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Prepublished online as a Blood First Edition Paper on September 26, 2002; DOI 10.1182/blood-2002-04-1240.
IMMUNOBIOLOGY
From the Divisions of Immunology and Virology, Aichi
Cancer Center Research Institute, and the Department of Hematology and
Chemotherapy, Aichi Cancer Center Hospital, Nagoya, Japan.
We determined cytotoxic T lymphocyte (CTL) epitopes through
screening with a computer-assisted algorithm and an enzyme-linked immunospot (ELISPOT) assay using in vitro-reactivated polyclonal Epstein-Barr virus (EBV)-specific CD8+ T cells as
responders. In addition, to confirm that the epitopes were generated
after endogenous processing and presentation of the EBV proteins, a
novel T-cell receptor (TCR) down-regulation assay was introduced, in
which a fluorescent tetrameric major histocompatibility complex
(MHC)/peptide complex was employed for detecting TCR
down-regulation after stimulation with the epitope presented on
antigen-presenting cells. Through such screening, 3 HLA
A*2402-restricted epitopes were identified: IYVLVMLVL, TYPVLEEMF, and
DYNFVKQLF, derived from LMP2, BRLF1, and BMLF1 proteins, respectively. TCR down-regulation assays disclosed that, in contrast to the other 2 epitopes, IYVLVMLVL was not presented on HLA A24-positive fibroblast
cells infected with recombinant vaccinia viruses expressing LMP2.
Furthermore, ELISPOT assays with an epitope-specific CTL clone
demonstrated that the presentation was partially restored by
pretreatment of the fibroblast cells with interferon- Epstein-Barr virus (EBV) is associated with
malignancies, such as a subset of Hodgkin disease, Burkitt lymphomas,
immunoblastic lymphomas seen in immunocompromised hosts, nasopharyngeal
carcinomas (NPCs), and some gastric carcinomas.1 Primary
infection is usually asymptomatic, although some individuals may suffer
from infectious mononucleosis, followed by a strong HLA class
I-restricted, virus-specific CD8+ cytotoxic T lymphocyte
(CTL) response.1,2 The latter is believed to play an
important role in controlling the virus both during primary infection
and in the long-term carrier state, whereby EBV persists for life in a
subset of B cells.3 Both lytic- and latent-cycle EBV
proteins are known to be recognized by EBV-specific CTLs targeting
short peptides presented on cell surfaces by HLA class I
molecules.2
The importance of EBV-specific T cells for control of latently infected
B cells has been re-emphasized by observations in patients with
immunoblastic lymphomas or lymphoproliferative disease (LPD) after bone
marrow transplantation.4,5 Adoptive transfer of
EBV-specific CTLs or unfractionated donor lymphocytes can result in
remission of the LPD.6,7 Withdrawal of immunosuppressive drugs may cause regression of EBV-associated LPD in some patients after
bone marrow transplantation.8-12
In contrast to immunoblastic lymphomas seen in immunocompromised hosts,
whereby the full panel of the EBV latent proteins is expressed, virus
protein expression in other EBV-associated malignancies is more
restricted. In Hodgkin disease, NPC, and EBV-positive gastric
carcinomas, the only EBV proteins expressed are EBNA1, latent membrane
protein 1 (LMP1), and LMP2.1,13,14 Of these, LMP1 and LMP2
have drawn attention as potential target antigens to establish
CTL-based immunotherapy.15-23 Indeed, T cells isolated
from biopsy samples of Hodgkin disease demonstrate MHC-restricted cytotoxicity to target cells expressing LMP2.17 Dendritic
cells, infected with recombinant adenovirus expressing
LMP2,18,19 or transfected with mRNA encoding the LMP2
gene,20 have been studied for effective and selective
stimulation of LMP2-specific CTLs.
