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1 Division of Immunology, Aichi Cancer Center Research Institute, Nagoya, Japan
2 Department of Cell Therapy, Aichi Cancer Center Hospital, Nagoya, Japan
Correspondence
Kiyotaka Kuzushima
kkuzushi{at}aichi-cc.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), is associated with several different lymphoid and epithelial malignancies, including Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin's disease (HD) and post-transplant lymphoproliferative disorder (PTLD). All EBV-positive malignant cells exhibit one of three latency types, distinguished from each other by the pattern of expressed EBV antigens. In latency type I, only EBV-encoded nuclear antigen 1 (EBNA1) is expressed, as in BL; latent membrane protein 1 (LMP1) and LMP2, as well as EBNA1, are expressed in latency type II, as in HD and NPC. In latency type III, highly immunogenic EBNA3 genes, EBNA3A, EBNA3B and EBNA3C, are expressed together with other EBV latent antigens, as in PTLD (Rickinson & Kieff, 2001).
EBNA1 is required for the maintenance and replication of the viral episome in EBV-transformed cells (Kieff & Rickinson, 2001). Because it is expressed in all EBV-associated tumours, EBNA1 is an attractive target for immunotherapy. However, CD8+ cytotoxic T-lymphocyte (CTL) responses are directed preferentially toward EBNA3s among latent-cycle proteins, and EBNA1 has been believed to be immunologically invisible because of studies indicating that there has been escape from recognition by CTLs (Callan et al., 1998; Khanna et al., 1992; Murray et al., 1992; Steven et al., 1996). A glycine–alanine-repeat domain (GAr) within EBNA1 was found to prevent antigen processing for CTL recognition (Levitskaya et al., 1995). Presence of this GAr was shown to prevent processing by the proteasome, the main catalytic machinery for generation of major histocompatibility complex (MHC) class I epitopes (Blake et al., 1997; Levitskaya et al., 1997). Moreover, the same domain was established to prevent EBNA1 mRNA translation (Yin et al., 2003).
To explore the possibility of targeting EBNA1, EBV-specific CD4+ T-cell responses have been examined and EBNA1-specific CD4+ T-cell responses have been shown to be mainly T helper type 1 in nature (Bickham et al., 2001), with direct recognition of EBV-transformed cells (Khanna et al., 1997; Munz et al., 2000). Several MHC class II-restricted EBNA1 epitopes have been identified (Khanna et al., 1995; Kruger et al., 2003; Leen et al., 2001; Paludan et al., 2002; Voo et al., 2002), implying that EBNA1-specific CD4+ T cells may play a role in controlling tumour growth in vivo. Surprisingly, recent studies demonstrated that EBNA1-specific CD8+ CTLs moderately lyse EBV-transformed lymphoblastoid cell lines (LCLs) and suppress LCL outgrowth in vitro (Lee et al., 2004; Tellam et al., 2004; Voo et al., 2004). Defective ribosomal products of EBNA1 were shown to be the sources of EBNA1 CTL epitopes presented on the cell surfaces.
In this study, we induced CD8+ EBNA1-specific CTL clones from peripheral blood mononuclear cells (PBMCs) by using GAr-containing EBNA1 mRNA-transfected dendritic cells (DCs) as antigen-presenting cells (APCs). Antigen-transduced DCs have been demonstrated to prime antigen-specific CTLs efficiently in vitro (Grunebach et al., 2003; Heiser et al., 2000; Muller et al., 2004; Nair et al., 1998) and in vivo (Heiser et al., 2002; Nair et al., 2000; Su et al., 2003; Zeis et al., 2003). Furthermore, we identified a novel human leukocyte antigen (HLA)-C-restricted CTL epitope and confirmed EBNA1 recognition by CTL clones using tetrameric MHC–peptide complexes (tetramer). Finally, we estimated frequencies of EBNA1-specific CTL precursors (CTLp) in PBMCs of healthy EBV-seropositive donors and assessed effects of the CTL clones on EBNA1-expressing cells in vitro.