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Tumor Biology |
Department of Medicine and Melanoma Center, University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15213 [H. M. Z., J. M. K., E. W.]; Department of Surgery and Molecular Genetics and Biochemistry, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15261[W. J. S.]; and Kent Ridge Digital Labs, Singapore 119613 [V. B.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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specifically in response to, and also lysed,
T2.DR4 cells pulsed with peptide NY-ESO-1119–143 and the
autologous tumor cell line, but not a DRB1*0401+ melanoma
cell line that does not express NY-ESO-1. Interestingly, the
NY-ESO119–143 peptide contains two overlapping putative
"core" epitopes recognized by non-cross-reactive
anti-NY-ESO-1119–143 CD4+ T-cell clones. Taken together,
these data support the use of this novel DR4-restricted tumor peptide,
NY-ESO-1119–143, or its two "sub-epitopes" in
immunotherapeutic trials designed to generate or enhance specific CD4+
T-cell responses against tumors expressing NY-ESO-1 in
vivo. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have recently designed a strategy to identify a novel DR4-restricted Melan-A/MART-1-derived peptide recognized by CD4+ T cells (14) . A peptide-binding algorithm (15 , 16) was used to select candidate DR4-binding epitopes, which were then screened for recognition by melanoma-reactive CD4+ T cells derived from normal donors or long-lived patients with melanoma. This strategy has now been applied to demonstrate that the NY-ESO-1 protein encodes at least two distinct, but overlapping, DR4-restricted epitopes capable of stimulating CD4+ T-cell responses in vitro. Both bulk and cloned CD4+ T cells recognized DR4+ antigen-presenting cells pulsed with the NY-ESO-1119–143 peptide, as well as DR4+, NY-ESO-1+ melanoma cell lines. These findings support the use of the NY-ESO-1119–143 peptide or analogues derived from this sequence as a cancer vaccine for DR4+ patients with NY-ESO-1+ tumors.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthesis of NY-ESO-1 HLA-DRB1*0401 Binding Peptides.
Peptides were synthesized using standard Fmoc chemistry by the
University of Pittsburgh Peptide Synthesis Facility (Shared Resource),
were >90% pure as indicated by analytical high-performance liquid
chromatography, and were validated for identity by mass spectrometry.
Lyophilized peptides were dissolved in PBS/10% DMSO or 100% DMSO at a
concentration of 2 mg/ml and stored at -20°C until use. Synthesis of
NY-ESO-1 peptides was based on the sequence of a NY-ESO-1
gene published by Chen et al. (Ref. 11
; GenBank
accession no. U87459).
The full-length NY-ESO-1 and SSX recombinant proteins were produced in baculovirus and were kindly provided by Drs Lloyd J. Old and Gerd Ritter (Ludwig Institute for Cancer Research, New York Branch, New York, NY).
Induction of CD4+ T Cells with Peptides.
PBMCs were isolated by density centrifugation on Ficoll-Hypaque
gradients (LSM; Organon-Teknika, Durham, NC) and used to prepare mature
DCs using the procedure of Jonuleit et al. (18)
with minor modifications. PBMCs were resuspended at
107/ml in AIM-V medium (Life Technologies, Inc.,
Grand Island, NY) and were incubated for 90 min in
75-cm2 tissue culture flasks or six-well plates
(37°C; 5% CO2). Nonadherent (T cell-enriched)
cells were gently washed out with HBSS and subsequently frozen. The
plastic adherent cells were cultured in AIM-V medium supplemented with
1000 units/ml rhGM-CSF and 1000 units/ml rhIL-4 (Schering-Plough,
Kenilworth, NJ). Six days later, the culture medium was removed, and
the immature DCs were cultured in AIM-V supplemented with 1000 units/ml
rhGM-CSF and 1000 units/ml rhIL-4, 1000 units/ml rhIL-6 (Sandoz), 10
ng/ml recombinant human tumor necrosis factor-
(R & D), and 10 ng/ml
IL-1ß (R & D). Mature DCs were harvested on day 8, centrifuged,
frozen, or used to stimulate autologous T cells. The stimulator cells
were resuspended in AIM-V at 106/ml supplemented
with each peptide (10 µg/ml) and incubated for 4 h at 37°C.
The peptide-pulsed DCs were then irradiated (50 Gy) and washed and
resuspended in culture medium (Iscove’s medium supplemented with 10%
human serum, L-arginine,
L-asparagine, and
L-glutamine). Autologous CD4+ T cells were
positively isolated from PBMCs with immunomagnetic beads (Miltenyi
Biotech, Germany) and added (106) to the
peptide-pulsed DCs (105) in a final volume of 2
ml of culture medium (24-well tissue culture plate) along with 1000
units/ml IL-6 and 10 ng/ml IL-12 (Genetics Institute, Cambridge, MA).
