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IMMUNOBIOLOGY
From the Division of Virology, Aichi Cancer Center
Research Institute, Nagoya, Japan; and the Department of Pediatrics,
Nagoya University School of Medicine, Japan.
Antigenic peptides recognized by virus-specific cytotoxic T
lymphocytes (CTLs) are useful tools for studying the CTL responses exclusively among those who own the major histocompatibility complex (MHC) class I molecules that present the peptides. For widening the
application, an efficient strategy to determine such epitopes in the
context of a given MHC is highly desirable. A rapid and efficient strategy is presented for the determination of CTL
epitopes in the context of given MHC molecules of interest through
multiple screenings consisting of a computer-assisted algorithm
and MHC stabilization and enzyme-linked immunospot assays. A
major cytomegalovirus (CMV)-specific CTL epitope,
QYDPVAALF, in the amino acid sequence of its lower matrix 65 kd phosphoprotein (pp65) presented by HLA-A*2402 molecules was
identified from 83 candidate peptides. The results indicate that the
CMV-specific CTL response is highly focused to pp65 in the context of
HLA-A*2402. Endogenous processing and presentation was confirmed using
a peptide-specific CD8+ T-cell clone as the effectors and
autologous fibroblast cells infected with recombinant vaccinia virus
expressing pp65 gene or CMV as antigen-presenting cells. Flow
cytometric analysis of intracellular interferon- Human cytomegalovirus (CMV) is a ubiquitous
CD8+ cytotoxic T lymphocytes (CTLs) are essential to
the control of CMV infection. In patients undergoing allogeneic BMT,
the delayed regeneration of CMV-specific CD8+ T lymphocytes
following transplantation has been correlated with CMV
disease.5,6 In addition, adoptive transfer of CMV-specific CD8+ T-cell clones has led to restoration of virus-specific
immunity in BMT patients with relatively low frequencies of CMV-related disease.7,8 In addition, recent data indicate that there is an inverse correlation between the CMV-specific CTL response and CMV
antigenemia after renal transplantation.9 This observation underlines the potential benefit of testing for CMV-specific CTL responses in immunocompromised patients in order to monitor and control
CMV infection.
Recently, we have shown that monitoring of the Epstein-Barr virus (EBV)
load and EBV-specific CD8+ T-cell frequencies, determined
by multiparameter flow cytometric analysis to detect rapid accumulation
of interferon Virus-specific CD8+ T cells recognize short
peptides We present here a rapid and efficient strategy for determination
of CTL epitopes in the context of HLA-A*2402 molecules through multiple
screenings consisting of a computer-assisted algorithm, an in vitro MHC
stabilization assay, and an enzyme-linked immunospot (ELISPOT) assay.
In many ethnic groups, HLA-A24 is one of the most common
alleles24,25 and, especially in our country, HLA-A24 is
the most frequently encountered HLA class I allele and the genotype is
almost exclusively A*2402.26 We here introduce an amino
acid sequence from the lower matrix 65-kd phosphoprotein (pp65) of CMV,
QYDPVAALF, as one of the the major CMV-specific CTL epitopes presented
by HLA-A*2402 molecules.
Donors
Virus and cell lines
AD169 strain CMV (VR-538, American Type Culture Collection, Manassas,
VA) was propagated in fibroblast cells infected at a multiplicity of
infection 0.1. Whole infected cultures were harvested when a 100%
cytopathic effect was evident. They were frozen, thawed once, and spun
at 3000 rpm. Aliquots of the supernatant with a titer of
5 × 106 plaque-forming units per milliliter were stocked
at The peptide transporter-negative B × T hybrid cell line 174 CEM.T2 (referred to as T2) was transfected with a plasmid expressing HLA-A*2402,27 a kind gift from Dr K. Itoh, Kurume
University, Japan. The transfected cell line was cloned by
limiting dilution and an A24+ clone screened by indirect
immunofluorescence using an anti-A24 monoclonal antibody (mAb)
(clone 0041HA, One Lambda, Canoga Park, CA), and antimouse fluorescein
isothiocyanate (FITC)-labeled antibodies, named T2-A24, were further
cultured in Iscoves modified Dulbecco medium (Gibco) supplemented with
2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin,
5 × 10 Peptides To identify the potential HLA-A24-binding peptides within CMV proteins (accession code X17403),28 a computer-based program was employed by access through the World Wide Web site BioInformatics and molecular analysis section (BIMAS) HLA peptide bonding predictions.29,30 Most peptides were synthesized with a Cleaved PepSet from Mimotope (Melbourne, Australia) dissolved in 100 µL dimethyl sulfoxide and further diluted in 40% acetonitrile, 0.1 M HEPES (pH 7.4), where necessary. The yield of each peptide was assumed to be 1 µM. HLA-A24-binding peptides RYLRDQQLL, derived from the HIV envelope protein,31 and TYGPVFMSL, derived from EBV latent membrane protein 2A,32 were synthesized (Sawady, Tokyo, Japan) and used as control peptides. The octamer QYDPVAAL of CMV pp65 used in the CTL assay was also synthesized (Sawady). Peptides used in this paper are listed in Table 1.