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Schultze et al., 1997). Briefly, PBMCs were cultured with irradiated human CD40L-transfected NIH3T3 cells (referred as to t-CD40L; kindly provided by Dr Gordon Freeman, Dana-Farber Cancer Institute, Boston, MA, USA), recombinant interleukin-4 (IL-4) (Genzyme) and cyclosporine A (Sandoz) in the culture medium. Expanding CD40-B cells were stimulated twice a week. LCLs were prepared by transforming PBMCs with B95-8 cell-culture supernatant as described previously (Kuzushima et al., 1999) and cultured in RPMI 1640 medium (Sigma) supplemented with 10 % fetal calf serum (FCS), 2 mM L-glutamine, 50 U penicillin ml–1, 50 µg streptomycin ml–1 and 50 µg kanamycin ml–1 (referred to as complete culture medium). HLA-Cw*0303-expressing gastric carcinoma cells [MKN45 (referred to as MKN45-Cw0303) cells] were generated from MKN45 cells by retroviral transduction using Phoenix GALV cells (kind gifts from Dr Kiem, Fred Hutchinson Cancer Research Center, Seattle, WA, USA, and Dr Nolan, Stanford University, Stanford, CA, USA). Retroviral transduction of HLA genes was performed as described previously (Akatsuka et al., 2002; Kondo et al., 2002). EBNA1 (without GAr)-expressing MKN45-Cw0303 cells (referred to as MKN45-Cw0303-
GA-EBNA1) were then generated from MKN45-Cw0303 cells by lentivirus transduction (Bai et al., 2003). For this, an EBNA1-coding sequence without GAr (EBNA1 codons 92–323) was inserted into the lentivirus self-inactivating vector (CSIICMV-MCS; kindly provided by Dr Hiroyuki Miyoshi, RIKEN BioResource Center, Tsukuba, Japan) (Bai et al., 2003). MKN45-Cw0303 cells expressing full-length EBNA1 (referred to as MKN45-Cw0303-full-EBNA1) were also generated from MKN45-Cw0303 cells by mRNA transduction. Transduction of in vitro-transcribed full-length EBNA1 mRNA was performed as described above. MKN45-Cw0303 and MKN45-Cw0303-GA-EBNA1 cells were cultured in complete culture medium with 1.0 µg puromycin ml–1.
Preparation of DCs.
DCs were prepared as described previously (Dauer et al., 2003; Romani et al., 1994; Sallusto & Lanzavecchia, 1994). Briefly, CD8+ T cells were isolated from PBMCs by using CD8 MicroBeads (Miltenyi Biotec) and stored at –135 °C. The CD8-depleted PBMCs were suspended in 4 ml RPMI 1640 medium supplemented with 5 % human serum (MP Biomedicals), 2 mM L-glutamine, 50 U penicillin ml–1, 50 µg streptomycin ml–1 and 50 µg kanamycin ml–1 (referred to as DC medium) and incubated for 2 h in six-well plates at 37 °C. Non-adherent cells were removed by gentle pipetting and adherent cells were cultured in DC medium in the presence of 50 ng granulocyte–macrophage colony-stimulating factor ml–1 (GM-CSF; Osteogenetics) and 10 ng IL-4 ml–1 (Osteogenetics). On days 2 and 4, half of the medium was replaced with fresh DC medium containing GM-CSF and IL-4. On day 6, DCs were collected and electroporated for mRNA transduction.
Production and transduction of in vitro-transcribed mRNA.
To generate in vitro-transcribed EBNA1 mRNA, a pcDNA/EBNA1 vector was constructed. The coding sequence for EBNA1 was obtained by extraction of total RNA from B95-8-transformed LCLs using an RNeasy kit (Qiagen) and, after reverse transcription, EBNA1 cDNA was amplified by PCR with specific primers as follows: EBNA1 forward primer, 5'-AAGCTTGCCACCATGTCTGACGAGGGGCCAGGTACAG; reverse primer, 5'-GAATTCTCACTCCTGCCCTTCCTCACCCTC. The full-length EBNA1 fragment was then ligated into pcDNA3.1(+) (Invitrogen) using its HindIII and EcoRI sites (pcDNA/EBNA1). Clones were sequenced to verify their identity. Resulting plasmid DNA was linearized and transcribed in vitro by using an mMESSAGE and mMACHINE kit (Ambion) according to the manufacturer's instructions. A 3'-poly(A) tail was added by using poly(A) polymerase (Ambion) followed by purification with an RNeasy kit. The resulting mRNA was visualized by using the Reliant RNA gel system (Cambrex). DCs and CD40-B cells were transfected with mRNA by electroporation. First, they were washed twice with serum-free RPMI 1640 medium and suspended at a final concentration of 2.5x107 cells ml–1. After mixing with 20 µg mRNA in 40 µl RPMI 1640 medium, they were then electroporated in a 2 mm cuvette by using an Electro Square Porator ECM 830 (Harvard Apparatus), under conditions of 450 V and 500 µS for DCs and 350 V and 350 µS for CD40-B cells. DCs were subsequently cultured in DC medium supplemented with GM-CSF and IL-4 for 3 h, followed with tumour necrosis factor alpha (PeproTech), IL-1
(PeproTech) and prostaglandin E2 (Cayman Chemical) for maturation. CD40-B cells were seeded immediately onto irradiated t-CD40L cells and, after 36–48 h, these cells were used as APCs.
