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* Department of Medicine and The Melanoma Center, and
Department of Immunology, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213;
Protein Engineering and Research Department, Commissariat à l’Energie Atomique-Saclay, Gif sur Yvette, France; and
Kent Ridge Digital Laboratories, Singapore, Singapore
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-5 ELISPOT assays and PBL from patients with NY-ESO-1-expressing tumors, we observed the existence of Th1-type circulating CD4+ T cells recognizing peptide NY-ESO-1 87–111 in the context of HLA-DP4 molecules. Taken together, these data represent the first report of an HLA-DR- and HLA-DP-restricted epitope from a tumor Ag. They also support the relevance of cancer vaccine trials with peptides NY-ESO-1 87–111 in the large number of cancer patients with NY-ESO-1-expressing tumors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we develop a strategy to identify pan-MHC class II peptide sequences, which can be broadly presented by multiple HLA-DR alleles and HLA-DP4 to stimulate CD4+ T cells. Our strategy is based on the combination of binding assays to multiple HLA-DR and HLA-DP4 molecules with in vitro stimulations with T cells and APCs from normal donors and melanoma patients. These pan-MHC class II peptides may be useful in vaccine trials for a large number of patients without prior HLA typing, because any patient will most likely express one or more allele molecules capable of presenting the peptide. In this report we apply this strategy to peptide NY-ESO-1 87–111. We demonstrate that peptide NY-ESO-1 87–111 not only binds to multiple HLA-DR and HLA-DP4 alleles, but also stimulates CD4+ T cell responses when presented in the context of these molecules. We also observed that this peptide sequence stimulated circulating Th1-type CD4+ T cells from PBL of melanoma patients. These findings support the use of the NY-ESO-1 87–111 peptide as a cancer vaccine for a large number of patients with NY-ESO-1-expressing tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The tissues and blood samples used for all studies reported in this manuscript were obtained under University of Pittsburgh Cancer Institute (UPCI) institutional review board approval. Patients UPCI-MEL 527 and UPCI-MEL 285 are long-lived patients who remain disease-free several years after successful therapy for disseminated NY-ESO-1-expressing metastatic melanoma. UPCI-MEL 527.1 and UPCI-MEL 285.1 cell lines were derived from one metastatic lesion of each patient. Patients UPCI-MEL 527 and UPCI-MEL 285 have been genotyped as HLA-DRB1*0401+/DRB1*1401+/DRB4*0101+/DPB1*0402+ and HLA-DRB1*0401+/DRB1*0101+/DRB4*0101+/DPB1*0401+, respectively. HLA-DR andHLA-DP genotyping of melanoma patients and normal donors was performed using commercial typing panels of PCR primers according to the manufacturer’s instructions (Dynal Biotech). Normal donor 1 (ND1) has been genotyped as HLA-DRB1*0701+/DRB1*1101+/DRB4*0101+/DPB1*0401+, respectively. HLA-DRB1*0401-transfected T2 cells, i.e., T2.DR4 cells, and HLA-DR-transfected mouse cells, i.e., L.DR cells, were previously described (5, 9). All cell lines were cultured in RPMI 1640 medium (Invitrogen Life Technologies) supplemented with 10% FCS, L-arginine (116 mg/l), L-asparagine (36 mg/l), and L-glutamine (216 mg/l). The HB55 and HB95 hybridomas, secreting L243 anti-HLA-DR (class II) mAb and W6/32 anti-HLA-A, -B, and -C (class I) mAb, respectively, were purchased from American Type Culture Collection. The B7/21 anti-HLA-DP (class II) mAb used in our blocking experiments was previously described (10).
Peptide synthesis
The NY-ESO-1-derived peptides were synthesized and stored as previously reported (5, 6). Synthesis of NY-ESO-1 peptides was based on the sequence of a NY-ESO-1 gene reported by Chen et al. (1) (GenBank accession no. U87459).
Recombinant proteins
The full-length NY-ESO-1 recombinant protein was produced in Escherichia coli and was provided by Drs. L. J. Old and G. Ritter (Ludwig Institute for Cancer Research, New York, NY). The LAGE-1 open reading frame 2 (ORF2) protein was also produced in E. coli as previously described (11).