MHC stabilization assay The synthesized peptides were used in an MHC stabilization assay using T2-A24 cells as described earlier.33 Briefly, T2-A24 cells (2 × 105) were incubated with 200 µL RPMI 1640 (Sigma, St Louis, MO) containing 0.1% FCS, 5 × 10 5 M  -mercaptoethanol, and each of
the peptides at a concentration of 10 µM at 26°C for 16 hours,
followed by incubation at 37°C for 3 hours. After the incubation,
surface HLA-A24 molecules were stained with the anti-A24 mAb and
antimouse FITC-labeled antibodies. Expression was measured by FACScan
(Becton Dickinson, San Jose, CA), and mean fluorescence intensity (MFI)
was recorded. Percent MFI increase was calculated as follows: Percent
MFI increase = (MFI with the given peptide  MFI without
peptide)/(MFI without peptide) × 100.
Generation of polyclonal and clonal CMV-specific CTLs CMV-specific polyclonal CTLs were generated as described earlier.34,35 Briefly, heparinized peripheral blood was obtained by venipuncture from a CMV-seropositive volunteer, and PBMCs were isolated by density gradient centrifugation over Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). The PBMCs (1 × 107) were cultured with 5 × 105 autologous fibroblast cells infected 2 hours previously with the AD169 strain of CMV at a multiplicity of infection of 5 in individual wells of a 6-well plate in a final volume of 6 mL RPMI 1640 supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 µg/mL streptomycin, 5 × 105 M -mercaptoethanol, and 10% FCS (referred
to as culture medium). After 7 days, the cultures were restimulated
with both autologous CMV-infected fibroblast cells and  -irradiated
(33 Gy) allogeneic PBMCs, and they were then supplemented with
recombinant IL-2 (20 U/mL) 2 days thereafter. The CTL line was further
stimulated weekly with autologous CMV-infected fibroblast cells,
-irradiated allogeneic PBMCs, and IL-2.
To generate CMV-specific CTL clones, CD8+ T cells were
enriched with the aid of magnetic beads (Dynal, Oslo, Norway) and
plated in wells of 96-well round bottom plates at 0.3 and 1 cell per well with 5 × 104 ELISPOT assay Flat-bottom, 96-well MultiScreen-HA plates with a nitrocellulose base (Millipore, Millipore Corporation, Bedford, MA) were coated with 10 µg/mL anti-IFN- mAb (R & D Systems, Minneapolis, MN) and incubated overnight at 4°C. After washing with
phosphate-buffered saline (PBS), plates were blocked with the culture
medium for 1 hour at 37°C. T2-A24 (5 × 104) cells were
pulsed with each peptide in 100 µL RPMI 1640 with 0.1% FCS and
5 × 105 M -mercaptoethanol in each well of the
plates for 30 minutes at room temperature. A total of 10 000
polyclonal CD8+ T cells suspended in culture medium
supplemented with additional FCS to 20% and 20 U/mL IL-2 were seeded
in each well. When the spots were too many to count, 1000 CD8+ T cells were used as responder cells, and the numbers
of spots were multiplied by 10 (Figure
1). For testing the specificity of
CMV-specific T-cell clones, indicated numbers of the T cells in figures
were used as responders. The peptide concentrations described in the
text and figures indicate those in the final assay volume. In some
experiments, HLA-A24+ fibroblast cells were infected 2 hours previously with CMV AD169 strain, recombinant vaccinia virus
expressing glycoprotein B, or pp65 of CMV (kind gifts from Dr S. R. Riddell, Fred Hutchinson Cancer Research Center, Seattle, WA) at a
multiplicity of infection of 5 and used as antigen-presenting cells
(10 000 cells/well). All assays were performed in duplicate. The
plates were incubated in a 5% CO2 incubator at 37°C for
20 hours and extensively washed with PBS containing 0.05% Tween 20. A
polyclonal rabbit anti-IFN- antibody (Genzyme, Cambridge, MA) was
added to individual wells and left for 90 minutes at room temperature,
followed by exposure to peroxidase-conjugated goat antirabbit
immunoglobulin G (Genzyme) for an additional 90 minutes. For
visualization of IFN--specific spots, 100 µL of 0.1 M sodium
acetate buffer (pH 5.0) containing 3-amino-9-ethylcarbazole
(Sigma) and 0.015% H2O2 was added to each
well. After 40 minutes, the reaction was stopped by washing with water
and the plates were dried. Diffuse large spots were counted under a
dissecting microscope.