EBNA1 staining.
EBNA1 mRNA-transfected CD40-B cells were collected and fixed with 4 % paraformaldehyde in PBS for 10 min at room temperature. After washing with PBS, cells were permeabilized with PBS containing 0.5 % Tween 20 and reacted with anti-EBNA1 rabbit polyclonal antibodies (kindly provided by Dr Tatsuya Tsurumi, Aichi Cancer Center Research Institute, Nagoya, Japan) for 30 min at 4 °C. After washing with PBS, cells were stained with fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit IgG (Beckman Coulter) for 30 min at 4 °C. The stained cells were analysed by FACSCalibur (BD Biosciences) using CELLQUEST software (BD Biosciences).
CTL induction.
The stored CD8+ T cells were thawed, washed and co-cultured with irradiated (33 Gy) autologous EBNA1 mRNA-transfected DCs in 200 µl RPMI 1640 medium supplemented with 10 % human serum, 2 mM L-glutamine, 50 U penicillin ml–1, 50 µg streptomycin ml–1 and 50 µg kanamycin ml–1 (referred to as CTL medium) in the presence of 5 ng IL-7 ml–1 (R&D Systems) and 5 ng IL-12 ml–1 (R&D systems) at 5 % CO2 in a humidified incubator. On days 8, 16 and 23, T cells were restimulated with EBNA1 mRNA-transfected and irradiated DCs. One day after each restimulation, IL-2 (Shionogi) was added to a final concentration of 20 U ml–1. To establish T-cell clones, limiting dilution of polyclonal CTLs was performed (Kuzushima et al., 2001). In brief, polyclonal CD8+ T cells were seeded at 1 cell per well in round-bottomed 96-well plates containing CTL medium with a monoclonal antibody (mAb) specific to CD3 (30 ng ml–1; Ortho Biotech), 1x105 irradiated (33 Gy) PBMCs and 2x104 irradiated (55 Gy) LCLs. The next day, IL-2 was added to each well (50 U ml–1). After 2 weeks culture, growing wells were split into two replicates and used as effectors in ELISPOT assays against either autologous EBNA1 mRNA-transfected CD40-B cells or autologous LCLs. Positive wells were transferred into flasks and expanded with anti-CD3 mAb, irradiated feeder cells and IL-2.
ELISPOT assays.
ELISPOT assays were performed as described previously (Kuzushima et al., 2003). Briefly, CD8+ T cells were co-cultured with various stimulators in wells of Multiscreen-HA plates (Millipore) coated with anti-human gamma interferon (IFN-
) mAb (Pierce Biotechnology). As stimulators, (i) autologous EBNA1 mRNA-transfected CD40-B cells or non-transfected CD40-B cells and (ii) autologous or allogeneic LCLs (1x105 cells per well) were seeded into each well. For peptide-titration assays and overlapping-peptide assay, serial concentrations of synthetic peptides were pulsed to autologous CD40-B cells for 1 h at room temperature. After probing with anti-human IFN- rabbit polyclonal antibodies (Pierce Biotechnology) followed by exposure to horseradish peroxidase-labelled anti-rabbit IgG antibody (Genzyme) and substrate, the plates were washed and dried. IFN- spots were enumerated under a dissecting microscope. The numbers of spots were confirmed by three investigators.
Peptides.
