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,§
*
Ludwig Institute for Cancer Research, Lausanne Branch,
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland;
Division of Clinical Onco-Immunology, Ludwig Institute for Cancer Research, Lausanne Branch, and
§
Multidisciplinary Oncology Center, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland;
¶
Ludwig Institute for Cancer Research, New York Branch, New York, NY 10021; and
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Department of Surgery, University of Basel, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NY-ESO-1 and LAGE-1 are two highly homologous genes encoding
tumor-specific Ags expressed in a significant proportion of tumors of
different histological types (7, 8). These genes are not
expressed in normal tissues, except testis and, to a much lower extent,
placenta, ovary, and uterus and thus belong to the so-called
cancer-testis gene family. NY-ESO-1 and LAGE-1 mRNAs and their
predicted protein products are summarized in Fig. 5
. Two forms of
LAGE-1 transcripts, LAGE-1S and
LAGE-1L, have been identified, the latter
deriving from retention of intron 2 (8). NY-ESO-1 and
LAGE-1S primary ORF code for homologous proteins
of 180 aa. The partially spliced LAGE-1L mRNA
contains an ORF encompassing most of intron 2 and encodes a putative
protein of 210 aa. In addition, LAGE-1 and NY-ESO-1 ORF2 code for two
putative products of 109 and 58 aa, respectively. Several NY-ESO-1- and
LAGE-1-derived CTL epitopes have been identified (3, 4, 9). In particular, an HLA-A2-restricted CTL epitope derived from
the primary ORF of NY-ESO-1 spanning residues 157–165 has been
recently described (9, 10). An identical sequence is
present in the LAGE-1S protein as predicted by
the primary ORF. In addition, a second HLA-A2-restricted CTL epitope
derived from a LAGE-1 gene product, named CAMEL, and encoded by ORF2 of
LAGE-1S and LAGE-1L, has
also been identified (4). The antigenic peptide has been
mapped to residues 1–11 of this putative protein. An identical
sequence is found in the putative product of NY-ESO-1 ORF2, and indeed,
CTL specific for CAMEL have been shown to recognize
NY-ESO-1-transfected COS-7 cells (4).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Frozen tumor samples from 62 stage II–IV melanoma patients were selected for analysis of NY-ESO-1 and LAGE-1 expression. Melanoma cell lines were established in our laboratory from surgically excised melanoma metastases and were cultured in RPMI 1640/10% FCS medium. The cells were characterized for surface expression of total HLA class I or HLA-A2 molecules by FACS analysis using W6-32 and BB7.2 Abs, respectively. All lines expressed the melanoma marker HMW-MAA and the adhesion molecule ICAM-1/CD54. The melanoma cell lines NA8-MEL, SK-Mel-37, 518/IL2.14, and FM6 were provided by Drs. F. Jotereau (U211, Institut National de la Santé et de la Recherche Médicale, Nantes, France), Y. T. Chen (Ludwig Institute for Cancer Research, New York Branch, New York, NY), P. Schrier (Leiden University Medical Center, Leiden, The Netherlands), and J. Zeuthen (Danish Cancer Society, Copenhagen, Denmark), respectively.
PCR analyses
RNA extraction from frozen tissue samples and cell lines was performed by the guanidinium thiocyanate/CsCl gradient method (11) and with TRIzol reagent (Life Technologies, Basel, Switzerland), respectively. cDNA synthesis was performed as previously described (11), and aliquots (equivalent to 100 ng of RNA) were used for different PCR using Qiagen DNA Taq polymerase (Basel, Switzerland). Amplification of NY-ESO-1 was performed for 35 cycles with primers ESO-1A (5'-ATGGATGCTGCAGATGCGG-3') and ESO-1B (5'-GGCTTAGCGCCTCTGCCCTG-3') with an annealing temperature of 60°C. Specific amplification of LAGE-1 sequences was performed as previously described (8). Both LAGE-1S and LAGE-1L are amplified by this PCR assay, yielding products of 399 and 628 bp, respectively. Amplification of actin for 21 cycles was performed on each cDNA sample to assess the quality and quantity of input RNA (11). Dilutions of input RNA from a reference cell line expressing both NY-ESO-1 and LAGE-1 (SK-Mel-37) during each cDNA synthesis allowed a semiquantitative analysis of NY-ESO-1 and LAGE-1 expression.