CTL assay CTL assays were performed employing 51Cr release as previously described.10,11 Briefly, 51Cr-labeled T2 or T2-A24 cells (2000 cells/well) were pulsed with each peptide in 100 µL RPMI 1640 with 0.1% FCS and 5 × 105 M -mercaptoethanol in each well of a
V-bottom 96-well plate for 30 minutes at room temperature. Desired
numbers of CTLs suspended in 100 µL of the culture medium
supplemented with additional FCS to 20% were seeded in each well. The
peptide concentrations described in the text and figures indicate those
in the final assay volume. Each assay was performed in triplicate.
After 5 hours of incubation, the supernatants were harvested and
radioactivity counted with a -counter. Percent specific lysis was
calculated as follows: Percent specific lysis = (experimental
lysis minimum lysis) × 100/(maximum lysis minimum
lysis). Minimum lysis was obtained by incubating the target cells with
the culture medium alone. Maximum lysis was obtained by exposing the
target cells to 1% Nonidet P-40.
Detection of IFN-
(Coulter) mAbs. Stained cells were analyzed by FACScan using Lysis II
software. Live gating of the CD8+ subset was performed, and
50 000 events were acquired for each analysis.
Tetramer production and staining MHC-peptide tetramers were produced as described previously.17-21,23 Briefly, BL21(DE3) pLysS (Novagen, Madison, WI) competent cells were transformed with pET11d plasmid (Novagen) encoding HLA-A*2402 heavy chain (a kind gift from Dr H. Takasu, Sumitomo Pharmaceutical, Osaka, Japan) or pET-3a plasmid (Novagen) encoding2-microglobulin (a kind gift from Dr
H. Takizawa, Otsuka Pharmaceutical, Tokushima, Japan) to produce the
recombinant proteins. Expression of the HLA heavy chain was limited to
the extracellular domain, and C-terminus of their domain was modified
by the addition of a substrate sequence for the biotinylating enzyme
BirA. Monomeric HLA-peptide complexes were folded in vitro by adding
the HLA protein to 2-microglobulin in the presence of
the 9-mer, QYDPVAALF. Proteins were dialyzed against water and then
concentrated. After purification on gel filtration, the MHC complex was
biotinylated by using recombinant BirA enzyme (Avidity, Denver, CO) and
then purified by gel filtration. HLA-peptide tetramers were made by
mixing the biotinylated MHC with phycoerythrin-labeled streptavidin
(Molecular Probes, Eugene, OR) at a molar ratio of 4:1. Tetramers were
purified by gel filtration on a Superdex 200 HR 10/30 (Amersham
Pharmacia Biotech, Uppsala, Sweden) and concentrated to 3 mg/mL of
total proteins and stored at 4°C until use. PBMCs
(2 × 106) or CTL clones (2 × 105) were
stained with the tetramer at a concentration of 0.1 mg/mL tetramers and
Tricolor anti-CD8 mAb (Caltag, Burlingame, CA) at 37°C for 15 minutes. After washing twice, stained cells were fixed with 0.5%
paraformaldehyde before analysis with FACScan.