Peptides were purchased from Bio-Synthesis Inc. The whole EBNA1 protein, excluding the GAr, deduced from the prototype B95-8 DNA sequence (GenBank accession no. V01555
[GenBank]
) was covered by 20 aa long synthetic peptides overlapping by 13 aa (total of 56 peptides). In addition, potential epitope peptides VYGGSKTSL (509–517), FVYGGSKTSL (508–517) and VFVYGGSKTSL (507–517) were synthesized, as predicted by the program SYFPEITHI (http://www.syfpeithi.de/) (Rammensee et al., 1999). The known EBNA1 epitope peptide HPVGEADYFEY (Blake et al., 1997) was also synthesized.
Tetramer production and staining.
HLA-Cw*0303 and -Cw*0304 cDNA clones were used as templates to amplify sequences encoding the extracellular domains of HLA-Cw*0303 and -Cw*0304 heavy chains with primers C03F (5'-AACCATGGGCAGCCATTCTATGCGCTATTTTTACACCGCTGTGTCCCGGCC-3') and C03R (5'-AAGGATCCTGGCTCCCATCTCAGGGTGAGG-3'). C03F contains several base changes designed to optimize protein expression in Escherichia coli BL21 (DE3) pLysS. The PCR product was digested with NcoI and BamHI and cloned into a vector containing a BirA biotinylation site in frame with the 3' end of the HLA sequence. Recombinant HLA-B*3501 protein was produced by using pGMT7-B35 (a kind gift of Dr McMichael, Weatherall Institute of Molecular Medicine, Oxford, UK). Recombinant MHC molecules were folded in vitro with 2-microglobulin and epitope peptides FVYGGSKTSL or VFVYGGSKTSL. Soluble complexes, purified by gel filtration, were biotinylated by using the BirA enzyme (Avidity LCC). Phycoerythrin (PE)-labelled tetramers were produced by mixing these biotinylated complexes with PE-labelled streptavidin (Molecular Probes). Tetramer staining was performed as follows. CTL clones (2x105) were incubated with tetramers at a concentration of 0.1 mg ml–1 and FITC–anti-CD8 mAb (Caltag) at 4 °C for 15 min. After washing twice, stained cells were fixed in 0.5 % paraformaldehyde and analysed by flow cytometry.
Mixed lymphocyte–peptide culture.
Mixed lymphocyte–peptide culture was performed as described by Coulie et al. (2001) with modifications. PBMCs were isolated by density-gradient centrifugation and suspended in CTL medium. An aliquot was used for enumeration of CD8+ cells. The cells were then distributed at 2x105, 1x105 or 5x104 cells per well in 96-well round-bottomed plates in 200 µl medium in the presence of epitope peptide (1 µg ml–1) and IL-2 (20 U ml–1). Thirty-two cultures of each PBMC density were set up. On day 7, half of the medium was replaced by fresh medium containing the peptide and IL-2. Tetramer staining was performed on day 14. We counted the tetramer-positive microcultures and estimated the anti-EBNA1 CTLp frequency by limiting-dilution evaluation using the L-Calc program (Stem Cell Technologies).
Outgrowth-inhibition assay.
Outgrowth-inhibition assays were performed as described by Lee et al. (2004) with modifications. Briefly, target LCLs were seeded into round-bottomed 96-well plates at 2x104 cells per well in triplicate. EBNA1-specific CTL clones (1x104 cells per well) or CTL medium alone (as a control) were added to target-cell cultures. All cultures were maintained weekly by changing half of the medium. After 4 weeks culture, the number of LCLs in the culture at each setting was counted. Cell growth (percentage of control) was calculated as [no. LCLs from the culture with CTLs (clone B5 or clone C6)]/[no. LCLs from the culture without CTLs (medium)]x100. B-cell identity of grown cells was confirmed by staining with PE–cyanin 5-labelled anti-CD19 and PE-labelled anti-CD8 mAbs (Beckman Coulter) and analysis by flow cytometry.
Detection of IFN--producing CD8+ T cells by flow cytometry.
EBNA1-specific CTL recognition was measured as described previously (Kuzushima et al., 2001) with slight modifications. Briefly, 5x105 B5 CTLs were incubated with 2x106 MKN45-Cw0303, MKN45-Cw0303-GA-EBNA1 or MKN45-Cw0303-full-EBNA1 cells in 1 ml complete culture medium in a culture tube at 37 °C for 6 h, in the presence of brefeldin A (Sigma) during the last 5 h. After the incubation, the cell suspensions were fixed with 4 % paraformaldehyde in PBS. After washing with PBS, cells were permeabilized with IC Perm (BioSource International) and stained with PE–cyanin-5.1-labelled anti-CD8 (Beckman Coulter), PE-labelled anti-CD69 (Immunotech Coulter) and FITC-labelled anti-human IFN- (BD Biosciences) mAbs. Stained cells were analysed by flow cytometry. Live gating of the CD8+ subset was performed and 50 000 events were acquired for each analysis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Transfection was then performed by electroporation and EBNA1 expression was detected in most CD40-B cells, although the mean fluorescent intensity appeared to be low (Fig. 1b).