Tetramers, mAbs, and flow cytometric immunofluorescence analysis
HLA-A2/peptide tetramers were synthesized as previously described (12, 13). The CAMEL1–11 sequence (MLMAQEALAFL) was used as the antigenic peptide. Cells were stained with tetramers (20 µl of 10 µg/ml in PBS, 2% BSA, and 0.2% NaN3) for 1 h at room temperature, then further incubated with anti-CD8-FITC (20 µl; Becton Dickinson, Mountain View, CA) at 4°C for 30 min. Cells were washed once with the same buffer and analyzed by flow cytometry. Data analysis was performed using CellQuest software.
MLC, isolation of CAMEL-specific CD8+ T cells, and cloning
For peptide stimulation experiments, CD8+ lymphocytes were positively selected by magnetic cell sorting from PBMC of HLA-A*0201 melanoma patients using a miniMACS device (Miltenyi Biotec, Sunnyvale, CA). Cells from the CD8- fraction were irradiated (3000 rad) and used as APC. CD8+ highly enriched lymphocytes (1 x 106/well) were stimulated with peptide (1 µM) and irradiated autologous APC in 2 ml of CTL medium (14) containing human rIL-2 (100 U/ml; Glaxo Welcome, Geneva, Switzerland; provided by Dr. M. Nabholz, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland) and human rIL-7 (10 ng/ml; R&D Systems Europe, Oxon, U.K.). Cells were cultured for 2 wk before A2/CAMEL peptide tetramer analysis. A2/CAMEL tetramer+ cells were isolated by FACS as previously described. CAMEL-specific CTL clones were derived from A2/CAMEL tetramer+-sorted cells by limiting dilution cultures in the presence of irradiated allogeneic PBMC, PHA, and rIL-2 as described previously (15).
Chromium release assay
Ag recognition was assessed using chromium release assay. Target cells were labeled with 51Cr for 1 h at 37°C and washed twice. Labeled target cells (1000 cells in 50 µl) were incubated in the presence of various concentrations of peptide (50 µl) for 15 min at room temperature before the addition of effector cells (50 µl). Chromium release was measured in the supernatant (100 µl) harvested after 4-h incubation at 37°C. The percent specific lysis was calculated as: 100 x [(experimental - spontaneous release)/(total - spontaneous release)].
Transient transfections and TNF release assay
Plasmids containing full-length NY-ESO-1, LAGE-1S, and LAGE-1L cDNAs were gifts from B. Lethé (Ludwig Institute for Cancer Research, Brussels, Belgium). Minigene-containing plasmids were based on the previously described pGFP/Ub plasmid (16). Sites SacII and AvaI were used to insert minigenes at the 3' end of the ubiquitin sequence. Minigenes encoding the NY-ESO-1157–165- and CAMEL 1–11-derived sequences were obtained by annealing of complementary synthetic oligonucleotides (Microsynth, Balgach, Switzerland) designed so as to reconstitute the SacII and AvaI sites and including a stop codon. NA8-MEL and COS-7 cells were transiently transfected using Lipofectamine Plus reagent according to the manufacturer’s instructions (Life Technologies) and as previously described (16). COS-7 cells were cotransfected with a plasmid encoding HLA-A2. Transfected cells were then tested for their ability to stimulate the release of TNF by the NY-ESO-1157–165- and CAMEL 1–11-specific CTL. In brief, CTL were added at the appropriate effector to stimulator cell ratio in 200 µl of IMDM supplemented with 10% human serum and 20 U/ml rIL2 (Glaxo Welcome). Where indicated, synthetic peptides (1 µM) were added. After a 24-h incubation at 37°C supernatants were collected, and the TNF content was determined in a functional assay using WEHI-164 clone 13 cells (17) as previously described (18).