Selection of potential HLA-A24-binding peptides within CMV proteins To identify potential HLA-A24-binding peptides within amino acid sequences of immediate early (IE), tegument and envelope proteins of CMV were analyzed with a computer program designed to predict HLA-binding peptides, based on estimation of the half-time dissociation of the HLA-peptide complex by access through the World Wide Web site BioInformatics and molecular analysis section (BIMAS) HLA peptide bonding predictions.29 A total of 83 peptides with estimated half-time dissociation scores above 100 were selected (Table 1). All the peptides share the HLA-A24-binding motifs as tyrosine at the second residue and phenylalanine or leucine at the ninth or tenth residue.37-39 Next, MHC stabilization assays were performed to test these peptides for HLA-A*2402 binding efficiency using T2-A24 cells. Most peptides increased the HLA-A24 expression on the cells, indicating that these peptides bound and stabilized the HLA complex on the cell surface, but peptides no. 20, 28, 32, 45, 50, and 53 gave negative values for the percent MFI (Table 1) and were excluded from further studies.Screening of peptides antigenic for an anti-CMV polyclonal CTL line by ELISPOT assay To identify peptides recognized by CMV-specific CTLs in the context of HLA-A*2402 molecules, the ELISPOT assay was performed using T2-A24 cells as antigen-presenting cells. We chose the assay for the screening because it can test many peptides simultaneously in a few plates using relatively small numbers of CTLs as responders. Moreover, because the sensitive nature of the assay enables 40 spots to be scored as positive, subdominant epitopes could be identified when T cells recognizing such epitopes were present at concentrations no less than 40 in 10 000 cells. As shown in Figure 1, the polyclonal CMV-specific CD8+ T cells established from PBMCs of a CMV-seropositive and HLA-A24+ donor produced significant numbers of IFN-
spots when incubated with T2-A24 cells pulsed with CMV peptides nos.
60, 80, 81, 82, and 83. Peptide no. 60 was derived from glycoprotein H,
and others were from pp65 (open reading frame name UL83). The
responders produced negligible spots when incubated with mock-infected
autologous fibroblast cells, while more than 1000 spots were detected
with CMV-infected autologous fibroblast cells. To ascertain the nature of the peptides recognized by the CMV-specific CD8+ T-cell
line, we performed ELISPOT assays with serial dilutions of the 4 peptides derived from pp65. Figure 2
shows that peptide no. 81 was still recognized at a concentration of 1 pM. The end points for recognition with the others were 10 pM for
peptide no. 83 and 10 nM for peptides no. 80 and 82. The 2 peptides,
no. 81 and 83, that were recognized at picomolar levels share the amino
acid sequence QYDPVAALF, with an additional C-terminal phenylalanine for no. 83.
Recognition of the 4 pp65 peptides by single CMV-specific CD8+ T-cell clones Notably, the sum of the number of T cells that reacted with each of the 5 peptides exceeded that reacting with CMV-infected fibroblast cells (Figure 1). This could be due to either weaker antigen presentation on the CMV-infected fibroblast cells, possibly by the mechanism of class I molecule down-regulation,40-44 or the same T-cell repertoires being reactive with some of the 5 peptides. To test these possibilities, we established T-cell clones from limiting dilution culture of the bulk CTL line stimulated with anti-CD3 mAb, appropriate feeder cells, and IL-2. The results of ELISPOT assays with 2 representative T-cell clones are shown in Figure 3. Both clones recognized all 4 peptides at high concentrations. The effect of peptide dilution was the same with bulk CD8 T cells and with the T-cell clones (Figures 2 and 3). Among a total of 54 clones established, 28 clones were reactive for all 4 peptides but not for peptide no. 60 at a concentration of 10 µMand effects of dilution on recognition were similar for all the
clones tested (data not shown). Thus, we conclude that the
spots in the wells containing each of the 4 pp65 peptides were produced
by the same T-cell repertoires.