Purification of HLA-DR molecules
HLA-DR molecules were purified from homozygous EBV cell lines by affinity chromatography using the monomorphic mAb L 243 coupled to protein A-Sepharose CL 4B gel (Amersham Biosciences) as previously described (12, 13).
HLA-DR peptide binding assays
Binding to multiple HLA-DR and HLA-DP4 molecules was performed as previously reported (12, 13, 14). Maximal binding was determined by incubating the biotinylated peptide with the MHC class II molecule in the absence of competitor. Binding specificity for each HLA-DR and HLA-DP4 molecule was ensured by the choice of biotinylated peptides as described previously (13, 14). Data were expressed as the concentration of peptide that prevented binding of 50% of the labeled peptide (IC50).
Induction of CD4+ T cells with peptides
The induction of CD4+ T cells and clones in vitro with the NY-ESO-1-derived peptides was performed as previously reported (6, 15).
In vitro sensitization with peptides
PBL from patients with active melanoma, long-term survivors, and normal donors (Table I) were used to purify CD8 and CD4+ T cells with immunomagnetic beads (Miltenyi Biotec). Two million non-CD8/CD4+ T cells were pulsed with 10 µg of peptide (4 h, 37°C), irradiated (3000 rad), washed, and used to stimulate 2 x 106 autologous CD4+ T cells in Iscove’s medium supplemented with 10% human AB serum (Sigma-Aldrich) without any cytokine. On day 7 of in vitro stimulation, the responder CD4+ T cells were harvested and analyzed in ELISPOT assays.
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and IL-5 ELISPOT assays
The recognition of APC pulsed with peptides, proteins, and tumor cells was assessed by ELISPOT assays specific for human IFN- and IL-5 as previously reported (5, 6). Alternatively, 5000 protein-loaded or cDNA-transfected dendritic cells (DCs) were added to 103 CD4+ T cell clones/well. The protein-loaded DCs were prepared as previously reported in the presence of the recombinant NY-ESO-1 ORF1 or LAGE-1 ORF2 proteins (30 µg/ml). Mature DCs were transfected with either pcDNA3-NY-ESO-1 (provided by Drs. E. Jager and A. Knuth (Department of Oncology,Krankenhaus Nordwest, Frankfurt, Germany)) or pcDNA3-MART-1 using human Nucleofactor (Amaxa). Shortly, 1 x 106 DCs were transfected with 5 µg of plasmid DNA in 100 µl of human DC Nucleofector solution. Electroporation was performed using the Nucleofector device (Amaxa). DCs were incubated, then collected and used for ELISPOT assays. In the blocking experiments, 3 µg of each purified anti-HLA mAb was added to each ELISPOT well. Spot numbers and spot sizes were determined with the use of computer-assisted video image analysis as previously described (16). For statistical evaluation, a t test for unpaired samples was used. A value of p < 0.05 was considered significant.
IFN- and IL-4 cytokine secretion assays
The recognition of autologous DCs pulsed with peptides (10 µg/ml) or proteins (30 µg/ml) was also assessed by MACS secretion assays for IFN- and IL-4 (Miltenyi Biotec) as previously described (11).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We evaluated the binding capacities of peptide NY-ESO-1 87–111 (LLEFYLAMPFATPMEAELARRSLAQ) as well as peptide NY-ESO-1 158–180 (LLMWITQCFLPVFLAQPPSGQRR) to 10 different HLA-DR molecules, including seven molecules encoded by the HLA-DRB1 genes (i.e., HLA-DRB1*0101, -DRB1*0301, -DRB1*0401, -DRB1*0701, -DRB1*1101, -DRB1*1301, and -DRB1*1501); three molecules encoded by the DRB3, DRB4, and DRB5 genes; and two molecules encoded by HLA-DPB1*0401 and 0402 (Table II). All these molecules are present at high frequencies in the Caucasian population. These peptide sequences have been chosen for analysis because they were previously predicted to contain HLA-DRB1*0401-binding sequences (6), as well as anchor residues to bind to the HLA-DPB1*0401 and 0402 molecules (residues are underlined).
