ABBS 2005,38(02): Identification of Melanoma-specific Peptide Epitopes by HLA-A2.1-restricted Cytotoxic T Lymphocytes

Abstract        HLA-A2.1-associated
peptides, extracted from human melanoma cells, were used to study epitopes for
melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) by epitope
reconstitution, active peptide sequence characterization and synthetic peptide
verification. CTL were ­generated from tumor-involved nodes by in vitro
stimulation, initially with autologous melanoma cells and subsequently with
allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and
allogeneic HLA-A2.1 positive melanomas, but not HLA-A2.1 negative melanomas or
HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by
anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1
molecules were purified from detergent-solubilized human melanoma cells by
immunoaffinity column chromatography and further fractionated by reversed phase
high performance liquid chromatography. The fractions were assessed for their
ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive
antigen-processing mutant T2 cells. Three reconstitution peaks were observed in
lactate ­dehydrogenase release assay. Mass spectrometry and ion-exchange high
performance liquid chromatography analysis were used to identify peptide
epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine
amino acid residues. The data from reconstitution experiments confirmed that
the synthetic peptides ­contained epitopes and that the peptides associated
with HLA-A2.1 and recognized by melanoma-specific CTL were present in these
different melanoma cells. These peptides could be potentially exploited in
novel peptide-based antitumor vaccines in immunotherapy for CTL.

Key words        melanoma cell line; cytotoxic T
lymphocyte; tumor antigenic peptide; HLA-A2.1-associated peptide

Lymphocytes from
patients with melanoma, when stimulated in vitro with recombinant human
interleukin (IL)-2 and autologous melanoma cells, develop a melanoma-specific
cytotoxic response [1]. Evidence that the response of cytotoxic T lymphocytes
(CTLs) to human melanoma is restricted by major histopathology complex (MHC)
class I molecules includes demonstration of cross-reactivity for allogeneic
melanoma cells that share restricting MHC class I molecules with the autologous
tumor. The human leucocyte antigen (HLA)-A2 molecules are effective restricting
elements for the melanoma-specific CTL response, and melanoma-specific
HLA-restricted CTLs lyse the majority of HLA-A2 positive melanomas. The ­lysis
of HLA-A2 positive melanomas transfected with the ­­HLA-A2.1 gene suggested
that these transfected melanomas present the epitopes recognized by HLA-A2-restricted
melanoma-specific CTL and that CTLs recognize HLA-A2-restricted epitopes shared
by the majority of melanomas, evidence for some CTL epitopes in a single HLA-A2
­positive melanoma has been presented [2]. Because HLA-A2 molecules are
frequently expressed in human populations, ­­HLA-A2-restricted
melanoma-specific CTL may have ­significant therapeutic potential for a large
number of patients. To optimize their therapeutic value, it is essential to
identify the antigenic epitopes that stimulate the CTL response [3].

Epitopes for CD8
positive CTL are believed to be short peptides of usually nine residues that
bind to a cleft on the surface of the MHC class I molecule. The peptides, ­generated
from proteolysis of endogenous proteins in the cytosol, are transported to the
endoplasmic reticulum, where they become associated with newly synthesized MHC
class I molecules. They are then transported to the cell surface [4]. The
definition of individual peptides that comprise specific CTL epitopes has proved
difficult, ­because of the complexity of the peptide mixture ­associated with
MHC class I molecules. A lot of human ­­tumor-associated antigen peptides have
been identified to date, most of which are derived from melanomas of several ­melanoma
antigens (tyrosinase, gp100/Pmel-17, ­Mart-1/Melan-A,­
NY-ESO-1, MAGE and gp75) that give rise to HLA-A2-restricted antigenic peptides
and represent ­melanoma-specific epitopes [5–7]. Most known HLA class I restricted peptides have been
isolated using a genetic method [9,10]. An alternate
approach for the characterization of CTL epitopes is to directly identify the
peptides. Virus-specific CTL epitopes have been reconstituted in vitro by
allowing exogenous viral peptides to bind to MHC molecules on the cell surface
of target cells [11]. To use this approach for the identification of clinically
important melanoma-specific epitopes, it is necessary to demonstrate that
peptides ­containing these epitopes can be extracted from melanoma cells and
that these epitopes are shared by different ­melanoma cell lines [12].

In the present report, ­HLA-A2.1­-associated
peptides purified from human melanoma cells using immunoaffinity chromatography
and fractionated with reversed phase high performance liquid ­chromatography
(HPLC) were used to reconstitute epitopes for ­HLA-A2.1-restricted
melanoma-specific CTL. The presence of a ­peptide-defined CTL epitope in human
melanoma cells was identified and the synthetic peptides comprising the epitope
of melanoma-specific CTL were verified. The obtained peptides could be
exploited in novel peptide-based ­antitumor vaccines in immunotherapy for CTL
[13,14].