Western blot analysis
Cells lysates were prepared with a Nonidet P-40 buffer and subjected to SDS-PAGE (15% gel) under reducing conditions. Western blotting was performed as previously described (19). The anti-NY-ESO-1 mouse mAbs used were ES121 (20) (A. Jungbluth et al., manuscript in preparation), a mouse mAb recognizing specifically the NY-ESO-1 protein, and B9.8, recognizing both NY-ESO-1 and LAGE-1s (21). Peroxidase-conjugated anti-mouse secondary Ab and ECL detection system were obtained from Amersham Pharmacia Biotech Europe (Duebendorf, Switzerland). The primary Ab specificities were confirmed by Western blot analysis of lysates of COS-7 cells transfected with NY-ESO-1, LAGE-1S, and LAGE-1L cDNAs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine the proportion of HLA-A*0201 melanoma patients
responsive to in vitro stimulation with the
CAMEL1–11 peptide MLMAQEALAFL, highly enriched
CD8+ T cells prepared from blood samples of 33
patients were cultured for 2 wk in the presence of peptide (1 µM),
autologous APCs, and cytokines as previously described
(14). To directly enumerate
CAMEL1–11-specific CD8+ T
cells in these cultures, we prepared fluorescent
A2/CAMEL1–11 peptide tetramers (12, 13). Tetramer+ CD8+
populations were clearly detected in three melanoma patients, LAU 50,
143, and 342 (Fig. 1A).
Interestingly, the tumor from patient LAU 50 expressed both NY-ESO-1
and LAGE-1 (see Table I). Gene expression
could not be assessed in the lesions from patients LAU 143 and 342 due
to lack of tumor material. A2/CAMEL1–11
tetramer+ cells were then isolated by cell
sorting followed by PHA-driven expansion, either in bulk or as clones
by plating at limiting dilution (14, 15). From the culture
derived from patient LAU 50 we isolated a polyclonal cell line highly
enriched in tetramer+ CD8+
T cells as well as several tetramer+
CD8+ T cell clones (Fig. 1B). In
addition, a polyclonal monospecific line, but no clones, was isolated
from the culture derived from patient LAU 342. Remarkably, whereas the
tetramer+ CD8+ cells
present in the unsorted LAU 342 culture displayed a bright fluorescence
signal (Fig. 1A), the sorted line was tetramer dull (Fig. 1B). This phenotype was maintained over several cycles of in
vitro stimulation with PHA. One representative clone from patient LAU
50 (clone LAU 50 CAMEL/4) and the line LAU 342 CAMEL (Fig. 1B) were selected for further analysis.
A2/CAMEL1–11 tetramer+
CD8+ T cells were also detected among
tumor-infiltrated lymph node cells (TILN) from HLA-A2 melanoma patients
cultured for 2 wk in the presence of exogenously added cytokines only
(Fig. 1C). These results underline the existence of natural
CAMEL-specific CD8+ T cell responses in several
melanoma patients.
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To assess whether the difference in the intensity of T cell
staining by specific MHC/peptide tetramers correlated with different
avidity of the Ag-specific T cells, as suggested by a recent study
(22), we performed the experiments illustrated in Fig. 2. Both clone LAU 50 CAMEL/4 (bright
tetramer staining) and line LAU 342 CAMEL (dull tetramer staining)
specifically lysed T2 cells in the presence, but not in the absence, of
a high dose (1 µM) of peptide CAMEL1–11.