Some viral proteins synthesized in infected cells are degraded,
primarily by the proteasome system, and the resultant peptides are
translocated into the endoplasmic reticulum by transporters associated
with antigen processing (TAPs). Stable major histocompatibility complex
(MHC) class I molecules then move to the cell surface for presentation
of peptides to CD8+ T cells.24 A number of
antigenic peptides have been identified within the amino acid sequences
of LMP2.15,16,21 These peptides can be pulsed on dendritic
cells to elicit a peptide-specific polyclonal CTL
response.22 In addition, such peptides have the potential
to elicit EBV-specific CTL responses in vivo when used as vaccines
before EBV challenge or as immunotherapeutic reagents for treatment of
EBV-associated malignancies.1 Another attribute of the
antigenic peptide is to produce fluorescent-labeled tetrameric MHC-peptide complexes to enumerate and/or to sort peptide-specific CTLs
for large-scale expansion.25-28 One limitation of using
such epitope peptides is that their presentation to T cells is HLA class I allele specific. In many ethnic groups, HLA A24 is one of the
most common alleles in Japan,29,30 being the most
frequently encountered HLA class I allele with a genotype almost
exclusively A*2402.31 An LMP2 epitope presented by HLA
A*2402 has been reported.16 To establish epitope-based
immunotherapy, however, multiple epitopes are preferred because of the
emergence of escaping mutant cells after single epitope
vaccination,32 and the nature of processing of such
epitopes needs to be extensively studied to obtain a maximal clinical response.
We have adopted an approach for determination of CTL epitopes through
screening with a computer-assisted algorithm and an enzyme-linked
immunospot (ELISPOT) assay by which a major human cytomegalovirus
epitope presented by HLA A*2402 molecules has been successfully
identified.33 In addition, we introduce here a novel assay
using relevant tetramers for detecting down-regulation of T-cell
receptor (TCR) expression after stimulation with HLA A24-positive
fibroblast cells expressing each of the EBV genes, in order to confirm
endogenous processing and presentation. In this paper, the
identification through such screening of 2 HLA A*2402-restricted
epitopes derived from EBV lytic-cycle proteins, and an unusually
presented LMP2 epitope, together with characterization of the epitope
processing is described in detail.
Cell lines
CD40-activated B cells (CD40-Bs) were generated from the peripheral
blood mononuclear cells (PBMCs) of HLA A24-positive donors as
previously described.34 Briefly, PBMCs were cultured with irradiated human CD40L-transfected NIH3T3 cells (kindly provided by Dr
Gordon Freeman, Dana-Farber Cancer Institute, Boston, MA), recombinant
interleukin 4 (IL-4) (Genzyme, Cambridge, MA), and cyclosporine A
(Sandoz, Basel, Switzerland) in the culture medium. Expanding B cells
were stimulated twice a week.
EBV-carrying lymphoblastoid cell lines (LCLs) were prepared by
transforming PBMCs with B95-8 cell culture supernatant as previously described11 and propagated in culture medium. Small
populations, between 0.3% and 4%, of the LCLs used for establishment
of CTLs, expressed the BZLF1 protein determined by indirect
immunofluorescent analysis.
EBV-specific polyclonal CTLs were generated as described
earlier.7,11 To establish EBV-specific T-cell clones,
CD8+ T cells were plated in wells of 96-well round-bottom
plates at 0.3 cell/well and 1 cell/well with Construction and transfection of plasmids
Each plasmid and one expressing HLA A*240233 were transfected using LIPOFECTAMINE PLUS (GIBCO) into HEK293T cells. T2-A24 cells were electroporated in the presence of each plasmid using a GENE PULSER II (Bio-Rad Laboratories, Hercules, CA). Transfected cells were incubated in medium with 10% FCS for 48 hours and applied as antigen-presenting cells (APCs) in the ELISPOT assay. Peptides To identify the potential HLA A24-binding peptides within B95-8 strain EBV proteins (accession no. V01555),35 a computer-based program was applied with access through the website of BioInformatics & Molecular Analysis Section (BIMAS) HLA Peptide Binding Predictions.36,37 Most peptides were synthesized with a Cleaved PepSet from Mimotope (Melbourne, Australia). There were 4 HLA A24-restricted CTL epitope peptides derived from EBNA3A,38 EBNA3B,2 LMP2,16 and HIV envelope protein39 that were synthesized by Toray Research Center (Kamakura, Japan). The peptides used in this study are listed in Table 1.