On day 7 and weekly thereafter, lymphocytes were restimulated with
autologous irradiated DCs pulsed with peptide in culture medium
supplemented with 10 units/ml IL-2 and 5 ng/ml IL-7 (Genzyme). The
stimulated CD4+ T cells were analyzed for specificity in ELISPOT assays
at day 18 and then every 10 days after the most recent stimulation.
Induction of CD4+ T Cells with Protein.
Immature DCs were prepared as indicated previously and incubated at
37°C, 5% CO2 for 2 days in AIM-V supplemented
with 1000 units/ml rhGM-CSF and 1000 units/ml rhIL-4, 1000 units/ml
rhIL-6 (Sandoz), 10 ng/ml recombinant human tumor necrosis factor-
(R & D), and 10 ng/ml IL-1ß (R & D) in the presence of the
recombinant NY-ESO-1 protein (30 µg/ml). DCs were harvested on day 8,
centrifuged, and frozen or used to stimulate autologous PBMCs. The
protein-loaded DCs were then irradiated at 50 Gy, washed, and
resuspended in culture medium (Iscove’s medium supplemented with 10%
human serum, L-arginine, L-asparagine, and
L-glutamine). Autologous PBMCs were added (3 x 106) to the protein-loaded DCs
(105) in a final volume of 2 ml of culture medium
(24-well tissue culture plate) along with 1000 units/ml IL-6 and 10
ng/ml IL-12 (Genetics Institute, Cambridge, MA). On days 7 and 14, the
lymphocytes were restimulated with autologous irradiated protein-pulsed
DCs in culture medium supplemented with 10 units/ml IL-2 and 5 ng/ml
IL-7 (Genzyme). The stimulated CD4+ T cells were analyzed for
specificity in ELISPOT assays at day 21.
The CD4+ T cells were cloned by limiting dilution using allogeneic PBLs
and EBV-B cells as feeders in the presence of IL-2 (100 units/ml) and
phytohemagglutinin (0.5 µg/ml) and subsequently tested for
specificity in IFN- ELISPOT and IFN- ELISA assays. The CD4+
T-cell clones were maintained by restimulation every 2 weeks by
alternating irradiated allogenic PBL and EBV-B cells or autologous
peptide-pulsed DCs as stimulators.
Assessment of T-Cell Responses to Peptides and Tumor Cells.
The recognition of APCs pulsed with peptides and tumor cells was
assessed by ELISPOT and ELISA assays specific for hu-IFN-. For
IFN- ELISPOT assay, multiscreen hemagglutinin antigen plates
(Millipore, Bedford, MA) were coated with 10 µg/ml of antihuman
IFN- mAb (1-D1K; Mabtech, Stockholm, Sweden) in PBS (Life
Technologies) overnight at 4°C. Unbound antibody was removed by four
successive washings with PBS. After blocking the plates with RPMI/10%
human serum (1 h at 37°C), CD4+ T cells were seeded in triplicates
(104
for bulk CD4+ T cells and
103
for CD4+ T-cell clones) in multiscreen
hemagglutinin antigen plates. Nonirradiated T2.DR4 cells (7.5 x 104
) or melanoma cells (25 x 103
) were added. Synthetic peptides were
then added at indicated concentrations into ELISPOT assays after
seeding APCs. Control wells contained unstimulated T cells, T cells in
the presence of unloaded T2.DR4 cells, or tumor cells alone.
Alternatively, 25 x 103
protein-loaded DCs were added to 5 x 103
CD4+ T-cell clones/well. Culture medium was
AIM-V at a final volume of 200 µl/well. After incubation at 37°C in
5% CO2 for 20 h, cells were removed by
washings with PBS/0.05% Tween 20 (PBS/T). Captured cytokine was
detected at sites of its secretion by incubation for 2 h with
biotinylated mAb anti-hu-IFN- (7-B6-1; Mabtech) at 2 µg/ml in
PBS/0.5% BSA. Plates were washed six times with PBS/T, and
avidin-peroxidase complex (diluted 1:100; Vectastain Elite kit; Vector,
Burlingame, CA) was added for 1 h at room temperature. Unbound
complex was removed by three successive washings with PBS/T and three
with PBS alone. Peroxidase staining was performed with
3-amino-9-ethyl-carbazole (Sigma) for 4 min and stopped by rinsing the
plates under running tap water. Spot numbers and spot sizes were
automatically determined with the use of computer-assisted video image
analysis as described previously (19)
.