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-producing cells were enumerated by ELISPOT assay. As shown in Fig. 2(a), CD8+ T lymphocytes of donor Y01 produced IFN- spots without in vitro stimulation. As CD8+ T lymphocytes of other donors did not produce significant spots on ex vivo analysis, the T cells were stimulated weekly with irradiated CD40-B cells that had been transfected with the full-length EBNA1 mRNA. After two rounds of stimulation, CD8+ T lymphocytes of another donor (K04) produced IFN- spots upon contact with autologous CD40-B cells transduced with EBNA1 mRNA in the ELISPOT assay (Fig. 2a). These data indicate that the full-length EBNA1 mRNA was translated and that CD8+ T-lymphocyte epitopes are processed and presented on APCs.
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specifically upon contact with autologous CD40-B cells transduced with EBNA1 mRNA in the ELISPOT assay. Thirty-two microcultures out of 36 wells were scored as EBNA1-specific (data not shown), and lymphocytes from two well-growing microcultures were cloned by limiting dilution. CTL clones B5 and C6 were thus established, recognizing EBNA1 mRNA-transfected autologous CD40-B cells and autologous LCLs, but not mock-transfected autologous CD40-B cells or HLA-mismatched allogeneic LCLs (Fig. 2
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Identification of the presenting HLA molecules
The donor was typed genetically as HLA-A*2402, -A*3101, -B*1507, -B*3501 and -Cw*0303. To identify the antigen-presenting HLA molecule, a panel of partially HLA-matched LCLs was used to stimulate clones B5 or C6 to produce IFN-. In addition to autologous LCLs, allogeneic LCLs expressing HLA-B*3501 were recognized by CTL clone C6 (Fig. 3a), and one LCL with HLA-Cw*0303 and one with -Cw*0304 were recognized by clone B5 (Fig. 3b), demonstrating that HLA-B*3501 is the putative restriction element for clone C6 recognition, whilst both HLA-Cw*0303 and -Cw*0304 act for clone B5.
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). Regarding the HLA-B*3501-restricted epitope, HPVGEADYFEY has been reported previously (Blake et al., 1997). As this epitope sequence is located in the centre of peptide 24 (aa 402–421) (Fig. 5a), we tested whether clone C6 might recognize HPVGEADYFEY-pulsed autologous CD40-B cells and confirmed a response to HPVGEADYFEY-pulsed stimulation (data not shown).
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), sharing the 13 aa sequence VFVYGGSKTSLYN [underlined in Fig. 5(b)]. To predict the optimal epitope binding to HLA-Cw*0303, the program SYFPEITHI was applied. Because the anchor leucine at the C terminus and the auxiliary anchors valine and tyrosine at the third position of epitopes were predicted by the program, we examined the 11mer (VFVYGGSKTSL) and the 10mer (FVYGGSKTSL) (Fig. 5b). Half-maximal recognition of the peptide-pulsed target cells was obtained with 5–10 nM of the 10mer peptide and 1–5 nM of the 11mer (Fig. 5c), suggesting that these two peptides may be the optimal epitopes. The 9mer, VYGGSKTSL, was not recognized, even at much higher concentrations.
Tetramers bind to the EBNA1-specific clone B5
As the peptide-dilution assay provided two optimal epitope candidates, we made fluorescently labelled tetramers incorporating the 10mer peptide FVYGGSKTSL or the 11mer VFVYGGSKTSL for further experiments. As shown in Fig. 5(d), these tetramers bound specifically to CTL clone B5. However, the tetramer incorporating the 10mer demonstrated higher avidity for the B5 clone than that incorporating the 11mer, suggesting the 10mer peptide FVYGGSKTSL to be the minimal and optimal epitope for the CTL. Moreover, clone B5 bound strongly to the HLA-Cw*0304 tetramer incorporating the 10mer, showing concordance with the results shown in Fig. 3(b). In addition, we characterized in vitro-expanded T cells from two donors by co-staining with MHC–peptide tetramer and CD62L. A proportion of 9.8 % of HLA-Cw*0303-FVYGGSKTSL tetramer-positive lymphocytes were CD62-positive in one donor, and 6.5 % in the other.
