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Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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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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To date, only a limited number of MHC class II-restricted tumor Ags have been identified. Several known MHC class I-restricted tumor Ags, such as tyrosinase, gp100,3 and MAGE-3, were demonstrated to contain MHC class II-restricted epitopes recognized by CD4+ T cells (11, 12, 13, 14, 15). Recently, a genetic approach was developed to identify unknown MHC class II-restricted tumor Ags by using tumor-specific CD4+ T cells (16). This has led to the identification of several mutated tumor Ags, including CDC27, triosephosphate isomerase, and LDLR-FUT (16, 17). Among them, triosephosphate isomerase is a mutated Ag that was independently identified by a biochemical approach (18).
The NY-ESO-1 gene was previously identified by Ab screening (19), and was recently identified as an MHC class I-restricted tumor Ag as well (20, 21). High titers of Abs against NY-ESO-1 were also detected from patients with cancer (22). The NY-ESO-1 cDNA encoded two gene products from two overlapping open reading frames (20). Because of its strict tumor-specific expression pattern, with the exception of expression in normal testis, as well as its high frequency of expression in many tumors, including melanoma, breast, prostate, lung, and other cancers (19, 20, 23), NY-ESO-1 is potentially an important immune target for the development of immunotherapies for a variety of cancer types (24).
Because both CTL and Ab immune responses against NY-ESO-1 were demonstrated in patients with cancer, identification of MHC class II-restricted T cell epitopes in the NY-ESO-1 protein could be important for the development of effective cancer vaccines. In this study, we report the identification of an HLA-DR4-restricted T cell epitope from the NY-ESO-1 Ag by using HLA-DR4 transgenic mice immunized with the purified NY-ESO-1 protein and in vitro stimulation of human PBMC with synthetic candidate peptides. We show that CD4+ T cells generated from human PBMC are NY-ESO-1 specific, and recognize NY-ESO-1 peptides pulsed on HLA-DR4+ EBV B cells as well as naturally processed peptides on melanoma cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To construct a bacterial expression vector encoding the full-length NY-ESO-1 gene, we generated a PCR fragment by using a pair of primers, ESO-5p (5'-GCTCCGGACATATGCAGGCCGAAGGCCGGGG) containing an NdeI site and ESO-3p (5'-AAGGGGCTCGAGGCTGGGCTTAGCGCCTCT) containing an XhoI site. After digestion with restriction enzymes and gel purification of the PCR product, a DNA fragment encoding NY-ESO-1 was fused to DNA encoding a polyhistidine peptide in frame in pET-28(+) (Novagen, Madison, WI). A similar strategy was also used to construct an expression vector for a truncated NY-ESO-1, ESO1–74, which contained only the first 74 aa residues. Escherichia coli strain BL21(DE3) bearing the correct plasmid construct was grown at 37°C to log phase, then induced for protein production by adding isopropyl ß-d-thiogalactoside to a final concentration of 0.5 mM and shaking for 3 h. Soluble fractions of bacterial extract were obtained; and NY-ESO-1 was purified by Ni2+ affinity chromatography. SDS-PAGE analysis of the purified protein was performed as previously reported (25). The N-terminal sequence of the purified protein was determined by automatic Edman degradation.
Serum and PBMC
Sera from patients with metastatic melanoma were stored at -80°C. Sera of normal donors were obtained from the Blood Bank at the Clinical Center of National Institutes of Health. The MHC class II genotype of patient TE with metastatic melanoma was HLA-DRß1*0401, ß1*1501. The patient was treated with the gp100:209–217(210 M) peptide plus high dose of IL-2, and experienced an objective tumor regression.
Detection of Abs against NY-ESO-1 protein
About 50 ng of purified NY-ESO-1 protein diluted in 50 µl PBST (PBS with 0.1% Tween-20) was adsorbed to each well of a 96-well MaxiSorp plate (Nunc, Roskilde, Denmark) overnight at room temperature. Control plates were coated with 150 ng BSA/well. Plates were blocked with 5% dry milk in PBST for at least 2 h, washed, and were loaded with 100 µl of diluted serum samples. All serum samples were diluted at 1/25, 1/250, and 1/2500 with 3% dry milk in PBST. Each sample at the three different dilutions was loaded onto NY-ESO-1-coated plates as well as BSA-coated plates. After 1-h incubation at room temperature, plates were washed and loaded with secondary Ab (goat anti-human IgG conjugated with HRP; Sigma, St. Louis, MO) diluted with 1% dry milk in PBST. Plates were developed after a 0.5-h incubation, and absorbance at 450 nm was read by using an ELISA reader (Dynatech, Chantilly, VA). A positive reaction was defined as an OD value against NY-ESO-1 that exceeded the mean OD value plus three times SDs of normal donors at serum dilutions of both 1/25 and 1/250. Western blot was performed as described (25) to confirm the specificity of the Ab in a few representative sera samples.