Alternatively, the IFN- secretion was assessed by ELISA. T2.DR4
cells were incubated for 6 h at 7.5 x 104
per flat-bottomed microwell with peptides at
indicated concentrations, and CD4+ T-cell clones at 2 x 103
cells/well were added to the assay for
20 h. Culture supernatants were then harvested, and IFN-
secretion by CD4+ T cells was measured using a commercial ELISA kit (R
& D systems; detection limit, 7 pg/ml).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The NY-ESO-1 protein sequence was obtained from the GenBank and
analyzed for HLA-DR4 binding peptides using a neural network algorithm
(15
, 16)
. High-scoring, nine amino acid-long "core"
peptide sequences were typically extended by three amino acids on
either flank using the corresponding genomic sequences and synthesized.
Alternatively, if multiple high-scoring sequences overlapped, a single
extended sequence encompassing the overlaps and two to three terminal
amino acid extensions was synthesized. Overall, three peptides of
23–26 amino acids in length were chosen for subsequent analyses (Table 1)
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ELISPOT assay was used to analyze the reactivity of freshly
isolated peripheral blood CD4+ T cells derived from patient UPCI-MEL
527 against T2.DR4 cells pulsed with each of the three potential
DR4-restricted NY-ESO-1 melanoma peptides. No immunoreactivity against
any of these peptides was observed in these responder cells that had
not been subjected to in vitro stimulation (data not shown).
CD4+ Bulk T Cells Recognize Peptide NY-ESO-1119–143
Presented on HLA-DR4.
In an independent series of in vitro experiments, we
"primed" CD4+ T cells from a DRB1*0401+
normal donor and from patient UPCI-MEL 527 against the potential
DR4-binding peptides predicted by the MHC binding algorithm (Table 1)
.
"Mature" DCs were incubated with each of the three different
peptides (10 µg/ml), irradiated, and used to stimulate autologous
CD4+ T cells (previously isolated from the peripheral blood, as
described in "Materials and Methods"). The individual responder
cell cultures were restimulated on a weekly basis with irradiated
autologous mature DCs loaded with the corresponding peptide used in the
primary stimulation. After at least three restimulations, the
immunoreactivity of the CD4+ T-cell cultures were analyzed in IFN-
ELISPOT assays. Bulk CD4+ T cells that were stimulated with the
NY-ESO-1119–143 peptide specifically recognized
T2.DR4 cells pulsed with the immunogenic peptide (Fig. 1)
. These CD4+ T cells also displayed reactivity against the
HLA-DR4-matched NY-ESO-1+ melanoma cell line
UPCI-MEL 527.1, which was inhibited by addition of anti-HLA-DR mAb
(L243) but not anti-HLA-A,B,C mAb (W6/32) to ELISPOT. No IFN- spots
were produced by the CD4+ T cells cultured with T2.DR4 cells alone or
pulsed with an irrelevant DR4-restricted peptide (i.e.,
Melan-A/MART-151–73; Ref. 14
) or
with the NY-ESO-1- cell line, UPCI-MEL 136.1. No
IFN- spots developed in wells containing UPCI-MEL 136.1 cells or
T2.DR4 cells in the absence of added CD4+ T cells (data not shown). No
immunoreactivity against the two other potential DR4-binding
peptides, NY-ESO-1149–168 and
NY-ESO-1158–180, was observed in CD4+ T-cell
cultures stimulated with autologous DCs pulsed with either
NY-ESO-1119–143,
NY-ESO-1149–168, or
NY-ESO-1158–180.
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. The recognition of UPCI-MEL 527.1 was inhibited by the addition of
anti-HLA-DR mAb (L243) but not anti-HLA-A,B,C mAb (W6/32) to ELISPOT or
ELISA wells. No IFN- spots were produced by clone 10/4 cultured with
T2.DR4 cells alone or pulsed with an irrelevant DR4-restricted peptide
(i.e., Melan-A/MART-151–73) or with
the NY-ESO-1- UPCI-MEL 136.1 cell line. However
clone 10/4 produced IFN- in the presence of the UPCI-MEL 136.1
cell-pulsed with NY-ESO-1119–143 peptide.
No IFN- spots were produced by UPCI-MEL 136.1 cells or T2.DR4 cells
in the absence of added CD4+ T cells (data not shown).