Frequencies of EBNA1 epitope-specific CD8+ T cells in PBMCs of healthy EBV-seropositive donors
We estimated frequencies of EBNA1 epitope-specific CD8+ T cells in healthy EBV-seropositive donors by the mixed lymphocyte–peptide culture method. PBMCs from two donors with HLA-B*3501, one donor with HLA-Cw*0303 and three donors with HLA-Cw*0304 were tested. We could compare the anti-EBNA1 CTLp frequency in two donors with both HLA-B*3501 and HLA-Cw*0303 (HLA-Cw*0304). Representative tetramer staining of negative and positive microcultures from mixed lymphocyte–peptide culture wells is shown in Fig. 6. EBNA1-specific CTLp frequencies of HLA-B*3501-positive donors were 7.2x10–6 and 1.8x10–4, and for HLA-Cw*0303 and -Cw*0304 were from 2.5x10–5 to >2.1x10–4. We did not find any hierarchy between the two EBNA1 epitopes in either of the donors with both HLA-B*3501 and HLA-Cw*0303 (Cw*0304) molecules (Table 1).
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, both CTL clones clearly inhibited outgrowth of not only autologous LCLs, but also allogeneic LCLs with restricting HLA, suggesting that these CTL clones have the ability to inhibit outgrowth of EBV-positive cells with latency type III.
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GA-EBNA1 and MKN45-Cw0303-full-EBNA1 to verify that EBV-positive gastric cancer cell lines present the FVYGGSKTSL epitope. To investigate recognition by clone B5, we applied flow cytometry to detect EBNA1-specific CTLs producing IFN-. As shown in Fig. 7(b), 2.55 % of B5 clone cells produced IFN- when co-cultured with MKN45-Cw0303-GA-EBNA1 cells, demonstrating specific recognition of the FVYGGSKTSL epitope on cells transduced with GAr-deleted EBNA1. Otherwise, B5 clone cells did not produce IFN- when co-cultured with MKN45-Cw0303-full-EBNA1 cells. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Tellam et al., 2004; Voo et al., 2004). In previous studies, such CTLs were generated from PBMCs of healthy donors after in vitro stimulation with autologous LCLs or EBNA1 peptide-pulsed PBMCs. As EBNA1 is expressed in all EBV-associated tumours and might be an important target for immunotherapy, we have explored the efficient induction of EBNA1-specific CTLs. Of the different methods used to obtain HLA class I-restricted epitopes on APCs for stimulation, we chose to employ full-length EBNA1 mRNA-transfected DCs. This strategy offers the following advantages: (i) the method is not dependent on knowledge of the HLA haplotype of each donor; (ii) there is complete deletion of antigenicity of the vector-backbone sequence; and (iii) the yield of mRNA in vitro transcription is highly reproducible and transduction is very efficient (Van Tendeloo et al., 2001). As an antigen, we used GAr-containing full-length EBNA1 instead of a GAr-deleted example to selectively activate CTL populations capable of reacting with epitopes that escape from the inhibitory mechanism governed by EBNA1 encoding GAr. Of note, even a low level of antigen delivery into DCs could induce antigen-specific CTL responses (Grunebach et al., 2003), suggesting that this strategy has the potential to induce a CTL response even when a low density of EBNA1 epitopes is presented on DCs. In this study, we generated HLA-B- and -C-restricted EBNA1-specific CTLs successfully from a single donor, demonstrating that this method is a useful tool for generating EBNA1-specific CTLs, allowing investigation of the contribution of EBNA1 to cell-mediated immune responses in EBV-associated malignancies. Moreover, EBNA1 may have antigenicity when expressed on APCs, even if containing GAr in vivo. Finally, this induction method may be applicable for preparing EBNA1-specific CTLs for immunotherapy.