Cell lines and Abs
Melanoma lines F049 and F050 were early cultures of fine needle asparate samples, provided by Adam Riker at the Surgery Branch of National Cancer Institute. All other melanoma lines and EBV B lines were generated and maintained in RPMI 1640 (Life Technologies, Rockville, MD) supplemented with 10% FCS (Biofluids, Gaithersburg, MD). 293IMDR1 and 293IMDR4 were genetically engineered to express human invariant chain, DMA, DMB, and DR molecules, and were cultured in RPMI 1640 supplemented with 10% FCS (16). Culture medium for murine lymphocytes was RPMI 1640 with 0.05 mM 2-ME, 5 CU/ml IL-2, plus 10% FCS, provided by HyClone (Logan, UT). Medium used for human T cell culture was RPMI 1640 with 0.05 mM 2-ME, 50 CU/ml IL-2, plus 10% human AB serum, provided by Sigma. Ab-blocking experiments were performed as previously described (15). Hybridoma HB55 and HB95 were obtained from American Type Culture Collection (Manassas, VA). Control Ab was purchased from PharMingen (San Diego, CA).
Transgenic animals and immunization procedures
HLA-DR4 transgenic (DR4-Tg) mice were murine class II deficient,
and expressed HLA-DR
-IE- and HLA-DR ß1*0401-IE-ß chimeric
molecules (26). Founder mice were obtained through Paul
Lehmann at Case Western Reserve University (Cleveland, OH). Mice were
inbred and maintained at Biocon (Rockville, MD). Female mice aged
between 6 and 10 wk were immunized with the full-length recombinant
NY-ESO-1 protein. About 50 µg of purified protein were emulsified in
CFA, divided evenly, and given to each mouse via s.c. injection into
rear footpads and the base of tail. Eleven days after the injection,
mice were sacrificed, and the bilateral hind limb popliteal and the
inguinal lymph nodes were harvested. Single cell suspensions were
obtained from the lymph nodes of two immunized animals, and followed by
in vitro stimulation.
Peptide synthesis
Synthetic peptides used in this study were made using a solid-phase method on a peptide synthesizer (Gilson, Worthington, OH) at the Surgery Branch of National Cancer Institute. The purity of each peptide was evaluated by mass spectrometry (Bio-synthesis, Lewisville, TX).
In vitro sensitization procedure and cytokine release assays
Peptides at a final concentration of 10 µM were mixed with 2.5 x 105 mouse lymphocytes for 1 wk before cytokine release assays were conducted. For in vitro sensitization of human PBMC, 2.5 x 105 cells were pulsed with peptides at 10 µM concentration and incubated in each well of a flat-bottom 96-well plate. After two in vitro stimulations, cells were tested against various targets and supernatants were harvested for cytokine release assays. Rapid expansion and cloning of human T cells were performed as described (20).
Peptide at a final concentration of 10 µM or protein at a final
concentration of 5 µg/ml was pulsed onto target cells. After 4-h
incubation, cells were washed in serum-free RPMI medium, and 
3
x 104 target cells were incubated with the same
number of TE4-1 cells overnight, and cytokine release was measured
using GM-CSF ELISA kits (R&D Systems, Minneapolis, MN) for human or
IFN-
kits (Endogen, Woburn, MA) for mouse. Other cytokines, such as
human IFN-, IL-10, TNF-, and IL-4, were measured using ELISA kits
from Endogen or R&D Systems, according to the manufacturer’s
instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NY-ESO-1-reactive Abs and CTL have been reported in patients with
cancer (19, 22). It thus appeared that NY-ESO-1-specific
CD4+ T cells might play a role in orchestrating
the development of Abs as well as CTLs against the NY-ESO-1 Ag. To
identify MHC class II-restricted CD4+ T cell
epitopes, we began by purifying NY-ESO-1 protein from a bacterial
expression system as the starting material. To facilitate NY-ESO-1
expression and protein purification, a cDNA fragment encoding NY-ESO-1
was fused to a polyhistidine tag in frame located at the N terminus in
the pET28 expression vector, and a high level production of recombinant
protein was obtained. Several milligrams of the NY-ESO-1 protein were
purified by using a Ni2+-charged affinity
chromatography column. The purified protein showed an apparent
molecular mass of 26 kDa on an SDS polyacrylamide gel (Fig. 1
A). To confirm the identity
of the purified protein, N-terminal microsequencing of protein was
performed by automatic Edman degradation. All 25 aa residues obtained
by Edman degradation matched the predicted amino acid sequences (data
not shown). A short version of NY-ESO-1 containing the first 74 aa
residues, ESO1–74, was also purified by the same approach (Fig. 1A).