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in the
presence T2.DR4 cells, preincubated with the
NY-ESO-1119–143 peptide at various
concentrations, was evaluated to determine the peptide-dose
"threshold" for effector T-cell recognition. Both ELISPOT and ELISA
assays provided similar information (Fig. 3)
, suggesting that half-maximal stimulation of
NY-ESO-1119–143 peptide-reactive CD4+ T cells
required peptide "loading" concentrations between 20 and 60
nM.
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; i.e., 122–130,
126–134, and 132–140) potential immunogenic "core epitopes,"
according to the algorithm. We synthesized a series of peptides
(i.e., 119–133, 123–137, and 129–143) from the internal
sequence of the immunogenic NY-ESO-1119–143
peptide. Peptide NY-ESO-1119–133, but not
peptides NY-ESO-1123–137 or
NY-ESO-1129–143, were able to sensitize T2.DR4
cells for recognition by the
NY-ESO-1119–143-reactive CD4+ T cell clone 10/4
as monitored by either IFN- ELISPOT and ELISA assays (Fig. 2)
.
Although full-recognition NY-ESO-1119–133
peptide was only a fraction of that observed against the
NY-ESO-1119–143 peptide, these data suggest that
one immunogenic epitope encoded by NY-ESO-1 is located between residues
119 and 133, likely encompassing the
NY-ESO-1122–133 core epitope identified by the
peptide-binding algorithm.
CD4+ T-Cell Clone 11/4 (Melanoma Patient) Recognizes the
NY-ESO-1123–137 Core Epitope within the
NY-ESO-1119–143 Sequence.
Clone 11/4 was obtained by limiting dilution from the CD4+ bulk T cells
of patient 527 that recognized the
NY-ESO-1119–143 peptide. Clone 11/4 specifically
recognized T2.DR4 cells pulsed with either the
NY-ESO-1119–143 peptide or the
NY-ESO-1123–137 peptide and the autologous
melanoma cell line UPCI-MEL 527.1 (Fig. 5)
. The recognition of the UPCI-MEL 527.1 was inhibited by the addition
of anti-HLA-DR mAb (L243) but not anti-HLA-A,B,C mAb (W6/32) to ELISPOT
or ELISA wells. No IFN- spots were produced by clone 11/4 cultured
with T2.DR4 cells alone or pulsed with an irrelevant DR4-restricted
peptide (i.e., Melan-A/MART-151–73)
or with the UPCI-MEL 136.1 cell line that fails to express NY-ESO-1 T
cells. The ability of clone 11/4 to produce IFN- in the
presence T2.DR4 cells, preincubated with various concentrations of
either the NY-ESO-1 119–143 peptide or the
NY-ESO-1123–137 peptide, was evaluated.
Half-maximal stimulation of clone 11/4 required peptide "loading"
concentrations between 1 and 10 nM (Fig. 6)
.
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. This lysis was inhibited by the addition of anti-HLA-DR mAb (L243)
but not anti-HLA-A,B,C mAb.
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ELISPOT
and chromium release assays (Figs. 5
6
7)
. These data indicate that a
second immunogenic epitope encoded by NY-ESO-1 is located between
residues 123 and 137 and is likely to contain the
NY-ESO-1126–134 core epitope identified by the
peptide-binding algorithm.
DCs Loaded with Recombinant NY-ESO-1 Protein Present the NY-ESO-1
Epitope Recognized by Clone 11/4 and Stimulate CD4+ T Cells That
Recognize the NY-ESO-1119–143 Peptide from a
DRB1*0401+ Normal Donor.
Autologous DCs were loaded with 30 µg/ml of either the NY-ESO-1
protein or the SSX protein (as indicated in "Materials and
Methods"). Twenty-five thousand protein-loaded DCs were added to
5 x 103
CD4+ T cells per
triplicate well in a 24-h ELISPOT assay. Only clone 11/4 but not clone
10/4 was able to recognize autologous DCs loaded with the NY-ESO-1
protein in ELISPOT assays (Fig. 8)
. Unloaded DCs or the DCs fed with the recombinant SSX protein served
as baseline and peptide controls, respectively.
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ELISPOT
assays. T cells that were stimulated with the protein-pulsed DCs
specifically recognized T2.DR4 cells pulsed with
NY-ESO-1119–143 peptide (Fig. 9)
. These CD4+ T cells also displayed reactivity against the
HLA-DR4-matched melanoma cell line UPCI-MEL 527.1, which was inhibited
by the addition of anti-HLA-DR mAb (L243) but not anti-HLA-A,B,C mAb.