However, lysis by clone LAU 50 CAMEL/4 was slightly more efficient than
that by line LAU 342 CAMEL at high E:T cell ratios (Fig. 2A). Moreover, peptide titration showed that clone LAU 50
CAMEL/4 recognized the peptide CAMEL1–11 about
10,000-fold more efficiently than it did line LAU 342 CAMEL (Fig. 2B). Using a single specific T cell clone, amino acid
sequence 1–11 from CAMEL has been previously identified as the optimal
antigenic peptide (4). To determine the length
requirements for TCR recognition by the
CAMEL1–11-specific T cells described in this
study we tested both N- and C-terminally truncated peptide analogues
(Fig. 2C). Indeed, the results of this analysis confirmed
the findings of Aarnoudse et al. (4). Truncation of the
N-terminal methionine residue resulted in 
10-fold decreased
recognition by clone LAU 50 CAMEL/4 and loss of recognition by line LAU
342 CAMEL. Further truncation of the leucine at position 2 resulted in
loss of T cell recognition by clone LAU 50 CAMEL/4. In addition,
truncations at the C terminus of the peptide completely abolished
recognition by both T cell populations.
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A highly correlated expression of NY-ESO-1 and LAGE-1 genes in a
small number of melanoma tumors has been reported, with five of six
positive tumors coexpressing the two genes (8). We
investigated extensively the expression of the two genes in a large
series of melanoma metastases (100 tumors from 63 patients) by RT-PCR
analysis. On a patient basis, 14 (23%) expressed NY-ESO-1, and 20
(33%) expressed LAGE-1 in their tumors at a level >1% of that found
in the melanoma cell line SK-Mel-37, which was used as a reference. In
addition, six and one patients expressed very low levels (equivalent to
<1% of those in SK-Mel-37) of NY-ESO-1 and LAGE-1, respectively.
Table I
summarizes the PCR results for the patients with NY-ESO-1-
and/or LAGE-1-positive tumors. Ten of the 14 patients whose tumors
clearly expressed NY-ESO-1 were also positive for LAGE-1 expression.
However, tumors from several patients (e.g., LAU 4, 119, 156, 203, 331,
and 332) selectively expressed only one of the two genes (4 and 10 for
NY-ESO-1 and LAGE-1, respectively). Multiple simultaneous and/or
subsequent metastases were available for analysis from 26 of the
patients studied. A conserved pattern of expression of NY-ESO-1 and
LAGE-1 was observed in the tumors of the majority of these patients, as
found for 16 NY-ESO-1- and LAGE-1-negative patients (not shown) and for
LAU 4, 53, and 156 (Table I). However, heterogeneous expression was
clearly observed in some patients (e.g., LAU 242 and 321). A
correlation between expression of NY-ESO-1/LAGE-1 and MAGE-1 and -3 has
been reported (8). RT-PCR analyses for MAGE-1 and -4 (the
latter being expressed in melanoma at a frequency similar to that
reported for NY-ESO-1 and LAGE-1) (11) were thus
performed. MAGE-1 and -4 were expressed in 57 and 50% of the NY-ESO-1-
and/or LAGE-1-positive tumor samples (n = 42),
respectively, but only in 38 and 14% of the negative ones
(n = 58; Table I and data not shown).
To study the correlation between expression of NY-ESO-1 and LAGE-1 by
tumor cells and recognition by CAMEL 1–11- and
NY-ESO-1157–165-specific CTL, we characterized
in detail the expression of these genes in 35 melanoma cell lines
derived from the patients analyzed above. Expression of one or both
genes was detected by RT-PCR in 17 cell lines, while the remainder were
negative. In the majority of cases (>75%) the gene expression profile
of cultured cells reflected that of the original tumor. The remaining
cases probably reflect a heterogeneous expression of NY-ESO-1/LAGE-1
within the tumor lesion. Indeed, staining of melanoma metastases with
mAb B9.8 revealed intratumor heterogeneity, both as the percentage of
positive cells and as staining intensity, in a fraction of the samples
analyzed (21). Table II
summarizes the results obtained for the positive melanoma cell lines.