Recognition of the nonamer QYDPVAALF at a lower concentration than the octamer or the decamer Because no. 81 and 83, which were recognized at picomolar levels, share the amino acid sequence QYDPVAALF and amino acid sequences of the peptides no. 80 (VYALPLKML) and 82 (QYVKVYLESF) were irrelevant, QYDPVAALF should be the core sequence of the T-cell epitope. Because class I molecules usually accommodate 8 to 10 amino acids in their antigen-binding sites, the octamer QYDPVAAL was simultaneously tested in CTL assays for its recognition by CTL clone no. 1. As shown in Figure 4, it far less effectively sensitized T2-A24 cells for CTL-mediated cell lysis than the nonamer or the decamer. We did not make and test the octamer YDPVAALF because tyrosine at the second residue is essential for binding to HLA-A24 molecules.37-39 Again, only the nonamer QYDPVAALF (peptide no. 81) could sensitize the target cells for CTL-mediated cell lysis at the concentration of 1 pM. In combination with the results of ELISPOT assays (Figures 2 and 3), this indicates that the nonamer QYDPVAALF is the minimal CTL epitope.
Recognition by the QYDPVAALF-specific CD8+ T-cell clone of the epitope in the context of HLA-A*2402 molecules To confirm the HLA restriction, CTL assays were performed using 51Cr-labeled T2 or T2-A24 cells pulsed with QYDPVAALF or the control peptide TYGPVFMSL, derived from EBV latent membrane protein 2A (Figure 5). Only T2-A24 cells pulsed with QYDPVAALF were effectively lysed by the CTL clone no. 1. The results indicate that the epitope QYDPVAALF is presented by HLA-A*2402 molecules.
Recognition by the QYDPVAALF-specific CD8+ T-cell clone of A*2402+ fibroblast cells infected either CMV or recombinant vaccinia virus expressing pp65 A set of experiments was performed to confirm that the epitope QYDPVAALF is processed within the cells infected with CMV. For that purpose, autologous and allogeneic HLA-A24+ fibroblast cells were mock-infected or CMV-infected and used as antigen-presenting cells for ELISPOT assays using the CTL clone no. 1 as responders. Figure 6 demonstrates that the CMV-infected cells stimulated for IFN- production by the T cells.
The T cells were cytolytic for the CMV-infected fibroblast cells (data
not shown). To confirm that endogenously produced pp65 proteins were processed and presented to the QYDPVAALF-specific CD8+ T
cells, autologous fibroblast cells were infected with recombinant vaccinia virus expressing pp65 or glycoprotein B of CMV. The results clearly showed only fibroblast cells infected with recombinant vaccinia
virus expressing pp65 to stimulate the T cells for IFN- production.
The data indicated that pp65 expressed within the cells is endogenously
processed to yield the epitope.
Frequencies of QYDPVAALF-specific CD8+ T cells in PBMCs of healthy CMV-seropositive donors We examined QYDPVAALF-specific CD8+ T-cell frequencies in PBMCs of healthy HLA-A24+, CMV-seropositive, and HIV-seronegative donors using tricolor flow cytometry analysis for detection of antigen-specific IFN--producing T
cells.10,13 First, the phycoerythrin-cyanin-5.1-labeled
anti-CD8 mAb was used for gating the population. Second, the
phycoerythrin-labeled anti-CD69 mAb was employed for enhancement of
precise detection of responding T cells. CD69 is up-regulated on
activated T cells prior to cytokine production and thus allows more
definitive clustering of the true responding
fraction.10,13 As a control peptide, we used
HLA-A24-binding peptide RYLRDQQLL, derived from the HIV envelope
protein.31
When PBMCs from long-term healthy HLA-A24+ and
CMV-seropositive individuals (n = 5) were stimulated with peptide
QYDPVAALF, 0.04% to 0.27% of total CD8+ T cells produced
IFN-
Tetramer staining Finally, we made fluorescent-labeled tetrameric MHC-peptide complexes (tetramer) using the nonamer peptide QYDPVAALF. The tetramers specifically bound to the peptide-reactive T-cell clone but not the unreactive clone (Figure 8A,B). QYDPVAALF-specific CD8+ T cells detected by the tetramers were 0.79% in peripheral blood from the donor 5 in Figure 7 and 0.0% in that from an HLA-A24+ and CMV-seronegative donor (Figure 8C,D).