No significant immunoreactivity could be observed against the other
potential DR4-binding NY-ESO-1 peptide (i.e.,
NY-ESO-1144–168 and
NY-ESO-1158–180).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in response to, and lysed, peptide-pulsed
DR4+ target cells, as well as
HLA-DR4+ melanoma cells constitutively expressing
the NY-ESO-1 protein. However, in patient 527 and two other
DR4+ melanoma patients that were analyzed in this
study, we could not detect any elevated "memory/effector " CD4+ T
cell frequency against the NY-ESO-1119–143
peptide in PBLs that were freshly isolated from the peripheral blood
and analyzed using the ELISPOT assay.
CD4+ T-cell clones recognizing the
NY-ESO-1119–143 peptide specifically secreted
the Th1-type cytokine IFN- in response to peptide-pulsed
DR4+ nonmelanoma target cells and HLA-DR4+
melanoma cells that constitutively expressed the NY-ESO-1 gene
product. Specific recognition of melanoma targets could be effectively
blocked by anti-HLA-DR antibody. Furthermore, HLA-DR4+ target cells
that lacked expression of the NY-ESO-1 gene product were not
recognized unless prepulsed with the
NY-ESO-1119–143 peptide.
These results may suggest a direct antitumor effector role for such CD4+ T cells against autologous NY-ESO-1+ melanoma cells that express HLA-DR4 class II molecules at their surface. Interestingly, the ESO-1 protein, like other MAGE proteins, is localized in the cytosol and nucleus, yet appears to be processed naturally via the endogenous class II pathway, giving rise to class II-restricted peptides presented at the surface of the tumor cells as recognized by our specific CD4+ T-cell clones (20) . Thus, our data are consistent with results reported for CD4+ T-cell recognition of alternate cancer-testis (CT) epitopes, such as the MAGE-3281–295 peptide presented by DR11 (3) . Despite the possibility of direct tumor presentation of these class II-binding epitopes, antitumor immunity may often be optimally promoted by "cross-priming" or "cross-presentation" of both CD8 and CD4 epitopes of tumor antigens by professional APCs (including DCs; Ref. 21 ). APCs may acquire tumor antigens from necrotic or apoptotic bodies in the tumor microenvironment (22) . This observation is consistent with other recent studies (23) that have documented the role of the CD4+ T cells in antitumor immunity of class II-negative tumors. This mechanism of immune-directed recognition of tumors would allow specific antitumor CD4+ T-cell responses to be beneficial in patients, irrespective of the MHC class II expression status of their tumors. Interestingly, we have also demonstrated that HLA-DR4+ DCs can process the NY-ESO-1 protein and present NY-ESO-1-derived epitopes able to stimulate in vitro CD4+ T cells that specifically recognize peptide NY-ESO-1119–143 and HLA-DR4+ tumor cells that naturally express the NY-ESO-1 protein.
Half-maximal CD4+ T effector cell stimulation required peptide loading concentrations as low as 1–60 nM. These results are in the range of values reported recently for CD4+ T-cell recognition of the HLA-DR13-restricted MAGE-3114–127 and HLA-DRB1*0101-restricted mutated TPI epitopes (2 , 5) .
We demonstrated that the original peptide NY-ESO-1119–143, used to generate the specific CD4+ T cells in vitro, encompasses two overlapping but non-cross-reactive epitopes (NY-ESO119–133 and NY-ESO-1123–137) recognized by distinct CD4 T-cell clones. Although the sensitivity of the different clones to the longer NY-ESO-1119–143 peptide seems to be similar for all of the ESO-1-reactive CD4+ T-cell clones evaluated, the reactivity of CD4+ T-cell clones to the shorter peptide sequences (i.e., NY-ESO119–133) may be inferior to that observed against the longer peptide (i.e., NY-ESO119–143). Although, we do not have a clear explanation for this phenomenon, one may hypothesize that although containing the DR4-binding core epitopes, the NY-ESO119–133 peptide in particular may exhibit a lower affinity for DR4 or a poorer solubility index than the NY-ESO119–143 peptide, perhaps because of the nature of its flanking sequences. We are currently evaluating this possibility. Alternatively, the shorter sequence may be subject to enhanced proteolytic degradation of the core epitope and hence neutralization of its "bioactivity" (24) .