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C). These data were consistent with results obtained by
other groups (22). To exclude the possibility that
patients’ sera reacted with a minor contaminant present in the
purified NY-ESO-1 protein, Western blot was performed using
representative sera samples. Fig. 1B showed that the
NY-ESO-1-reactive sera from a patient reacted only with cell lysates
from NY-ESO-1-expressing bacteria and the purified NY-ESO-1 protein,
but not with extracts from bacteria containing the control vector.
A nonreactive serum sample was also tested (Fig. 1B,
lanes 4, 5, and 6). Identification of putative MHC class II-restricted epitopes from HLA-DR4 transgenic mice
To identify CD4+ T cell epitopes, DR4
transgenic mice were immunized in the tail base and rear footpads with
50 µg of full-length NY-ESO-1 protein in CFA. Eleven days after
the injection, single cell suspensions obtained from bilateral hind
limb popliteal and inguinal lymph nodes of two immunized mice were
prepared and used for in vitro sensitization with synthetic peptides
derived from the NY-ESO-1 protein based on the predicted
peptide-binding properties of the HLA-DR4 molecules
(27).
Eight high-binding peptides containing amino acid sequence
segments predicted to bind to HLA-DR4 were used for the in vitro
sensitization experiments. Six days after the initial in vitro
sensitization, murine lymphocytes were tested for cytokine release
against human HLA-DR4-positive 1359EBV B cells alone and 1359EBV B
pulsed with the corresponding peptide used for stimulation. Three
peptides were recognized by murine T cells based on cytokine secretion
from T cells, while other five peptides showed no recognition (Fig. 2). The ESO p116–135 showed the
strongest activity among the positive peptides, suggesting that this
peptide might contain an epitope presented by the HLA-DR4 molecule for
T cell recognition. This peptide was thus chosen for further
analysis.
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PBMCs from patient TE, who had high titered Abs against
NY-ESO-1 (Fig. 1C), were used for in vitro stimulation with
the ESO p116–135 peptide. After 1 wk of in vitro stimulation, PBMC
from patient TE showed marked expansion. IL-2 was added in the second
week of stimulation. The cell line thus established was named TE4-1,
which continued growth for more than 2 wk in the presence of 20 CU/ml
IL-2. The TE4-1 T cells were 90% CD4+ T cells
based on FACS analysis. TE4-1 contained Th1-type
CD4+ T cells as they secreted GM-CSF, IFN-,
and TNF-, but not IL-10 or IL-4 (data not shown). After depletion of
a few percent of CD8+ T cells, the purified
population of CD4+ T cells still retained its
reactivity. Some T cell clones derived from TE4-1 cell line were also
shown to recognize the ESO p116–135 peptide (data not shown).
TE4-1 recognized EBV B cells pulsed with the full-length NY-ESO-1
protein as well as the ESO p116–135 peptide in the context of HLA-DR4,
but not with the truncated NY-ESO-1 protein containing the first 74 aa
(Fig. 3A). The TE4-1 cell line
was also reactive specifically with DR4-positive dendritic cells
infected with adenovirus encoding NY-ESO-1, but not adenovirus encoding
the green fluorescence protein (data not shown).