The identification of new CTL epitopes is generally time-consuming and labor-intensive and consists of (1) establishing virus-specific CTL clones; (2) testing a panel of allogeneic virus-infected target cells that share a part of MHC class I molecules with the CTLs to determine the MHC restriction; (3) transfecting or transducing cells expressing the appropriate MHC class I molecules with each of the segregated virus genes for the target of CTL; (4) making overlapping peptides, eg, 20 amino acid peptides overlapping by 10 amino acids to span a given antigen length for testing the specificity of the T cells; and, finally, (5) mapping the sequences with truncated peptides to determine the minimal epitope. We show in this article a rapid and efficient strategy to find new CTL epitopes, especially presented by MHC molecules of interest. An important step in this process is the use of a computer-based program designed to predict potential HLA-binding peptides within viral proteins.29,33,45 Analysis of amino acid sequences of various proteins of CMV AD169 strain revealed a number of potential HLA-A24-binding peptides, and most of them then functionally stabilized HLA-A*2402 molecules that had been expressed on the peptide transporter-deficient cell line T2. Next and equally important is screening of the peptides by ELISPOT assay for their recognition by bulk CMV-specific CTLs, established from PBMCs of a CMV-immune donor positive for HLA-A24 typing. The ELISPOT assay has many advantages in this regard. First, it allows results with a number of different peptides to be generated simultaneously in a few plates within 3 days. Second, the assay demands relatively small numbers of CTLs, eg, 25 to 100 000 in a well, depending on the concentration of specific T cells. Third, the sensitive and specific nature of the assay enables it to detect minor populations of antigen-specific T cells. For example, 40 spots in 100 000 CD8+ T cells in a well could be scored positive as long as the numbers of background spots were sufficiently low. Fourth, it needs no radioisotopes. Target antigens of CMV-specific CD8+ T cells have been reported to be restricted to IE proteins,46,47 glycoprotein B,34,46 and tegument proteins such as pp65.35,47,48 Thus, we first searched candidate peptides that have the HLA-A24-binding motif by a computer-assisted algorithm among proteins in the IE gene family, glycoproteins, and tegument proteins to find as many epitopes as possible. Among 83 peptides synthesized, 6 were unable to stabilize HLA-A*2402 molecules expressed on T2 cells at all and were excluded from further analysis. The ELISPOT assay using CMV-specific CD8+ T cells maintained by weekly stimulation with CMV-infected autologous fibroblast cells and IL-2 revealed that the T cells recognized almost exclusively peptides derived from pp65 (Figure 1). The results underline the observations of McLaughlin-Taylor et al,35 who showed that pp65 is a target antigen for CMV-specific CTLs in most seropositive donors tested, and the finding of Wills et al47 that 70% to 90% of CMV-specific CTL precursors are pp65-specific. It has been reported that pp65-specific CTL clones recognize CMV-infected target cells in the absence of viral gene expression.34,35 The capacity of CMV to interfere with class I MHC presentation at an early time after infection40-44 might explain why the specificity of the CTL response should be focused on structural proteins, which are relatively abundant immediately after infection, derived from input virus. The pp65 is a major constituent of the virus tegument43 and consequently available to the class I processing pathway immediately after infection. Our data confirm and extend the previous observations in the context of HLA-A*2402 molecules. Both the bulk CMV-specific CTL line and the CD8+
T-cell clones simultaneously recognized peptides no. 80 (VYALPLKML), 81 (QYDPVAALF), 82 (QYVKVYLESF), and 83 (QYDPVAALFF) at concentrations
higher than 10 nM. However, only peptides no. 81 and 83, which share a
core sequence QYDPVAALF, were able to sensitize T cells at
concentrations lower than 100 pM (Figures 2 and 3). Actually, peptides
no. 80 (VYALPLKML) and 82 (QYVKVYLESF), which were resynthesized
independently, were not able to sensitize the T cells even at a
concentration of 10 µM (data not shown). Thus, we reason that the
recognition of peptides no. 80 and 82 could be due to