Our findings add two new candidate CD4 "helper T cell" epitopes for future peptide-vaccine trials. Vaccination to both epitopes might be best accomplished using peptide NY-ESO119–143 that encompasses the two overlapping sequences. Given the role of the CD4+ T cells in maintaining CD8+ T-cell responses and the potential direct antitumor effector function of CD4+ T cells, it will be important to broaden clinical strategies to target the in vivo induction of both tumor-specific CD4+ T cells and CD8+ T cells. This might be most readily accomplished by vaccination with protein or multivalent peptides including class I and class II-restricted epitopes encoded by cancer-testis (i.e., the NY-ESO-1157–165 and NY-ESO-1119–143) and/or melanocyte-specific genes (i.e., the Melan-A/MART-127–35 and Melan-A/MART-151–73 peptides) in HLA-A2+, DR4+ patients with melanoma.
NY-ESO-1 is expressed by 30% of metastatic melanoma and by
non-small cell lung cancer, bladder, prostate, and head and neck tumors
(25)
. Although nearly 18% of American Caucasians express
the HLA-DR4 allele (26)
, we have observed an HLA-DR4
allele frequency of 34% (15 of 44) in a recent serological assessment
of melanoma patients treated at the University of Pittsburgh Cancer
Institute.4
Because the DRB1*0401, DRB1*0404, and DRB1*0408
subtypes are expressed by 
80% of the DR4+ population
(27, 28, 29)
, NY-ESO-1119–143 peptide
will likely prove to be clinically relevant in 4.4–8.2% of Caucasian
melanoma patients. Recent studies also demonstrate that HLA-DR4
molecules are part of a larger HLA class II supertype
(30)
, including several other common DR types. Because the
identification of a broadly cross-presented epitope would clearly
expand the potential population coverage, we are currently determining
the ability of the NY-ESO-1119–143 peptide to
bind to a broader range of HLA-DR alleles. Interestingly, in a recent
study by Jager et al. (31)
, three
DRB4*0101–0103 (DR53)-restricted epitopes recognized by melanoma
reactive CD4+ T cells have been identified. Two of these had sequences
similar to the two epitopes reported in our study,
NY-ESO-1115–132 and
NY-ESO-1121–138, respectively. This supports the
relevance of the NY-ESO-1119–143 peptide and its
two "sub epitopes" as potential immunogens for cancer vaccines
designed to treat HLA-DR4+ and
HLA-DR53+ patients with
NY-ESO-1+ tumors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grants CA 57840
(to W. J. S.) and CA 56774 (to J. M. K.), a Clinical Investigator
Award from the Cancer Research Institute (to W. J. S.), the
Competitive Medical Research Fund of the University of Pittsburgh
Medical Center, and a Cancer Research Institute/Elaine R. Shepard
Memorial Fellowship (to H. M. Z.). 
2 To whom requests for reprints should be
addressed, at University of Pittsburgh Cancer Institute, Biomedical
Science Tower, E 1051, 211 Lothrop Street, Pittsburgh, PA 15213-2582.
Phone: (412) 624-7796; Fax: (412) 624-7794; E-mail: zarourhm{at}msx.upmc.edu
3 The abbreviations used are: mAb, monoclonal
antibody; PBMC, peripheral blood mononuclear cell; DC, dendritic cell;
rhGM-CSF, recombinant human granulocyte/macrophage-colony stimulating
factor; rhIL, recombinant human interleukin; PBL, peripheral blood
lymphocyte; APC, antigen-presenting cell.
4 J. M. Kirkwood, personal communication.
Received 3/23/00. Accepted 6/23/00.