Cell lines SK-Mel-37, 518/IL-2.14, and FM6, used in previous reports
(4, 9), were also included as a reference. Western blot
analyses of lysates of melanoma cell lines with Abs recognizing
NY-ESO-1 only or both NY-ESO-1 and LAGE-1 proteins confirmed the
expression of the genes at the protein level (Fig. 3). Overall, there appeared to be a
correlation between the degree of expression at the RNA and protein
levels (Fig. 3 and Table II).
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We initially compared high and low avidity
CAMEL1–11-specific CTL for their capacity to
kill melanoma cells expressing NY- ESO-1/LAGE-1. Remarkably, only
high avidity CTL were able to specifically lyse Ag-expressing tumors,
although both high and low avidity cells efficiently lysed
HLA-A2+ NY-ESO-1/LAGE-1-
targets in the presence of exogenously added peptide
CAMEL1–11 (Fig. 4
and data not shown).
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) was tested in parallel. The two T cell
clones showed similar patterns of tumor recognition (Table II). The
reference melanoma cell lines SK-Mel-37, FM6, and 518/IL2.14
(NY-ESO-1+, LAGE-1+,
HLA-A2+) were efficiently lysed in both the
absence and the presence of the antigenic peptides. The melanoma cell
line NA8-MEL (NY-ESO-1-,
LAGE-1-, HLA-A2+) was
efficiently lysed only in the presence of antigenic peptide, while no
significant lysis was detected on melanoma line Me 242.B.1
(NY-ESO-1+ and LAGE-1+ but
HLA-A2-).
Among the melanoma cell lines generated in our laboratory, the majority
of the NY-ESO-1/LAGE-1+ lines were specifically
lysed by both the NY-ESO-1/LAGE-1 ORF1- and ORF2-specific CTL in the
absence of exogenously added peptide, although the level of specific
lysis varied from line to line. An exception was represented by cell
line Me 257, which was not significantly lysed in the absence of
antigenic peptide despite clearly positive NY-ESO-1 and LAGE-1 gene
expression (Fig. 3). However, Me 257 cells (which express the
melanocyte differentiation Ags Melan-A/MART-1 and tyrosinase) were
specifically lysed by Melan-A/MART-1 and tyrosinase-specific CTL clones
in the absence of exogenously added antigenic peptides, thus excluding
a general defect in the presentation of endogenously derived peptides
(data not shown). Two melanoma cell lines expressing NY-ESO-1 but not
LAGE-1 (Me 333 and Me 252) were also specifically lysed by both CTL
clones, although to a limited extent. Finally, of six melanoma lines
expressing LAGE-1 but not NY-ESO-1, three were efficiently lysed (Me
237, Me 285.A, Me 290), while the remainders were not (Me 323, Me 324,
T343A). It should be noted that among the latter, Me 323 and T343A
cells, although they were LAGE-1 PCR positive, expressed very low
levels of LAGE-1 protein (Fig. 3). In addition, Me 323 cells
(Melan-A/MART-1+,
tyrosinase+) also failed to present endogenously
derived Melan-A/MART-1 and tyrosinase peptides to the corresponding CTL
clones (data not shown). Finally, all the HLA-A2+
melanoma lines tested (expressing, or not, NY-ESO-1 and LAGE-1) were
efficiently lysed by either CTL clone in the presence of the
appropriate antigenic peptide.
Recognition of NA8-MEL and COS-7 cells transiently transfected with NY-ESO-1- and LAGE-1-encoding constructs by specific CTL
To unambiguously assess recognition of NY-ESO-1- or
LAGE-1-expressing cells by ORF1- and ORF2-specific CTL, melanoma cells
NA8-MEL (NY-ESO-1-,
LAGE-1-, HLA-A2+) and
COS-7 cells (NY-ESO-1-,
LAGE-1-, HLA-A2-) were
transiently transfected with plasmids encoding full-length NY-ESO-1,
LAGE-1S, and LAGE-1L cDNAs.
COS-7 cells were cotransfected with a plasmid encoding HLA-A2.