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In sharp contrast, peptide NY-ESO-1 87–111 binds not only to HLA-DRB1*0401, but also to HLA-DRB1*0101, -DRB1*0701, -DRB1*1101, -DRB1*1501, and -DRB5*0101. Importantly, peptide NY-ESO-1 87–111 also binds well to HLA-DPB1*0401 and -0402. NY-ESO-1 87–111 contains at least one HLA-DPB1*0401/0402-binding motif, i.e., NY-ESO-1 91–99 (YLAMPFATP), that is most likely responsible for the DP4-binding activity of the peptide. This is confirmed by our binding data, which show that peptide sequence NY-ESO-1 87–101, but not 97–111 or 92–105, was capable of binding to HLA-DPB1*0401 and -0402. Moreover, peptide NY-ESO-1 87–101, but not 97–111 or 92–105, appears to encompass the promiscuous HLA-DR-binding region contained in the peptide NY-ESO-1 87–111. The IC50 values of peptide NY-ESO-1 87–101 were very similar to those of peptide NY-ESO-1 87–111, except for HLA-DR15. Overall, the NY-ESO-1 87–111 peptide exhibits broad binding capacity to multiple HLA-DR molecules as well as to the HLA-DPB1*0401 and -0402 molecules.
Peptide NY-ESO-1 87–111 is presented by multiple HLA-DR and HLA-DPB1*0401 and -0402 molecules and stimulates tumor-reactive CD4+ T cells
In an independent series of in vitro experiments, we primed CD4+ T cells from one normal donor and two melanoma patients against peptide NY-ESO-1 87–111. Melanoma patients and normal donor were typed as HLA-DRB1*0401+/DRB1*1401+/DRB4*0101+/DPB1*0402+ (patient 1), DRB1*0101+/DRB1*0401+/DRB4*0101+/DPB1*0401+ (patient 2), and DRB1*0701+/DRB1*1101+/DRB4*0101+/DPB1*0401+ (donor 1), respectively. Mature DCs were incubated with peptide NY-ESO-1 87–111 (10 µg/ml), irradiated, and used to stimulate autologous CD4+ T cells, as previously described (6, 15). The CD4+ T cells were restimulated on a weekly basis with irradiated autologous mature DCs pulsed with peptide. After at least three restimulations, the immunoreactivity of the CD4+ T cell cultures was analyzed in IFN- ELISPOT assays. We used as APCs in IFN- ELISPOT assays, L cells that have been genetically engineered to express HLA-DR1 (L.DR1), HLA-DR4 (L.DR4), HLA-DR7 (L.DR7), HLA-DR 53 (L.DR53), or HLA-DP4 (L.DP4).
Bulk CD4+ T cells isolated from patient 1 stimulated with NY-ESO-1 87–111 peptide specifically recognized L.DR4 cells and L.DP4 cells pulsed with peptides NY-ESO-1 87–111 or NY-ESO-1 87–101, but not NY-ESO-1 97–111 (Fig. 1A). These CD4+ T cells also displayed reactivity against the NY-ESO-1+ autologous melanoma cell line, UPCI-MEL527.1, that was partially inhibited by addition of the anti-HLA-DR mAb (L243) or anti-HLA-DP mAb (B7/21), but not the anti-HLA-A, -B, or -C mAb. No significant reactivity was noted against an HLA-DRB1*0401+, NY-ESO-1– melanoma cell line, UPCI-MEL 136.1. Of note, the CD4+ T cells produced IFN- not only in the presence of NY-ESO-1 87–111 peptide-pulsed DCs, but also in the presence of autologous DCs loaded with the NY-ESO-1 protein (Fig. 1B). No significant amount of IFN- was produced by CD4+ T cells in the presence of peptide NY-ESO-1 87–111 alone. The autologous DCs pulsed with an irrelevant peptide or protein were not recognized. L.DR cells expressing allogenic HLA-DR molecules (i.e., L.DR1, LDR7, and L.DR11) and pulsed with peptide NY-ESO-1 87–111 were not recognized (data not shown).