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REFERENCES |
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K. Hasegawa, Y. Noguchi, F. Koizumi, A. Uenaka, M. Tanaka, M. Shimono, H. Nakamura, H. Shiku, S. Gnjatic, R. Murphy, et al. In vitro Stimulation of CD8 and CD4 T Cells by Dendritic Cells Loaded with a Complex of Cholesterol-Bearing Hydrophobized Pullulan and NY-ESO-1 Protein: Identification of a New HLA-DR15-Binding CD4 T-Cell Epitope Clin. Cancer Res., March 15, 2006; 12(6): 1921 - 1927. [Abstract] [Full Text] [PDF]
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F. Neumann, C. Wagner, K.-D. Preuss, B. Kubuschok, C. Schormann, S. Stevanovic, and M. Pfreundschuh Identification of an epitope derived from the cancer testis antigen HOM-TES-14/SCP1 and presented by dendritic cells to circulating CD4+ T cells Blood, November 1, 2005; 106(9): 3105 - 3113. [Abstract] [Full Text] [PDF]
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M. Mandic, F. Castelli, B. Janjic, C. Almunia, P. Andrade, D. Gillet, V. Brusic, J. M. Kirkwood, B. Maillere, and H. M. Zarour One NY-ESO-1-Derived Epitope That Promiscuously Binds to Multiple HLA-DR and HLA-DP4 Molecules and Stimulates Autologous CD4+ T Cells from Patients with NY-ESO-1-Expressing Melanoma J. Immunol., February 1, 2005; 174(3): 1751 - 1759. [Abstract] [Full Text] [PDF]
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H. A. Vaughan, S. Svobodova, D. MacGregor, S. Sturrock, A. A. Jungbluth, J. Browning, I. D. Davis, P. Parente, Y.-T. Chen, E. Stockert, et al. Immunohistochemical and Molecular Analysis of Human Melanomas for Expression of the Human Cancer-Testis Antigens NY-ESO-1 and LAGE-1 Clin. Cancer Res., December 15, 2004; 10(24): 8396 - 8404. [Abstract] [Full Text] [PDF]
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 M. Matsuo, Y. Nagata, E. Sato, D. Atanackovic, D. Valmori, Y.-T. Chen, G. Ritter, I. Mellman, L. J. Old, and S. Gnjatic IFN-{gamma} enables cross-presentation of exogenous protein antigen in human Langerhans cells by potentiating maturation PNAS, October 5, 2004; 101(40): 14467 - 14472. [Abstract] [Full Text] [PDF]
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I. D. Davis, W. Chen, H. Jackson, P. Parente, M. Shackleton, W. Hopkins, Q. Chen, N. Dimopoulos, T. Luke, R. Murphy, et al. Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4+ and CD8+ T cell responses in humans PNAS, July 20, 2004; 101(29): 10697 - 10702. [Abstract] [Full Text] [PDF]
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F. Korangy, L. A. Ormandy, J. S. Bleck, J. Klempnauer, L. Wilkens, M. P. Manns, and T. F. Greten Spontaneous Tumor-Specific Humoral and Cellular Immune Responses to NY-ESO-1 in Hepatocellular Carcinoma Clin. Cancer Res., July 1, 2004; 10(13): 4332 - 4341. [Abstract] [Full Text] [PDF]
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N. A. Danke, D. M. Koelle, C. Yee, S. Beheray, and W. W. Kwok Autoreactive T Cells in Healthy Individuals J. Immunol., May 15, 2004; 172(10): 5967 - 5972. [Abstract] [Full Text] [PDF]
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E. Maraskovsky, S. Sjolander, D. P. Drane, M. Schnurr, T. T. T. Le, L. Mateo, T. Luft, K.-A. Masterman, T.-Y. Tai, Q. Chen, et al. NY-ESO-1 Protein Formulated in ISCOMATRIX Adjuvant Is a Potent Anticancer Vaccine Inducing Both Humoral and CD8+ T-Cell-Mediated Immunity and Protection against NY-ESO-1+ Tumors Clin. Cancer Res., April 15, 2004; 10(8): 2879 - 2890. [Abstract] [Full Text] [PDF]
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M. J. Palmowski, L. Lopes, Y. Ikeda, M. Salio, V. Cerundolo, and M. K. Collins Intravenous Injection of a Lentiviral Vector Encoding NY-ESO-1 Induces an Effective CTL Response J. Immunol., February 1, 2004; 172(3): 1582 - 1587. [Abstract] [Full Text] [PDF]
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M. Mandic, C. Almunia, S. Vicel, D. Gillet, B. Janjic, K. Coval, B. Maillere, J. M. Kirkwood, and H. M. Zarour The Alternative Open Reading Frame of LAGE-1 Gives Rise to Multiple Promiscuous HLA-DR-restricted Epitopes Recognized by T-helper 1-type Tumor-reactive CD4+ T Cells Cancer Res., October 1, 2003; 63(19): 6506 - 6515. [Abstract] [Full Text] [PDF]
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T. Tatsumi, C. J. Herrem, W. C. Olson, J. H. Finke, R. M. Bukowski, M. S. Kinch, E. Ranieri, and W. J. Storkus Disease Stage Variation in CD4+ and CD8+ T-Cell Reactivity to the Receptor Tyrosine Kinase EphA2 in Patients with Renal Cell Carcinoma Cancer Res., August 1, 2003; 63(15): 4481 - 4489. [Abstract] [Full Text] [PDF]