Transfected cells were then tested for their ability to specifically
stimulate TNF release by NY-ESO-1157–165 (ORF1)
and CAMEL1–11 (ORF2)-specific CTL. As summarized
in Table III and in good agreement with
the tumor recognition data shown above, both NY-ESO-1 and high avidity
CAMEL-specific CTL efficiently recognized NA8-MEL and COS-7 cells
transfected with plasmids encoding NY-ESO-1,
LAGE-1S, or LAGE-1L.
Importantly, both NY-ESO-1 and CAMEL-specific CTL clones exhibited a
similar pattern of recognition, indicating that the relevant antigenic
peptides were simultaneously generated with comparable efficiency. In
addition, these results, together with those on tumor cells shown
above, formally prove that the epitope recognized by
NY-ESO-1157–165-specific CTL is also generated
from the homologous LAGE-1S protein.
Interestingly, the comparable efficiencies in stimulating TNF secretion
observed with LAGE-1S and
LAGE-1L transfectants suggest that most of the
primary LAGE-1L transcripts undergo complete
splicing (see Fig. 5). Low avidity CAMEL-specific CTL specifically
secreted TNF only upon stimulation with transfected COS-7, but not
NA8-MEL, cells. Because COS-7 cells are more efficiently transfected
and express higher levels of the transfected gene than NA8-MEL cells
(23) (our data, not shown), these results indicate that
low avidity CAMEL-specific CTL can indeed recognize the endogenously
expressed Ag, but only when present at very high (and presumably not
physiological) levels.
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CAMEL-specific CTL secreted TNF above background levels only upon
stimulation with NA8-MEL transfected with plasmids encoding the
CAMEL1–11 pGFP/Ub minigene. Cells transfected
with a NY-ESO-1157–165 pGFP/Ub minigene, used as
an internal control, failed to stimulate specific TNF secretion by
clone LAU 50 CAMEL/4, but were efficiently recognized by clone LAU 156
ESO/5 (Table III). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The identification of T cell epitopes from tumor-associated Ags
recognized by CD8+ lymphocytes in association
with frequently expressed HLA class I alleles is a prerequisite for the
design of peptide-based immunotherapy of cancer. Several T cell
epitopes derived from the primary ORF of melanoma-associated Ags have
been identified, including a number derived from CT Ags. Among the
latter, NY-ESO-1 has recently received attention, because Ab responses,
indicative of strong immunogenicity, have been detected in 50% of
late stage melanoma patients bearing NY-ESO-1-positive tumors
(20). An NY-ESO-1/ORF1-derived CTL epitope recognized by
HLA-A31-restricted TIL derived from a melanoma lesion has been
described previously (3). More recently, an epitope
located in region 157–165 of NY-ESO-1 has been shown to be recognized
by a tumor-reactive CTL line from a melanoma patient in association
with HLA-A2 (an allele expressed by 50% of Caucasians)
(9). We have recently studied CD8+ T
cell responses to this epitope in HLA-A2 melanoma patients by using
fluorescent HLA-A2/NY-ESO-1157–165 peptide
tetramers. Specific CD8+ T cell responses were
readily detected in peptide-stimulated CD8+ T
cell cultures from 50% (5 of 10) of the patients analyzed
(10). Similarly, Jäger et al. have detected
CD8+ T cell responses to the same epitope (as
measured by IFN-
enzyme-linked immunospot assay) in 10 of 11
patients with NY-ESO-1 Abs, but not in seronegative patients or
patients with NY-ESO-1-negative tumors (24).