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B,
both peptide 116–135 and peptide 111–130 were recognized by TE4-1 in
the context of HLA-DR4. Interestingly, peptide 116–135 was also
capable of stimulating cytokine secretion from T cells when pulsed onto
293IMDR1 cells. No activity was detected with 293IMDR4 pulsed with the
control ESO p91–110 peptide (Fig. 3B). The recognition of
ESO-p116–135-pulsed 293IMDR4 was completely inhibited by an
anti-HLA-DR Ab (HB55), but not by the control and anti-HLA
class I Abs (HB95) (Fig. 3C). A gp100-specific
CD8+ T cell line (CTL-C3G1) and an
HLA-DR1-restricted CD4+ T cell line (T3–80) were
used as specificity controls for the Ab blocking. Recognition of tumor cells by TE4-1
Although peptide-specific CD4+ and
CD8+ T cell activities can often be generated
against a putative tumor Ag, in many cases tumor reactivity could not
be demonstrated due to either the low affinity of the T cells or the
failure of presentation of naturally processed peptides on the tumor
cell surface (2). To test whether TE4-1 could recognize
NY-ESO-1 epitopes naturally processed and presented by tumor cells,
several melanoma lines were used as targets. The expression of NY-ESO-1
in each line was determined by RT-PCR, while the expression of HLA-DR
alleles was determined by FACS analysis (data not shown). As shown in
Fig. 4, TE4-1 was capable of recognizing
NY-ESO-1/HLA-DR4-positive tumors (1359 mel and F049 mel), but failed to
recognize tumor cell lines 397 mel and 624.38 mel
(NY-ESO-1+/HLA-DR-), nor
526 mel
(NY-ESO-1-/HLA-DR4+). T
cell reactivity was also detected on F050 mel
(DR1+/NY-ESO-1+), but not
on 1300 mel expressing DR1 and a low level of NY-ESO-1. One possible
explanation is that CD4+ T cells may recognize
the same peptide presented by DR1 molecule when the peptide
concentration is high. Alternatively, ESO p116–135 may bind to DR1 and
subsequently released and picked up by TE4-1 and presented to each
other as TE4-1 cells express DR4 molecules. The recognition of F049 mel
could be specifically blocked in the presence of anti-HLA-DR Ab,
but not the anti-MHC class I Ab (data not shown). These studies
suggested that the TE4-1 cell line recognized a naturally processed
peptide on the tumor cell surface.
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Because the two reactive peptides shared 15 aa (LPVPGVLLKEFTVSG),
the minimal length of peptide was determined by testing a series of N-
and C-terminal truncated peptides. Peptides were pulsed onto
DR4+ 1088 EBV B cells and tested for their
ability to stimulate TE4-1 cells. The valine residue at position 128
was found to be critical for T cell recognition (Fig. 5A). The peptides with the
N-terminal deletions up to leucine residue at position 123 did not
affect T cell recognition, but the peptide with further deletions
partially lost its ability to stimulate T cells. The leucine residue at
position 123 may be a P1 anchor residue because the P1, P4, P6, and P7
residues contributed to the peptide binding to MHC class II molecules.
Further deletions are required to determine the critical residues for
binding to MHC class II molecules.
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B, no or little T cell
activity was observed at 33 nM or lower concentrations of the ESO
p119–130 peptide; high activities were detected at 0.33 µM peptide
concentration, and the T cell activity did not reach a plateau at a 33
µM peptide concentration. The control peptide was not recognized by
TE4-1 even at a 33 µM peptide concentration. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, B and
C), while a very low percentage of patients developed high
titers of Abs against the MAGE Ags or differentiation Ags such as
tyrosinase, gp100, TRP-1, and TRP-2 (data not shown) (22).
It is possible that Abs reacting with glycosylated proteins, such as
gp100, TRP-1, and TRP-2, may not react with bacteria-derived
unglycosylated proteins. However, these studies strongly suggest that
NY-ESO-1-reactive CD4+ T cells may be involved in
Ab production and CTL proliferation. In this study, we identified the
HLA-DR4-restricted T cell epitope derived from NY-ESO-1 by the use of
HLA-DR4 transgenic mice and in vitro stimulation of human PBMC with
candidate peptides. To our knowledge, this is the first demonstration
that T cell epitopes from NY-ESO-1 were shown to be presented by
HLA-DR4 (DRß1*0401) molecule to CD4+ T cells.
Because NY-ESO-1-specific Abs and CTL were detected in patients with
different HLA genotypes, other CD4+ T cell
epitopes presented by HLA class II molecules other than HLA-DR4
probably exist. Indeed, after this work was submitted, a study
describing the identification of NY-ESO-1 epitopes presented by
DRß4*0101–0103 was reported (28).