Material and Methods

Cell lines

The CS96-Mel and
Chap-Mel melanoma cell lines were established in our laboratory from biopsies
of metastatic lesions from patients with cutaneous melanoma. The cells were
cultured in a monolayer culture in complete medium consisting of RPMI 1640
supplemented with 10% ­­heat-inactived fetal calf serum (FCS) and antibiotics.
The ­624-Mel and 397-Mel melanoma cell lines were obtained from the laboratory
of Dr. Steven A. ROSENBERG (National Institutes of Health,

Phenotypic analysis

After trypsin digestion,
the collected cells (human ­melanoma cells, JY and T2) were washed twice in ­­ice-cold
Hanks balanced salt solution, then resuspended in ­0.­­1%
bovine serum albumin (BSA)/phosphate-buffered saline (PBS). We incubated 5´105 cells at 4
ºC for 30 min with 50 ml (0.05
mg/ml) of purified IgG of HLA-A2.1-specific monoclonal antibody (MAb), MA2.1, and
isotype-matched mouse ­antibodies served as the negative control. Staining was
carried out with 100 ml of 1:1000
fluorescein isothiocyanate-conjugated rabbit antimouse IgG antibody (Capple,
Irvine, USA) at 4 ºC for 30 min. The cells were fluorescently analyzed using a
FACScan flow cytometer (Becton Dickinson,

Cytotoxicity assay

Cell-mediated killing
was determined using in vitro ­lactate dehydrogenase (LDH) release
assays. Briefly, 5´103 ­target
cells/well on a 96-well round bottom plate were mixed with various numbers of
effector cells in 200 ml of ­serum-free
AIM-V medium, centrifuged at

The measurements were
conducted in triplicate. In the experiments, the spontaneous LDH release was
assessed by incubating target cells in the absence of effector cells, or
effector cells in the absence of targets cells. Maximum release was determined
in the presence of lysis solution.

Inhibition of
cytotoxicity with MAb

We pre-incubated 5´103 target
cells with different ­specific MAb at 4 ºC for 30 min at a final concentration
of 100 mg/ml in the medium, prior to the assay for cytotoxicity.
The MAbs used were anti-CD3, anti-HLA class I, anti-HLA-A2.1 and anti-HLA class
II (Genzyme,

Peptide isolation and
fractionation

The human melanoma cells
624-Mel, Chap-Mel and JY were cultured in Triple flask (Nunc,

Western blot analysis

Western blot analysis of
HLA-A2.1 molecules purified by immunoaffinity was carried out using SDS-PAGE ­­(­4%–20% gradient gels).
Ten microliters of sample buffer (­1.6% SDS, 10% glycerol, 4%
2-mercaptoethanol,

Reversed phase HPLC

The peptide extracts
were fractionated by reversed phase HPLC. The whole extracts were concentrated
to 40 ml on a
SpeedVac, loaded onto an Aquapore C-18 column (

Reconstitution
experiments

T2 cells were
pre-incubated at 37 ºC for 2 h with ­peptide fractions (5 U for reconstitution
assays or various doses from 0.25 to 5 U for titration assays) in 96-well plates then
added to effector cells at an effector-to-target ratio of 10:1 or 40:1.
Cytotoxicity was determined by 4 h LDH release assay. Wells containing peptides
and target cells only were used as a control to rule out toxicity of peptides
themselves.

Peptide sequencing by
mass spectrometry and ion-exchange HPLC

Matrix-assisted laser
desorption ionization-time-of-fight (MALDI-TOF) mass spectrometry was carried
out on fraction 30 from the 624-Mel sample HPLC separation. MALDI-TOF samples
were prepared by mixing 0.5 ml ­aliquot of
analytic reagents. Each resulting solution was spotted onto the stainless steel
MALDI sample target and allowed to air dry. Spectra were acquired on a VG
organic TofSpec SE mass spectrometer (Micromass,

Synthetic peptides

Peptides were
synthesized by the solid-phase method with standard tert-butoxycarbonyl
chemistry on an ­Applied Biosystems