The HLA-A2-restricted CAMEL-derived CTL epitope (aa 1–11) encoded by ORF2 of both LAGE-1 and NY-ESO-1 was originally identified as the target of a tumor-reactive CTL clone isolated from PBMC of a melanoma patient stimulated with IL-2-transfected autologous tumor cells (4). Few other human CTL epitopes derived from gene products encoded by alternative ORF have been described to date. These include a CTL epitope encoded by ORF3 of the wild-type TRP1/gp75 differentiation Ag and an epitope from NY-ESO-1 ORF2 (2, 3), both restricted by HLA-A31, an allele expressed by only 6% of Caucasians. More recently, an HLA-B*702-associated epitope derived from ORF2 of intestinal carboxyl esterase mRNA recognized by TIL from a renal cell carcinoma patient has been identified (5). Little is known on CTL responses to these alternative ORF-derived epitopes. In fact, most of the information available is still limited to single patients. In the present study we have detected A2/CAMEL1–11+ tetramer+ CD8+ T cells in peptide-stimulated PBMC from 3 of 33 (9%) A2+ patients as well as in TILN. Thus, a response to this alternative ORF-derived epitope is not an exception resulting from a unique treatment/stimulation procedure (25). The proportion of patients with circulating CAMEL1–11-specific CD8+ T cells appeared to be lower than that for HLA-A2 NY-ESO-1 ORF1-specific CD8+ T cells observed by us and others (10, 24). Nonetheless, this proportion might have been underestimated due to the stimulation protocol used in this study. This involved a single in vitro peptide stimulation in the presence of autologous APC, which is thus likely to give detectable responses only when a relatively high frequency of specific CTL precursors is present in the patient’s PBMC. At any rate, the low percentage of responders to in vitro peptide stimulation does not necessarily imply the inability of nonresponders to mount specific anti-CAMEL CTL responses upon vaccination. While studying responses to the primary ORF-derived NY-ESO-1157–165 epitope, we have observed that a specific CTL response could also be detected, albeit at a low level, in melanoma patients whose tumor lesion had no evidence of NY-ESO-1 expression (10). Future studies are planned to investigate, in a comparative setting, the incidence of responses to NY-ESO-1157–165 and CAMEL1–11 in groups of patients with NY-ESO-1/LAGE-1-positive or -negative tumors.
Upon tetramer-guided cell sorting, we derived dull and bright A2/CAMEL1–11 tetramer+ lymphocytes. The two populations exhibited a 10-fold difference in tetramer staining intensity at saturating tetramer concentrations. In agreement with a recent report (22), a correlation was observed between tetramer staining and T cell recognition avidity. Indeed, to obtain 50% maximal lytic activity on HLA-A2-positive target cells, A2/CAMEL1–11 tetramerdull lymphocytes required a dose of antigenic peptide 10,000-fold higher than that required by A2/CAMEL1–11 tetramerbright lymphocytes. Importantly, only A2/CAMEL1–11 tetramerbright lymphocytes were able to efficiently recognize tumor cells endogenously expressing NY-ESO-1 and/or LAGE-1. These data indicate that fluorescent A2/CAMEL1–11 peptide tetramers can be used to selectively identify high avidity tumor-reactive CAMEL-specific CTL.
The availability of isolated CTL clones specific for NY-ESO-1 and LAGE-1 sequences encoded by ORF1 and -2 made it possible to functionally assess the capacity of tumor cells to generate the corresponding epitopes. We characterized and tested a large panel of A2-positive NY-ESO-1- and/or LAGE-1-expressing melanoma cell lines. Several conclusions could be drawn from these experiments. 1) For the majority of the cell lines tested, there was a good correlation between NY-ESO-1/LAGE-1 mRNA and protein expression and specific CTL lysis. However, a few exceptions of melanoma cells expressing detectable levels of NY-ESO-1 or LAGE-1 but not recognized by specific CTL in the absence of exogenous antigenic peptides were detected. Lack of recognition of two of these lines could be explained by a particularly low level of protein expression, whereas other mechanisms are probably involved in the remaining cases. Such mechanisms could include a general defect in the generation of antigenic peptides by the intracellular processing machinery as well as specific alterations in NY-ESO-1/LAGE-1 protein metabolism and remain to be studied. 2) ORF1- and ORF2-specific CTL showed a remarkably similar pattern of tumor recognition, both qualitatively and quantitatively. Thus, although it is generally assumed that nonprimary ORF-encoded products are produced much less efficiently than primary ORF-derived proteins, we observed that NY-ESO-1/LAGE-1 ORF2-derived A2/peptide complexes are produced at a level functionally detectable to a comparable extent to that of NY-ESO-1/LAGE-1 ORF1-derived A2/peptide complexes. 3) The CAMEL1–11/ORF2-derived epitope is efficiently generated by melanoma cells individually expressing NY-ESO-1 and LAGE-1, as shown by chromium release assay. This finding confirms, in a physiological context, the original observation by Aarnoudse et al. that COS-7 cells transfected with either LAGE-1 or NY-ESO-1 cDNAs can stimulate TNF release by a specific T cell clone (4). 4) The HLA-A2-restricted NY-ESO-1/ORF1-derived epitope (aa 157–165) is efficiently generated by melanoma cells expressing LAGE-1 only. Although not surprising, given the sequence identity of NY-ESO-1 and LAGE-1 in the region containing this epitope, this finding provides formal proof that a similar processing occurs for the two homologous products. It also provides the rationale for the inclusion of patients with LAGE-1-positive but NY-ESO-1-negative tumors in future NY-ESO-1157–165-specific immunization protocols.