Recently, two groups reported the identification of MHC class
II-restricted T cell epitopes from the known MHC class I-restricted
tumor Ag, MAGE-3. CD4+ T cell clones generated
from PBMC stimulated with DC pulsed with purified MAGE-3 protein
recognized peptide or protein pulsed on HLA-DR13-matched EBV B cells,
but not MAGE-3+/DR13+ tumor
cells (13). However, in another study,
CD4+ T cells generated from PMBC stimulated with
peptides predicted by a computer-assisted algorithm were capable of
recognizing both peptide pulsed on EBV B cells and
MAGE-3+/DR11+ tumor cells
(15). In the case of NY-ESO-1, we show in this study that
CD4+ T cells can recognize the NY-ESO-1 protein
or peptide pulsed on DR4-matched EBV B cells as well as tumor cells
expressing NY-ESO-1 (Figs. 3 and 4). Utilization of HLA-DR transgenic
mice may have advantages in identifying putative peptides because
immunized transgenic mice presumably have a high precursor frequency of
specifically reactive T cells. Once candidate peptides were identified,
CD4+ T cells could be generated from PBMC
stimulated with synthetic candidate peptides. Therefore, the combined
use of transgenic mice immunized with the whole protein and stimulated
with the peptides predicted by a computer-assisted algorithm may avoid
the need to stimulate human PBMC with a large number of peptides and
several rounds of in vitro stimulation. Furthermore, candidate peptides
identified by using the immunized transgenic mice are likely to be
peptides that are naturally processed and presented on the cell
surface. This may increase the likelihood that peptide-specific
CD4+ T cells can recognize tumor cells as well.
Finally, the use of PBMC from a patient (TE), who developed a high
titer of Ab and a high precursor frequency of CTL against NY-ESO-1, may
make it easier to generate tumor-specific CD4+ T
cells because both Ab production and CTL require the help of
CD4+ T cells. This approach has been used to
identify a number of MHC class II-restricted T cell epitopes from known
autoantigens involved in autoimmune disease (29).
Therefore, the strategy used in this study may be applicable to many
other known MHC class I-restricted tumor Ags, while other strategies
such as a direct gene-cloning approach may facilitate the
identification of unknown MHC class II-restricted tumor Ags.
In this study, we observed that ESO p116–135 was recognized by TE4-1
when pulsed on 293IMDR1 cells at a relatively high peptide
concentration (Fig. 3B). T cell recognition was
significantly reduced when peptide concentrations decreased from 33 to
3.3 µM for the peptide titration experiment (data not shown). In a
separate experiment, the peptide ESO p116–135 as well as the whole
NY-ESO-1 protein were weakly recognized when pulsed on
DR1+ 586 EBV B cells. The recognition was
specifically blocked by anti-DR Ab, but not anti-class I Ab and
control Ab (data not shown). TE4-1 cells also only recognized
DR1-positive tumor cell line F050 mel expressing a high level of
NY-ESO-1, but not DR1-positive1300 mel expressing a low level of
NY-ESO-1 (Fig. 4). Thus, this T cell recognition may be explained by
two possibilities. The peptide ESO p116–135 may be promiscuous and can
be recognized by TE4-1 in the context of either DR1 or DR4, but with
different affinities. It has been shown that T cells can degenerately
recognize peptides presented by multiple HLA class II alleles
(30, 31). Alternatively, ESO p116–135 might bind to DR1
molecule. The carried-over peptide is subsequently released and
presented to each other by DR4-positive TE4-1 T cells. T cell-T cell
presentation has been reported in CD4+ TIL1363
(17).
Clinical trials using peptides derived from tissue-specific differentiation Ags such as gp100 showed some evidence of therapeutic efficacy in the treatment of patients with melanoma (3). Although no significant toxic side effects were observed in the patients treated with the modified gp100 peptides, vitiligo or depigmentation was often found in patients who responded to therapy (32), suggesting that antitumor immunity induced by immunization with self Ags may cause autoimmunity. In animal studies using TRP-1 as an immune target, similar results (antitumor immunity and coat depigmentation) were also obtained (33, 34, 35). Interestingly, antitumor immunity and autoimmunity mediated by gp75/TRP-1 appeared to involve CD4+ T cells and Abs (36). Immunization of mice with human TRP-2 (37), but not murine TRP-2 (38), broke tolerance to the self Ag, and the antitumor immunity required the participation of both CD4+ and CD8+ T cells (36). These studies suggested that antitumor immunity could be mediated by either Abs or CD8+ T cells, but both require the critical help of CD4+ T cells (24, 36).