MHC stabilization assay MHC stabilization assays were performed as described earlier.23,33 Briefly, T2-A24 cells were pulsed with each of the peptides (10 µM) at 26°C for 16 hours, followed by incubation at 37°C for 3 hours. Surface HLA A24 molecules were then stained with a specific mAb. Expression was measured by FACSCalibur (Becton Dickinson, San Jose, CA) and mean fluorescence intensity (MFI) was recorded. Percentage MFI increase was calculated as (MFI with the given peptide MFI without peptide)/(MFI without peptide) × 100.
ELISPOT assays ELISPOT assays were performed as previously described.37 The following APCs in 100 µL culture medium were seeded into each well: (1) T2-A24 cells (5 × 104 cells/well) pulsed with each peptide (the peptide concentrations described in the text and figures are those in the final assay volume); (2) HLA A24-positive or -negative LCLs (1 × 105 cells /well); (3) HLA A24-positive fibroblast cells (1 × 104 cells/well) infected 2 hours earlier with recombinant vaccinia virus expressing each of the EBV genes at a multiplicity of infection of 10 (some of the cells had been cultured in the presence of 400 U/mL interferon- (IFN-) (Strathmann Biotech GmbH, Hamburg, Germany)
for 6 days prior to the vaccinia infection); (4) HEK293T or T2-A24
cells (5 × 104 cells/well) transfected 48 hours earlier
with plasmids expressing each of the cloned genes.
As responders, polyclonal or monoclonal EBV-specific CD8+ T cells suspended in 100 µL culture medium were seeded into each well. All assays were performed in duplicate. CTL assays CTL assays were performed as previously described.33 Briefly, CTLs were suspended in fresh culture medium at the desired cell concentration and seeded in wells of V-bottomed 96-well plates (Costar, Cambridge, MA) containing 51Cr-labeled LCLs (2500 cells/well). In some wells, the target cells were pulsed with antigenic peptides to a final concentration of 100 ng/mL. After 5 hours of incubation, the supernatants were harvested and radioactivity counted with a-counter. Percentage specific lysis was calculated as previously
described.33 Each assay was performed in triplicate.
Tetramer-assisted TCR down-regulation assay MHC/peptide tetramers were produced as previously described.33,40,41 To quantify engagement-dependent TCR down-regulation,42,43 we set up several stimulation systems preceding the tetramer staining: (1) 1000 to 2000 tetramer-positive cells (analyzed by FACS beforehand) for a given EBV-specific T-cell line were stimulated by incubation with T2-A24 cells (1 × 106) in the presence of the same peptide used for the tetramer construction or a control peptide at a final concentration of 100 ng/mL in 1 mL culture medium supplemented with 20 U/mL IL-2 in 16 × 125-mm culture tubes at 37°C for 16 hours; (2) 1000 to 2000 tetramer-positive cells were incubated with 1 × 106 HLA A24-positive CD40-Bs, or HLA A24-positive or -negative LCLs in 1 mL culture medium supplemented with 20 U/mL IL-2 in the culture tubes at 37°C for 16 hours; (3) 3000 to 10 000 tetramer-positive cells in 2 mL culture medium supplemented with 20 U/mL IL-2 were placed on a full sheet of HLA A24-positive fibroblast cells (approximately 5 × 105/well) infected 2 hours earlier with recombinant vaccinia viruses expressing each of the EBV genes at a multiplicity of infection of 10 in each well of 6-well plates. After the plates were incubated at 37°C for 16 hours, the cells were harvested, washed, resuspended in 1 mL phosphate-buffered saline (PBS) with 0.04% EDTA (ethylenediaminetetraacetic acid) and incubated at room temperature for 10 minutes before staining with the indicated tetramers. For staining, CTLs or PBMCs (2 × 106) were incubated with 40 µg/mL of the tetramer and a TRI-COLOR-labeled anti-CD8 mAb (Caltag, Burlingame, CA) at 37°C for 15 minutes before analysis with FACSCalibur (Becton Dickinson, Mansfield, MA).