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not only in the presence of NY-ESO-1 87–111 peptide-pulsed DCs, but also in the presence of autologous DCs loaded with the NY-ESO-1 protein, as shown in Fig. 1D. No significant amount of IFN- was produced by CD4+ T cells in the presence of peptide NY-ESO-1 87–111 alone. The autologous DCs pulsed with an irrelevant peptide or protein were not recognized. L.DR cells expressing allogenic HLA-DR molecules (i.e., LDR7 and L.DR11) and pulsed with peptide NY-ESO-1 87–111 were not recognized (data not shown).
Bulk CD4+ T cells isolated from donor 1 previously stimulated with the NY-ESO-1 87–111 peptide specifically recognized L.DR7 cells, L.DR11 cells, or L.DP4 cells pulsed with peptides NY-ESO-1 87–111 or NY-ESO-1 87–101 (Fig. 1E). CD4+ T cells failed to produce a significant amount of IFN- in the presence of APCs pulsed with either NY-ESO-1 97–111 or NY-ESO-1 119–143, which was used as an irrelevant peptide. Importantly, these CD4+ T cells recognized autologous DCs loaded with the NY-ESO-1 ORF1 protein, but not with an irrelevant protein, LAGE-1 ORF2 (11). No significant amount of IFN- was produced by CD4+ T cells in the presence of peptide NY-ESO-1 87–111 alone. L.DR cells expressing allogenic HLA-DR molecules (i.e., L.DR1 and L.DR4) and pulsed with peptide NY-ESO-1 87–111 were not recognized (data not shown).
From the NY-ESO-1 87–111-specific bulk CD4+ T cells presented in Fig. 1, we have generated CD4+ T cell clones by limiting dilution. Clone 40/56 was obtained from the NY-ESO-1 87–111-specific CD4+ T cells from patient 1 and was capable of producing IFN- in the presence of not only L.DR4 cells pulsed with peptide NY-ESO-1 87–111, but also autologous DCs previously loaded with the NY-ESO-1 protein (Fig. 2A). Of note, no significant amount of IFN- production was observed in the presence of peptide NY-ESO-1 87–111 alone, autologous DCs alone, DCs fed with an irrelevant protein (Fig. 2A), or other L.DR cells pulsed with peptides (data not shown).
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in the presence of not only L.DR1 cells pulsed with peptide NY-ESO-1 87–111, but also autologous DCs fed with the NY-ESO-1 protein (Fig. 2B). No significant amount of IFN- production was observed in the presence of peptide NY-ESO-1 87–111 alone, DCs alone, autologous DCs fed with an irrelevant protein (Fig. 2B), or other L.DR cells pulsed with peptides (data not shown).
Clone 42/5 was obtained from the NY-ESO-1 87–111-specific CD4+ T cells from patient 2 and produced IFN- not only in the presence of L.DR7 cells pulsed with peptide NY-ESO-1 87–111, but also autologous DCs fed with the NY-ESO-1 protein (Fig. 2B). No significant amount of IFN- production was observed in the presence of peptide NY-ESO-1 87–111 alone, DCs alone, autologous DCs fed with an irrelevant protein (Fig. 2C), or other L.DR cells pulsed with peptides (data not shown).
These data corroborate our binding data and demonstrate the ability of peptide NY-ESO-1 87–111 to stimulate CD4+ T cells and CD4+ T cell clones capable of recognizing peptide NY-ESO-1 87–111 in the context of the HLA-DRB1*0101, -DRB1*0701, and -DRB1*1101 and HLA-DPB1*0401 and -0402 molecules. These bulk CD4+ T cells were also determined to be tumor reactive and capable of recognizing autologous DCs loaded with the relevant protein.