The MHC class II-restricted NY-ESO-1 peptides identified in this study may be useful in clinical applications because CTL and Abs against NY-ESO-1 were detected in patients with cancer. Immunization with both MHC class I- and II-restricted peptides or with a purified NY-ESO-1 protein may induce NY-ESO-1-specific CD4+, CD8+ T cells as well as Abs. Alternatively, patients could be immunized with dendritic cells loaded with both class I and II peptides or infected with recombinant viruses encoding the NY-ESO-1 gene. Because testicular germ cells do not express MHC class I and II molecules (39), immune responses against NY-ESO-1 should be specific for tumor cells, and thus generate little or no autoimmune responses. Similar studies using MHC class I-restricted peptides of MAGE-3 or peptides pulsed on dendritic cells indicated that while antitumor immunity (CTL responses) and slow tumor regression were demonstrated, no depigmentation/vitiligo or other significant side effects were observed (5, 6). Antitumor immunity may be enhanced by providing tumor-specific CD4+ T cell help.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rong-Fu Wang at the current address: ALKEK Building N1120, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77050.
3 Abbreviations used in this paper: gp, glycoprotein; TRP, tyrosinase-related protein; LDLR-FUT, low density lipid receptor (LDLR) and GDP-L-fucose ß-D-galactoside 2--L-fucosyltransferase (FUT) fusion protein; CU, cetus unit.
Received for publication February 4, 2000. Accepted for publication May 8, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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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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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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K. S. Voo, T. Fu, H. E. Heslop, M. K. Brenner, C. M. Rooney, and R.-F. Wang Identification of HLA-DP3-restricted Peptides from EBNA1 Recognized by CD4+ T Cells Cancer Res., December 15, 2002; 62(24): 7195 - 7199. [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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G. Zeng, Y. Li, M. El-Gamil, J. Sidney, A. Sette, R.-f. Wang, S. A. Rosenberg, and P. F. Robbins Generation of NY-ESO-1-specific CD4+ and CD8+ T Cells by a Single Peptide with Dual MHC Class I and Class II Specificities: A New Strategy for Vaccine Design Cancer Res., July 1, 2002; 62(13): 3630 - 3635. [Abstract] [Full Text] [PDF]
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 H. Y. Wang, J. Zhou, K. Zhu, A. I. Riker, F. M. Marincola, and R.-F. Wang Identification of a Mutated Fibronectin As a Tumor Antigen Recognized by CD4+T Cells: Its Role in Extracellular Matrix Formation and Tumor Metastasis J. Exp. Med., June 3, 2002; 195(11): 1397 - 1406. [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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R. Lapointe, R. E. Royal, M. E. Reeves, I. Altomare, P. F. Robbins, and P. Hwu Retrovirally Transduced Human Dendritic Cells Can Generate T Cells Recognizing Multiple MHC Class I and Class II Epitopes from the Melanoma Antigen Glycoprotein 100 J. Immunol., October 15, 2001; 167(8): 4758 - 4764. [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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C. Meyer zum Buschenfelde, J. Metzger, C. Hermann, N. Nicklisch, C. Peschel, and H. Bernhard The Generation of Both T Killer and Th Cell Clones Specific for the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells J. Immunol., August 1, 2001; 167(3): 1712 - 1719. [Abstract] [Full Text] [PDF]
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V. Deffrennes, J. Vedrenne, M.-C. Stolzenberg, J. Piskurich, G. Barbieri, J. P. Ting, D. Charron, and C. Alcaide-Loridan Constitutive Expression of MHC Class II Genes in Melanoma Cell Lines Results from the Transcription of Class II Transactivator Abnormally Initiated from Its B Cell-Specific Promoter J. Immunol., July 1, 2001; 167(1): 98 - 106. [Abstract] [Full Text] [PDF]
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T. Matsutake and P. K. Srivastava The immunoprotective MHC II epitope of a chemically induced tumor harbors a unique mutation in a ribosomal protein PNAS, March 27, 2001; 98(7): 3992 - 3997. [Abstract] [Full Text] [PDF]
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G. Zeng, X. Wang, P. F. Robbins, S. A. Rosenberg, and R.-F. Wang CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production PNAS, March 16, 2001; (2001) 61507398. [Abstract] [Full Text]
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G. Zeng, X. Wang, P. F. Robbins, S. A. Rosenberg, and R.-F. Wang CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA DP4 allele: Association with NY-ESO-1 antibody production PNAS, March 27, 2001; 98(7): 3964 - 3969. [Abstract] [Full Text] [PDF]
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