Selection of potential HLA A24-binding peptides within EBV proteins To identify antigenic peptides presented by HLA A*2402 molecules, EBNA2, EBNA3A, EBNA3B, EBNA3C, LMP1, and LMP2 were selected from the EBV latent-cycle proteins because they are reported to be sources of CTL epitopes presented by other HLA class I molecules.2,15,16,23 In addition, BZLF1, BRLF1, BMLF1, BMRF1, and BALF2 were selected from the lytic-cycle proteins because they are targeted by CD8+ CTLs restricted by HLA class I molecules other than A*2402.2,44 To search for potential HLA A24-binding peptides, amino acid sequences of the B95-8 EBV strain35 were analyzed with a computer program.36,37 A total of 42 peptides with estimated t1/2 dissociation scores of more than 100 were selected (Table 1). All share HLA A24-binding motifs with tyrosine as the second residue and phenylalanine or leucine as the ninth or tenth residues.45-47 Next, MHC stabilization assays were performed to test these peptides for HLA A*2402 binding efficiency using T2-A24 cells. All peptides increased the HLA A24 expression on the cells, indicating that they bound and stabilized the HLA complex on the cell surface (Table 1).Screening of peptides antigenic for EBV-specific polyclonal CD8+ T-cell lines by ELISPOT assay To identify peptides recognized by EBV-specific CD8+ T cells in the context of HLA A*2402 molecules, the ELISPOT assay was performed using T2-A24 cells as APCs. Figure 1 summarizes the results using 4 polyclonal EBV-specific CD8+ T cells established from PBMCs of EBV-seropositive and HLA A24-positive donors as responders.
EBV-specific CD8+ T cells from donors 1 and 3 produced
significant numbers of IFN- In addition to these reported epitope peptides, CD8+ T
cells from donor 1 produced significant numbers of IFN- Tetramer-assisted TCR down-regulation assay Peptides at high concentrations such as 10 µM in the ELISPOT assay can stimulate irrelevant T-cell repertoires with low-affinity TCR48 and there seems to be no evidence that these reacting peptides are endogenously processed and presented. We have adopted the novel strategy of confirming that the peptides are endogenously processed and presented with the aid of MHC/tetramer staining. For this purpose, we generated HLA A*2402 tetramers incorporating the 5 candidate peptides 11, 13, 21, 24, and 29, which were relatively commonly recognized in the ELISPOT assay, and 2 known epitopes, RYSIFFDYM (peptide no. 43) derived from EBNA3A,38 and TYSAGIVQI (peptide no. 44) derived from EBNA3B.2 Actually, the nonamer peptide RYSIFFDYM was used throughout this study instead of the reported octamer RYSIFFDY, because the nonamer gave high affinity to HLA A*2402 molecules, demonstrated by the MHC stabilization assay, and was recognized at lower concentrations by specific CD8+ T cells (data not shown).No cells in any EBV-specific CD8+ T cell line were stained with tetramers incorporating peptide no. 13 (Tet-13) and Tet-29 (data not shown). The reasons were not clear, but Tet-13 and Tet-29 were thus excluded from further studies. Tet-11 stained some proportion of CD8+ T cells from donor 1, Tet-21 stained those from all the donors, Tet-24 stained those from donors 1 and 3, Tet-43 stained those from donor 1, and Tet-44 stained those from donor 3 (data not shown). Thus, combinations of the CD8+ T cells from donor 1 and Tet-11, -21, -24, or -43, and those from donor 3 and the Tet-44 were used in further studies to demonstrate ligation-dependent TCR down-regulation. It has been shown that stimulation with cognate peptides leads to rapid
down-regulation of the TCR.42,43 To confirm the phenomenon
for the epitopes identified here, CD8+ T cells from donor 1 were stimulated with peptides 11, 21, 24, and 43 for 16 hours and
stained with Tet-11, -21, -24, and -43, respectively. CD8+
T cells from donor 3 were stimulated with peptide no. 44 and stained
with Tet-44. All aliquots of responder T cells were simultaneously stimulated with irrelevant peptides which can bind to HLA A*2402 molecules for control staining. The results are shown in Figure 2. The percentage of tetramer-positive
cells was specifically decreased in total CD8+ T cells that
had been stimulated with the same peptide incorporated in the
individual tetramer used for staining. These data not only confirmed
the previous observation of rapid stimulation-induced TCR
down-regulation but also provided evidence that the down-regulation occurs in an epitope-specific manner.
