or negative for EBNA6. Culture supernatants (2575 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- 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 120180° 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).
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Results |
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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 1) 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 1
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 C
). 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. 2EF
). 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 1
) 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 2). The results of peptide binding to the LCL surface HLA-DR are given in Fig. 3
(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 1
), 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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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 1).
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-DRpeptide 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 3. In good correlation with the results of the binding assay (Fig. 3
), 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 3
). Glu at position 12 is completely buried in pocket 4 of HLA-DR1 (Table 3
, 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 3
, row 1,3) and pocket 4 cannot be occupied by Glu (Table 3
, 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 3
, rows 47).
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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- 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. 4A
) and secreted IL-2 (Fig 4B
) 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. 4
). 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. 4B
). 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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Discussion |
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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 Gly9Glu12 alignment (Table 3, 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 peptideMHCTCR 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 CD40CD40 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.
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Acknowledgments |
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Abbreviations |
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ACIF anti-complement immunofluorescence |
APC antigen-presenting cell |
BL Burkitt's lymphoma |
CTL cytotoxic T lymphocyte |
DC dendritic cell |
EBNA EBV nuclear antigen |
EBV EpsteinBarr 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 |
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Notes |
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Received 30 May 1999, accepted 10 November 1999.
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References |
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