cross-contamination between peptides in adjacent wells during
synthesis, transfer, storage, or handling and that QYDPVAALF(F) is the
T-cell epitope. Because the octamer QYDPVAAL was far less effectively
recognized by the T cells (Figure 4), we concluded that the nonamer
QYDPVAALF is the minimal epitope. This also indicates that
phenylalanine at the C-terminal residue of the nonamer is important for
binding to HLA-A24 molecules.38 We did not
synthesize octamer YDPVAALF for testing because tyrosine at the second
residue is reported to be critical for binding to HLA-A24
molecules.37-39 We do not know yet which is the naturally
processed peptide, QYDPVAALF or QYDPVAALFF, or whether they both could
be. To answer this question, peptides eluted from HLA-A*2402 molecules
of CMV-infected cells should be analyzed for their sequences or
molecular weights after fractionating on high-performance liquid
chromatography.49 However, the minimal epitope QYDPVAALF
can stimulate specific T cells for IFN- The ELISPOT assay demonstrated numbers of CD8+ T cells
reacting with the minimal epitope, QYDPVAALF (no. 81), to exceed half of those reacting with autologous CMV-infected fibroblast cells (Figure
1), indicating that CMV-specific CD8+ T-cell responses in
the donor are highly focused on pp65 and especially the
HLA-A*2402-binding peptide, QYDPVAALF. This was also confirmed by the
evidence that, among 54 CD8+ T-cell clones established with
anti-CD3 mAb stimulation, 28 clones turned out to be specific for the
epitope. Thus, as far as the donor is concerned, HLA-A*2402 molecules
are dominant presenters of CMV antigens, and QYDPVAALF is a definitely
major epitope of CMV-specific CD8+ T cells. To extend this
observation, we performed flow cytometric analysis for detecting
IFN- Peptide no. 60, NYLDLSALL, derived from glycoprotein H was recognized by bulk CTL at the concentration of 10 µM but not by the pp65-specific CTL clones at the same concentration, indicating that it is a minor epitope presented by HLA-A*2402 molecules; however, this could not be confirmed because CTL clones that specifically recognize NYLDLSALL have not been established so far. In conclusion, we have identified a possible major CMV-specific CTL
epitope from the amino acid sequence of pp65 presented by HLA-A*2402
molecules. Minimal CMV-specific CTL epitopes so far reported are
exclusively within the pp65 (Table 2).
Our data are in line with the highly focused reaction of CMV-specific
CTLs to pp65 in the context of HLA-A*2402 molecules, as is the case with HLA-A*020118,20,21,48 and HLA-B*0702.17
The newly identified peptide QYDPVAALF could be used for detection of
CMV-specific CD8+ T-cell responses as a representative CTL
antigen. In addition, it might be a candidate reagent for peptide
vaccines for prevention of CMV disease or expansion of CMV-specific
CTLs in vitro for the purpose of adoptive transfer treatment of
immunocompromised patients positive for HLA-A*2402.
Submitted January 31, 2001; accepted May 14, 2001.
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 Promotion of Science (No. 12670802 and RFTF 97L00703).
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 Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chigusa-ku, Nagoya 464-8681, Japan; e-mail:kkuzushi{at}aichi-cc.pref.aichi.jp.
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Top
Abstract
Introduction
) and 83 (
), that were recognized at
picomolar levels share the amino acid sequence QYDPVAALF, with an
additional C-terminal phenylalanine for no. 83. The amino acid
sequences of peptides no. 80 (
) and 82 (
) are VYALPLKML and
QYVKVYLESF, respectively. A total of 1000 polyclonal CMV-specific
CD8+ T cells established from PBMCs of a CMV-seropositive
and HLA-A24+ donor were cocultured with 50 000 T2-A24
cells in each well in the presence of each peptide at the indicated
concentrations.
indicates TYGPVFMSL
derived from EBV latent membrane protein 2A. CTL assays were performed
using 51Cr-labeled T2-A24 cells as targets at an
effector-target ratio of 5:1 in the presence of each peptide
at the indicated concentrations.
) as target cells, incubated with CMV-specific CD8+ CTL
clone no. 1 at the indicated effector-target ratios in the presence of
100 nM of each peptide.
), 50 (
) CD8+ T cells were cocultured with
10 000 fibroblast cells in each well. Each bar represents the
average number of spots in duplicate wells.