Next we prepared HLA A24-positive or -negative LCLs and CD40-Bs
(EBV-negative) to study whether they could induce TCR down-regulation in epitope-specific T cells. As demonstrated in Figure
3, the percentage of CD8+ T
cells stained with either of the tetramers assembled with the 5 peptides decreased when stimulated with only HLA A*2402-positive LCLs,
but not with HLA A24-negative LCLs or HLA A24-positive CD40-Bs. These
data suggest specific TCR down-regulation to be caused by ligation of
the TCR and HLA A*2402-EBV peptide complexes. In other words, the data
suggest all the peptides are presented on the cell surfaces of HLA
A24-positive LCLs, recognizable by specific T cells.
Finally, for the TCR down-regulation assay we prepared HLA
A24-positive fibroblast cells infected with recombinant vaccinia viruses expressing each of the EBV genes as APCs. As shown in Figure
4, the percentage of CD8+ T
cells stained with Tet-21, -24, -43, and -44 decreased when stimulated
with HLA A24-positive fibroblast cells infected with vBRLF1, vBMLF1,
vEBNA3A, and vEBNA3B, respectively (upper panels). Each of the 4 genes
encodes sequences of the epitope peptide assembled in the tetramer used
for given staining. Stimulation with APCs expressing irrelevant EBV
proteins did not cause TCR down-regulation (lower panels). Thus,
peptide ligand-specific TCR down-regulation took place only when
stimulated with APCs expressing the separate EBV gene of interest. This
strongly indicates that the 4 EBV proteins are endogenously processed
and presented for recognition by T cells. Taken together, the data
demonstrated in Figures 2, 3, and 4 indicate that the newly identified
peptide no. 21, TYPVLEEMF from BRLF1 and peptide no. 24, DYNFVKQLF from
BMLF1, in addition to the peptides reported previously, no. 43, RYSIFFDYM, from EBNA3A and no. 44, TYSAGIVQI, from EBNA3B, are
endogenously processed and presented by HLA A*2402-positive
EBV-infected target cells. In contrast, the TCRs specific to HLA
A*2402-peptide no. 11 complexes, identified by staining with Tet-11,
were down-regulated when stimulated with T2-A24 cells pulsed with the
same peptide (Figure 2) or HLA A24-positive LCLs (Figure 3), but not
with the HLA A24-positive fibroblast cells infected with vLMP2
encoding the peptide no. 11 sequence (Figure 4).