Results

Lysis of HLA-A2.1
positive melanoma cells by ­specific CTL

Flow cytometric analysis
using specific MAb showed that HLA-A2.1 expression in the 624-Mel melanoma cell
line and two other cell lines (T2 and JY) was relatively stronger compared with
that in CS96-Mel and Chap-Mel cells, but not detectable in Depro-Mel, 1714-Mel,
­­­586-Mel and 397-Mel melanoma cell lines. We, therefore, ­analyzed the
presentation of HLA-A2.1 of tumor cells and used them as targets in a standard
4 h LDH release assay. In assays against panel 9 targets, the autologous and ­allogeneic
HLA-A2.1 melanoma cells were lysed, while HLA-A2.1 ­negative melanoma cells and
HLA-A2.1 ­positive tumor of other cell types were not. Lysis efficiencies of
624-Mel, ­Chap-Mel and CS96-Mel by CTL were ­79.0%, 25.1% and 21.3%
respectively, at an effector-to-target ratio of 20:1 (Table 1). The
lysis of HLA-A2.1 positive melanoma cells were blocked by anti-HLA class I ­antibodies
and anti-CD3 antibody. The cytolytic activity of CTL against HLA-A2.1 positive
melanoma cells was significantly eliminated by anti-HLA class I antibodies. A
similar result was obtained by anti-CD3 antibodies, but not by anti-HLA class
II ­antibodies (Table 2). The data suggested that the CTL ­recognized
the targets expressing the antigen in an HLA class I-restricted manner. Thus,
CTLs are population of melanoma-specific human CTLs, restricted by HLA-A2.1
molecules, as other class I MHC-restricted, human ­melanoma-specific CTL lines
that they reportedly lyse most of HLA-A2.1 positive melanomas, but fail to lyse
­autologous nonmelanoma cells, HLA-A2 positive nonmelanomas, or HLA-A2 negative
melanomas [12].

Identification of
peptide fractions that reconstitute melanoma-specific epitopes

The T2 cells, derived
from the fusion of a T cell line, and a human B cell mutant, LCL 721.174,
express ­HLA-A2.1 molecules with an antigen-processing defect that is
associated with enhanced presentation of exogenous ­peptides [15]. The cells
were used in the present study to test reconstitution of melanoma-specific
epitopes by soluble exogenous melanoma-derived peptides. HLA-A2.1 molecules
were isolated from detergent solubilized human melanoma cells and purified by
two affinity columns. The purity of HLA-A2.1 molecules was determined by
Western blotting (data not shown). HLA-A2.1 molecules extracted from 624-Mel
did reconstitute melanoma-specific epitopes on T2 cells (Fig. 1).
Maximal lysis of 34% was achieved at 2 U of peptide. Reconstitution of CTL
epitopes with unfractionated peptides indicated the ­presence of one or more
peptides in the extract that reconstituted melanoma-specific epitopes.

To define the components
responsible for ­reconstituting CTL epitopes, the mixture of peptides extracted
from ­­HLA-A2.1 on 624-Mel, Chap-Mel and JY cells was fractionated by reversed
phase HPLC respectively (data not shown), and 5 U of each fraction was added to
T2 cells. We ­observed one prominent peak of reconstitution (fraction 30) and
two small peaks (fractions 19 and 25) (Fig. 2). The reconstitution
pattern with 624-Mel-derived peptides was compared with that of HLA-A2.1
positive Chap-Mel, which was weakly expressed in
melanoma cells. The ­result showed that the HLA-A2.1 extracted from 624-Mel and
Chap-Mel was able to specifically reconstitute CTL epitopes with HPLC
fractions. JY, HLA-A2.1 molecules expressed in the Epstein-Barr virus-transformed
lymphoblastoid cells, were characterized by their inability in reconstituting
the CTL epitopes with HPLC fractions of the negative control extracts. Fig.
3 shows that the reconstitution for the prominent peak, at fraction 30,
using HLA-A2.1-associated peptides from 624-Mel was dose dependent within the
range ­0.25–5.00 U/well.
The pattern of three peaks of reconstitution was ­observed in two different
experiments with the same ­peptide extracts using 0.25–5.00 U of peptide/well. These data demonstrate that
peptides present in three HPLC ­fractions at the concentration of 5 U, common
to ­melanoma cells and reconstitute epitopes for melanoma-specific CTL.

Characterization of
melanoma-specific epitopes ­using HLA-A2.1-restricted CTL

The amino acid sequence
of peptides in HPLC fraction 30 from 624-Mel extracts was analyzed using mass
spectrometry. The peptides observed at mass-to-charge ratio ­of 948 have the
amino acid sequence of Ala-Leu-Trp-Leu-Phe-Phe-Gly-Val-Leu (Fig. 4),
which is a sequence motif common to HLA-A2.1-associated peptides, including
nine amino acid residues in length, with Leu at position 2, and an aliphatic
hydrocarbon side chain (Val, Leu, Ile or Ala) at position 9, as reported
previously [2].