Several mechanisms have been described for the generation of alternative ORF polypeptides, including translational initiation codon scan-through, ribosomal frameshifting, reinitiation of translation, and abnormal splicing events (1, 5, 26). The specific mechanism responsible for the efficient production of NY-ESO-1 and LAGE-1 ORF2-derived peptides remains to be elucidated, as is the actual extent of CAMEL synthesis. Defective ribosomal products have been recently shown to constitute up to 30% of all newly synthesized proteins and have been proposed to be a major source for MHC class I antigenic peptides (27). To what degree alternative ORF polypeptides, such as CAMEL, can be assimilated to defective ribosomal products is unknown.
In conclusion, our findings demonstrate that both primary and nonprimary ORF-derived CTL epitopes can simultaneously and efficiently be generated by tumor cells and support the use of nonprimary ORF-derived CTL epitopes as vaccines to induce tumor specific cell-mediated immunity against cancer.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: ORF, open reading frame; TILN, tumor-infiltrated lymph node cells.
Received for publication June 22, 2000. Accepted for publication September 15, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E. H. Slager, C. E. van der Minne, M. Kruse, D. D. Krueger, M. Griffioen, and S. Osanto Identification of Multiple HLA-DR-Restricted Epitopes of the Tumor-Associated Antigen CAMEL by CD4+ Th1/Th2 Lymphocytes J. Immunol., April 15, 2004; 172(8): 5095 - 5102. [Abstract] [Full Text] [PDF]
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M. Ayyoub, D. Rimoldi, P. Guillaume, P. Romero, J.-C. Cerottini, D. Valmori, and D. Speiser Tumor-reactive, SSX-2-specific CD8+ T Cells Are Selectively Expanded during Immune Responses to Antigen-expressing Tumors in Melanoma Patients Cancer Res., September 1, 2003; 63(17): 5601 - 5606. [Abstract] [Full Text] [PDF]
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M. Probst-Kepper, V. Stroobant, R. Kridel, B. Gaugler, C. Landry, F. Brasseur, J.-P. Cosyns, B. Weynand, T. Boon, and B. J. Van den Eynde An Alternative Open Reading Frame of the Human Macrophage Colony-stimulating Factor Gene Is Independently Translated and Codes for an Antigenic Peptide of 14 Amino Acids Recognized by Tumor-infiltrating CD8 T Lymphocytes J. Exp. Med., May 21, 2001; 193(10): 1189 - 1198. [Abstract] [Full Text] [PDF]
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, clone LAU 50 CAMEL/4; 
, line LAU 342 CAMEL.
C, Recognition of CAMEL-truncated peptides by clone LAU
50 CAMEL/4 and line LAU 342 CAMEL was assessed in a 4-h chromium
release assay at the L/T ratio of 10:1.