Peptide no. 11 is presented on HLA 24-positive LCLs,
IFN- As demonstrated in Figure 5, neither CTL
clone efficiently killed autologous LCLs unless cognate peptides were
pulsed, which is not uncommon for some EBV-specific CTL clones
stimulated with autologous LCLs for growing.49,50 This
could be due to the insensitive nature of the 51Cr-release
assay if small percentages of the target cells present sufficient
peptide/MHC complexes to stimulate cytolysis. On the other hand, assays
applying cytokine release could be useful when no less than 1% of the
stimulating LCLs display them, especially performed at higher
stimulator-to-CTL ratios.49 Thus, further studies were
performed applying the ELISPOT assay. Figure
6 demonstrates that both IYV-CTL and
RYS-CTL produced IFN-
Minigene constructs present peptide no. 11 in TAP-negative T2-A24 cells As a final set of experiments to characterize processing of peptide no. 11, we constructed plasmids encoding the minimal epitopes, peptide no. 11, IYVLVMLVL (pcIYV), and peptide RYSIFFDYM (pcRYS). Both the minigenes start with methionine, which should be cleaved by aminopeptidase in the endoplasmic reticulum52 and end with a stop codon just after the C-terminus amino acid. HEK293T or T2-A24 cells were independently transfected with pcDNA3 encoding full-length LMP2 (pcLMP2), the EBNA3A (pcEBNA3A) gene, or minimal epitopes. The plasmid expressing HLA A*2402 was cotransfected into HEK293T cells. The results are summarized in Figure 7. IYV-CTL produced IFN- spots when stimulated with HEK293T cells
transfected with pcIYV but not with pcLMP2 (Figure 7, upper left box),
indicating there is a defect in processing but not presentation of the
IYVLVMLVL, probably as is the case with fibroblast cells not treated
with IFN-. In contrast, RYS-CTL produced IFN- spots when
incubated with HEK293T cells transfected with either pcEBNA3A or pcRYS
(Figure 7, lower left box). Next we used TAP-deficient T2-A24 cells as APCs to study whether TAPs are necessary for the epitope presentation. IYV-CTL produced IFN- spots when incubated with T2-A24 cells transfected with pcIYV, indicating that IYVLVMLVL can be presented TAP-independently (Figure 7, upper right box). IYV-CTL did not produce
IFN- spots when incubated with T2-A24 cells transfected with pcLMP2
(Figure 7, upper right box), indicating some defect in processing to
yield the peptides in T2-A24 cells, as is the case with HEK293T cells.
In addition, IYV-CTL did not produce IFN- spots when incubated with
T2-A24 cells infected with vLMP2 (data not shown). In contrast, RYS-CTL
produced few IFN- spots when incubated with T2-A24 cells transfected
with either pcRYS or pcEBNA3A, indicating that presentation of
RYSIFFDYM is TAP dependent (Figure 7, lower right panel).
Antigenic peptides recognized by virus-specific CTLs are not only
useful tools for studying CTL responses but also potential reagents for
immunotherapy of those who have presenting MHC class I molecules. For
widening the application, efficient strategies to determine such
epitopes are a high priority. Meij et al21 recently
described an efficient approach using an IFN- We have employed LCL-stimulated polyclonal EBV-specific CD8+ T cells as effector cells in the ELISPOT assay to ensure a concentration of T cells specific to naturally processed peptides presented by HLA A*2402 molecules on the LCLs used for stimulation. In addition, a novel approach to confirm endogenous processing and presentation was introduced, consisting of (1) production of HLA A*2402 tetramers incorporating each of the candidate peptides; (2) staining the polyclonal EBV-specific CD8+ T-cell lines with tetramers; and (3) detecting decline of tetramer staining on the same T-cell lines, which had been stimulated with HLA A*2402-positive cells, whereby EBV genes encoding the epitopes were translated, processed, and presented. EBV has 2 types of replication cycles, namely, lytic infection, where infectious virions are produced, and latent infection represented by the majority of LCLs.1 This is why the percentages of Tet-21- and Tet-24-staining cells (specific to BRLF1 and BMLF1 proteins, respectively) in polyclonal CTL lines are relatively low and our stimulation protocol is unlikely to be optimal for reactivating and then growing lytic-cycle-specific CTLs. On the other hand, TCRs stained with Tet-21 and Tet-24 were specifically decreased in intensity after stimulation with HLA A24-positive LCLs. Thus, both T cells specific for latent proteins (stained with Tet-43 and Tet-44) and those specific for lytic proteins should be effectively stimulated for TCR down-regulation in our protocol. Most importantly, experiments using recombinant vaccinia viruses demonstrated that TCR down-regulation took place only on CD8+ T cells stimulated with APCs that expressed EBV genes encoding sequences of peptides incorporated in the tetramers used for TCR detection. Staining patterns using Tet-11 were unique because the TCRs specific to
the HLA A*2402-peptide no. 11 complexes were down-regulated with HLA
A24-positive LCL stimulation, but not with HLA A24-positive fibroblasts infected with vLMP2 encoding the peptide no. 11 sequence. This observation indicates the peptide to be present on the LCLs but
not on vLMP2-infected fibroblasts. The defective presentation of
vLMP2-infected fibroblast cells was confirmed using a CD8+
T-cell clone specific to peptide no. 11, IYVLVMLVL, designated as
IYV-CTL. Defective processing of peptide no. 11 was also demonstrated in the experiment using other APCs, such as HEK293T and T2-A24 cells
transfected with pcDNA3 expressing LMP2. Interestingly, IYV-CTL
produced IFN- EBV LMP2 is a multiple membrane-spanning molecule, within which a
number of epitopes recognized by human CD8+ CTLs are
included. Recently, Lautscham et al54 reported a clear correlation between hydrophobicity of the LMP2 epitope sequence and TAP
independence. Our data for the newly identified HLA A*2402-restricted epitope, IYVLVMLVL, is in accordance with the conclusions they drew.