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S. Gnjatic, D. Atanackovic, E. Jager, M. Matsuo, A. Selvakumar, N. K. Altorki, R. G. Maki, B. Dupont, G. Ritter, Y.-T. Chen, et al. Survey of naturally occurring CD4+ T cell responses against NY-ESO-1 in cancer patients: Correlation with antibody responses PNAS, July 22, 2003; 100(15): 8862 - 8867. [Abstract] [Full Text] [PDF]
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E. Davila, R. Kennedy, and E. Celis Generation of Antitumor Immunity by Cytotoxic T Lymphocyte Epitope Peptide Vaccination, CpG-oligodeoxynucleotide Adjuvant, and CTLA-4 Blockade Cancer Res., June 15, 2003; 63(12): 3281 - 3288. [Abstract] [Full Text] [PDF]
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E. H. Slager, M. Borghi, C. E. van der Minne, C. A. Aarnoudse, M. J. E. Havenga, P. I. Schrier, S. Osanto, and M. Griffioen CD4+ Th2 Cell Recognition of HLA-DR-Restricted Epitopes Derived from CAMEL: A Tumor Antigen Translated in an Alternative Open Reading Frame J. Immunol., February 1, 2003; 170(3): 1490 - 1497. [Abstract] [Full Text] [PDF]
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P. F. Robbins, M. El-Gamil, Y. F. Li, G. Zeng, M. Dudley, and S. A. Rosenberg Multiple HLA Class II-Restricted Melanocyte Differentiation Antigens Are Recognized by Tumor-Infiltrating Lymphocytes from a Patient with Melanoma J. Immunol., November 15, 2002; 169(10): 6036 - 6047. [Abstract] [Full Text] [PDF]
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H. Kobayashi, R. Omiya, M. Ruiz, E. Huarte, P. Sarobe, J. J. Lasarte, M. Herraiz, B. Sangro, J. Prieto, F. Borras-Cuesta, et al. Identification of an Antigenic Epitope for Helper T Lymphocytes from Carcinoembryonic Antigen Clin. Cancer Res., October 1, 2002; 8(10): 3219 - 3225. [Abstract] [Full Text] [PDF]
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J. A. Hural, R. S. Friedman, A. McNabb, S. S. Steen, R. A. Henderson, and M. Kalos Identification of Naturally Processed CD4 T Cell Epitopes from the Prostate-Specific Antigen Kallikrein 4 Using Peptide-Based In Vitro Stimulation J. Immunol., July 1, 2002; 169(1): 557 - 565. [Abstract] [Full Text] [PDF]
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 M. A. Haque, P. Li, S. K. Jackson, H. M. Zarour, J. W. Hawes, U. T. Phan, M. Maric, P. Cresswell, and J. S. Blum Absence of {gamma}-Interferon-inducible Lysosomal Thiol Reductase in Melanomas Disrupts T Cell Recognition of Select Immunodominant Epitopes J. Exp. Med., May 20, 2002; 195(10): 1267 - 1277. [Abstract] [Full Text] [PDF]
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H. M. Zarour, B. Maillere, V. Brusic, K. Coval, E. Williams, S. Pouvelle-Moratille, F. Castelli, S. Land, J. Bennouna, T. Logan, et al. NY-ESO-1 119-143 Is a Promiscuous Major Histocompatibility Complex Class II T-Helper Epitope Recognized by Th1- and Th2-Type Tumor-reactive CD4+ T Cells Cancer Res., January 1, 2002; 62(1): 213 - 218. [Abstract] [Full Text] [PDF]
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H. Kobayashi, J. Lu, and E. Celis Identification of Helper T-Cell Epitopes That Encompass or Lie Proximal to Cytotoxic T-Cell Epitopes in the gp100 Melanoma Tumor Antigen Cancer Res., October 1, 2001; 61(20): 7577 - 7584. [Abstract] [Full Text] [PDF]
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X.-F. Yang, C. J. Wu, S. McLaughlin, A. Chillemi, K. S. Wang, C. Canning, E. P. Alyea, P. Kantoff, R. J. Soiffer, G. Dranoff, et al. CML66, a broadly immunogenic tumor antigen, elicits a humoral immune response associated with remission of chronic myelogenous leukemia PNAS, June 19, 2001; 98(13): 7492 - 7497. [Abstract] [Full Text] [PDF]
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 J. M. Kirkwood, J. G. Ibrahim, J. A. Sosman, V. K. Sondak, S. S. Agarwala, M. S. Ernstoff, and U. Rao High-Dose Interferon Alfa-2b Significantly Prolongs Relapse-Free and Overall Survival Compared With the GM2-KLH/QS-21 Vaccine in Patients With Resected Stage IIB-III Melanoma: Results of Intergroup Trial E1694/S9512/C509801 J. Clin. Oncol., May 1, 2001; 19(9): 2370 - 2380. [Abstract] [Full Text] [PDF]
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) and used as target cells in
a 20-h IFN-