EBNA1 is generally immunologically invisible and only a small number of CTL epitopes have been identified (Blake et al., 1997, 2000; Voo et al., 2004). Of these, five epitopes are HLA-B-restricted and one is presented in the context of HLA-A. We detected two, one from HLA-B and another from HLA-C, from a single donor. To our knowledge, this is the first demonstration of an HLA-C-restricted EBNA1 epitope. To determine the minimal epitope, we compared the 11mer (VFVYGGSKTSL) and 10mer (FVYGGSKTSL) in a peptide-titration assay and found peptide concentration with half-maximal recognition of the target cells to be almost the same. However, clone C6 bound more strongly to tetramers incorporating the 10mer and we speculate that the N-terminal valine of the 11mer might be trimmed efficiently to yield 10mer in ELISPOT assay medium containing FCS. Moreover, the 10mer FVYGGSKTSL epitope was presented by HLA-Cw*0303 and -Cw*0304 molecules. As these two HLA-C alleles are possessed by >35 % of Japanese, >20 % of Northern Han Chinese (Hong et al., 2005) and >25 % of Caucasians, this new epitope should enable us to analyse cellular immunity to EBNA1 in a broad population. Indeed, we estimated CD8+ T-cell frequencies specific to either FVYGGSKTSL or HPVGEADYFEY in PBMCs of healthy EBV-seropositive donors by the mixed lymphocyte–peptide culture method followed by tetramer staining and found that EBNA1-specific CTLp frequencies of HLA-B*3501- or HLA-Cw*0303 (and -Cw*0304)-positive donors were between 1x10–5 and 1x10–4. These data provide useful information for understanding cellular immunity to EBNA1. For determination of frequencies of EBNA1 epitope-specific CTLs, the ex vivo ELISPOT assay (Blake et al., 2000) is simple and readily applicable, because frequencies can be predicted at the level of 1x10–4 CD8+ lymphocytes.
Adoptive immunotherapy with CTLs has proved feasible for preventing and treating EBV-associated PTLD, HD and NPC (Bollard et al., 2004; Gottschalk et al., 2005; Straathof et al., 2005). With respect to the targets for EBV-specific CTLs, EBNA3s and LMP2 are major EBV latent antigens; EBNA3s are immunodominant and LMP2 is recognized frequently, but is subdominant. In contrast, CTL responses to other antigens (EBNA2, EBNA-LP, LMP1 and EBNA1) seem to be less frequent (Rickinson & Moss, 1997), although EBNA1 can be immunodominant in some EBV-seropositive donors (Blake et al., 2000). Indeed, LCL-activated EBV-specific CTL lines from NPC patients for adoptive immunotherapy demonstrate stronger responses to the immunodominant EBNA3s than against LMP1 and LMP2 (Straathof et al., 2005). In addition, tetramer and functional analyses have shown that LMP2-specific CTLs are present in the infused CTLs used for adoptive immunotherapy and might have antiviral activity in patients with a good response to immunotherapy for HD (Bollard et al., 2004). Interestingly, the CTL line from one NPC patient who attained a complete response was shown to contain a relatively large T-cell population for an EBNA1-derived CTL epitope (Straathof et al., 2005). This suggests that increased attention should be focused on the contribution of EBNA1-specific CTLs to EBV cellular immunity. In this study, we showed two EBNA1-specific CTL clones to cause strong, specific inhibition of LCL outgrowth in vitro, which is consistent with recent observations with HLA-B8- and HLA-B*3501-restricted CTL clones (Tellam et al., 2004; Voo et al., 2004). C6 CTLs failed to respond to an HLA-Cw*0303-expressing gastric cancer cell line transduced with full-length EBNA1, although they produced IFN- when GAr-depleted EBNA1 was transduced (Fig. 7b). These data suggest differential antigen-processing machinery and presentation on class I molecules between LCLs and gastric cancer cells.
In conclusion, we have established EBNA1-specific CTL clones from PBMCs of a healthy donor by using EBNA1 mRNA-transfected DCs, and identified a novel CTL epitope of EBNA1 presented by HLA-Cw*0303 and -Cw*0304 molecules. The induction method adapted may be useful for generating EBNA1-specific CTLs and for investigating cellular immunity against EBNA1. Finally, the induced EBNA1-specific CTLs recognized EBNA1-expressing gastric carcinoma cells in the context of HLA-Cw*0303 in vitro, suggesting that EBNA1 is an important antigen for the further development of CTL therapy for EBV-associated malignancies.
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ACKNOWLEDGEMENTS |
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Received 31 August 2006;
accepted 7 November 2006.
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