The amino acid sequence has a high hydrophobicity value of
8.7,55 and lies within the fourth membrane-associate
domain of LMP2. The hydrophobicity value of the control peptide
RYSIFFDYM is 0.67. TAP-independent presentation of IYVLVMLVL was
confirmed using a TAP-negative cell line, T2-A24, transfected with
plasmid encoding the minimal peptide. In addition, to our knowledge
IYVLVMLVL is the first LMP2 epitope that is not presented on
HLA-matched fibroblast cells unless treated with IFN- Tetramers incorporating peptide no. 29 (Tet-29) were found to be
unstable, as disclosed by the final gel filtration profile (data not
shown). We could not deny the possibility that peptide no. 29 is an
epitope. It is derived from BMRF1, which is produced abundantly in the
early lytic cycle of EBV infection.53 Even though peptide
no. 29 demonstrated a low affinity for HLA A*2402 molecules, resulting
in a short half-life for MHC-peptide complexes on the cell surface, a
large amount of peptides might compensate to sustain the ligand density
sufficient to elicit CTL responses. To answer the question of whether
peptide no. 29 is really processed and presented, CTL clones specific
to TYTSGEACL need to be established for testing reactivity to target
cells with expression of HLA A*2402 and BMRF1 genes. This has not been
achieved so far. On the other hand, there were no problems regarding
gel filtration profiles when making tetramers with peptide no. 13 (Tet-13), suggesting stable assembly into MHC molecules. Nevertheless,
EBV-specific CD8+ T-cell lines established from PBMCs of
donors 2, 3, and 4, which had been shown to produce IFN- In conclusion, we have identified an LMP2 epitope presented by HLA
A*2402 molecules in addition to 2 lytic-cycle EBV-specific CTL epitopes
from the amino acid sequences of BRLF1 and BMLF1. The LMP2-derived
peptide, IYVLVMLVL, is processed and presented in a manner that appears
IFN-
The authors thank Dr Toshitada Takahashi for critically reading the manuscript.
Submitted May 2, 2002; accepted September 22, 2002.
Prepublished online as Blood First Edition Paper, September 26, 2002; DOI 10.1182/blood-2002-04-1240.
Supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (no. 13218152) and the Japan Society for the Promotion of Science (no. 12670802).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kiyotaka Kuzushima, Division of Immunology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chigusa-ku, Nagoya 464-8681 Japan; e-mail: kkuzushi{at}aichi-cc.jp.
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Top
Abstract
Introduction
5M 
-mercaptoethanol (referred to as
culture medium) in the presence of 800 µg/mL G418 (GIBCO). HEK293T
cells (American Type Culture Collection, Manassas, VA) and HLA
A24-positive dermal fibroblast cell lines cultured in Dulbecco
modified Eagle medium (GIBCO) supplemented with 10% FCS, 2 mM
L-glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin.
), RYSIFFDYM (
), or in the absence of
peptide (
) at the indicated effector-target ratio.
]; 50 [
]; or 25 [
]) were cultured with the indicated APCs in each well. Each bar
represents the average number of spots in duplicate wells.
]; or 25 [