To further analyze the
peptide sequence of ­melanoma-specific epitopes, a synthetic 9-amino acid
peptide with mass-to-charge ratio 948 was used in reconstitution experiments.
T2 cells were preincubated at 37 ºC for 2 h with the synthesized peptide at
various concentrations, or irrelevant synthetic 9-amino acid peptides in which
the Phe at position 5 was replaced by Gly, then added to CTL at an
effector-to-target ratio of 10:1 to assay their ­ability to lyse the targets.
The CTL efficiently recognized T2 cells coated with the right synthetic
peptide, but they did not recognize T2 cells coated with irrelevant synthetic
peptides. The CTL lysed target cells in a dose-dependent fashion, indicating
that the nine amino acid sequence was a melanoma-specific epitope by
HLA-A2.1-restricted CTL (Fig. 5).

Discussion

The present report
demonstrates the extraction of HLA-A2.1-associated peptides from human melanoma
cells, the reconstitution of epitopes for melanoma-specific CTL ­using the
peptides, and characterization of these peptides. We showed that the CTL
preparation in the present study was cytotoxic to two allogeneic HLA-A2.1
positive melanoma cell lines as well as fresh autologous melanoma cells. But it
did not lyse six other target cell lines, including four HLA-A2.1 negative
melanoma cell lines and two HLA-A2.1 positive cell lines. Based on Inhibition
of cytotoxicity with MAb showing that the lysis of HLA-A2.1 positive melanoma
cells was inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1, it is
suggested that the T cell epitopes are ­HLA-A2.1-associated and identical to,
or cross-reactive with, one or more of the CTL epitopes present on other
HLA-A2.1 ­positive ­melanoma cells. The observation that most, but not all,
HLA-A2.1 melanomas are lysed is consistent with the ­reports of other investigators
[19,20]. To characterize the peptides that are shared
among melanomas and recognized by this melanoma-specific CTL, the epitopes of
the CTL were reconstituted by adding peptide extracts to T2 cells. A minimum of
approximately 2 U of peptide was required for reconstitution on 5000 target
cells, and this could be extracted from approximately 3´107 melanoma
cells. The extracts are complex mixtures of peptides that bind to ­­HLA-A2.1
molecules. Therefore each peptide may be expected to compete for binding. The
required dose for each ­peptide is due to numerous variables, including the ­complexity
of the mixture, the incubation time, the rate of dissociation of endogenous
peptides from A2.1 molecules, the affinity of these exogenous peptides for A2.1
molecules, and the proportion of peptides that bind to the assay wells.

The activities of
reversed phase HPLC fractions ­suggest the presence of at least three distinct
peptide epitopes. It should be pointed out that some peptide epitopes could be
missed due to following three reasons. The HPLC ­conditions used may be unable
to identify each peptide efficiently within individual fractions. Because the
CTL line was stimulated initially with autologous tumor, then maintained on
allogeneic HLA-A2.1 positive melanoma, it is possible that the autologous
melanoma cells may ­express additional epitopes not expressed by 624-Mel. Since
CTL is an uncloned line and the preparation presumably
contains CTLs with different fine specificity, selective clonal outgrowth
during in vitro culture may cause a loss of certain ­specificity [21].

Furthermore, the study
using mass spectrometry and ion-exchange HPLC analysis demonstrated an epitope ­sequence
of nine residues, ALWLFFGVL, which does not match any other peptide in the
database at the We synthesized the
epitope-containing peptide and a peptide analogue of nine residues (irrelevant
peptide), and tested their binding to T2 cells and recognition by ­melanoma-specific
HLA-A2.1-restricted CTL. ­Quantitative assessment of peptide recognition
indicated that the most efficiently recognized synthetic peptide was ALWLFFGVL.
Of the peptides tested, the peptide titration data were in agreement with the
natural peptide from ­624-Mel ­melanoma cells. It has been confirmed that the
9-amino acid peptide is an HLA-A2.1-associated peptide epitope by ­melanoma-specific
CTL. Further searching for the candidate ­peptides with CTL immunoreactivity
will be potentially useful in immunotherapy [23,24].

In conclusion, our
results showed that ­melanoma-specific HLA-A2.1-associated peptides can be
extracted from human melanoma cells and used to reconstitute epitopes for CTL.
Determination of the amino acid residues of these peptides and identification
of the synthetic peptides will provide a better understanding of the mechanism
of CTL response to tumor and a possible therapeutic use of ­synthetic peptides
in inducing dendritic cells in vivo or peptide-MHC complexes to elicit
CTL response to ­melanoma with ­expression of the HLA-A2.1 molecules on the
cell surface. It is also useful for the development of novel peptide-based
synthetic tumor vaccines, and for the ­improvement of existing immunotherapy
regimens for melanoma [25].

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