Peptide NY-ESO-1 87–111 stimulates Th0 and Th2-type CD4+ T cell clones isolated from PBL of patients with melanoma that are capable of recognizing autologous protein-loaded DCs
Several CD4+ T cell clones were obtained by limiting dilution from the blood of patient UPCI-MEL527 after in vitro stimulation with peptide NY-ESO-1 87–111. One representative clone, 40/1, specifically produced IL-4, IL-5, and IFN- in the presence of autologous DCs pulsed with peptide NY-ESO-1 87–111, loaded with the NY-ESO-1 ORF1 protein, or transfected with NY-ESO-1 cDNA in ELISPOT and cytokine release assays as described in Materials and Methods (Fig. 3, A–C). Unloaded DCs, DCs pulsed with an irrelevant peptide (i.e., NY-ESO-1 119–143 or LAGE-1 ORF2 85–102), and DCs fed with the LAGE-1 ORF2 protein served as baseline and controls. Clone 40/1 did not recognize the autologous tumor cell line, UPCI-MEL 527.1 (data not shown). Of note, the same CD4+ T cell clone produced only IL-5 spots in the presence of L.DP4 cells pulsed with peptide NY-ESO-1 87–111 (Fig. 3D). No IL-5 spots were produced in the presence of L.DR cells pulsed with peptide NY-ESO-1 87–111 (data not shown). The ability of CD4+ T cell clone 40/1 to produce IL-5 in the presence of L.DP4 cells, preincubated with the NY-ESO-1 87–111 peptide at various concentrations, was evaluated to determine the peptide-dose threshold for effector T cell recognition. The half-maximal stimulation of clone 40/1 required peptide loading concentrations of 
50–60 nM (Fig. 3D).
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and IL-5 in ELISPOT assays in the presence of L.DP4 cells or autologous DCs pulsed with peptide NY-ESO-1 87–111. Clone 40/10 also produced IFN- and IL-5 in the presence of autologous DCs loaded with the NY-ESO-1 ORF1 protein or transfected with NY-ESO-1 cDNA (Fig. 4, A and B). IFN- and IL-5 production by clone 40/1 was inhibited by addition of anti-HLA-DP mAb (B7/21), but not anti-HLA-DR mAb (L243). The half-maximal stimulation of clone 40/10 required peptide loading concentrations of 20 nM in IFN- ELISPOT assays (Fig. 4C). IL-5 production by clone 40/10 in the presence of titrated doses of peptide NY-ESO-1 87–111 is represented as a dome-shaped curved with maximal production of IL-5 at peptide concentrations ranging from 20–200 nM (Fig. 4D).
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Taken together, these data demonstrate the capability of peptide NY-ESO-1 87–111 to stimulate Ag-specific CD4+ T cells whose phenotype (i.e., Th2 and Th0) may depend on the type of APCs used to stimulate the autologous responder T cells.
Detection of peptide NY-ESO-1 87–111-specific CD4+ T cells in peripheral blood of melanoma patients
We next investigated the blood of 21 patients with melanoma, four long-term survivors, and six normal donors for the presence of CD4+ T cells capable of recognizing peptide NY-ESO-1 87–111 (Table I). CD4+ T cells were first isolated from PBLs and stimulated with peptide-pulsed APCs for 7 days before the performance of IFN- and IL-5 ELISPOT assays, as previously described in Materials and Methods. We observed that the PBL of 5 of 12 HLA-DP4 patients with melanoma contained CD4+ T cells were capable of recognizing L.DP4 cells pulsed with peptide NY-ESO-1 87–111 (Fig. 5A). Five patients developed Ag-specific Th1-type CD4+ T cells, whereas one had Th0-type CD4+ T cells (producing a low number of IL-5 spots). One of the three HLA-DPB1*0401+ long-term survivors with NY-ESO-1-expressing tumors and none of the four HLA-DP4+ normal donors had detectable levels of NY-ESO-1 87–111-specific HLA-DP4-restricted CD4+ T cells. As a control and as shown in Fig. 5B, we also observed that 5 of 12 HLA-DP4 patients had NY-ESO-1 157–170-specific DP4-restricted CD4+ T cells: four had Th1-type CD4+ T cells, and one had Th0-type CD4+ T cells (with a high number of IL-5 spots), respectively. Two of three long-term survivors with NY-ESO-1-expressing tumors developed Th1- and Th0-type HLA-DP4-restricted CD4+ T cells recognizing peptide NY-ESO-1 157–170, respectively. No normal donor displayed NY-ESO-1 157–170-specific CD4+ T cells. Altogether 8 of the 12 patients with NY-ESO-1-expressing tumors developed CD4+ T cells responses against either NY-ESO-1 87–111 or NY-ESO-1 157–170. Of note, four patients developed HLA- DP4-restricted Th1-type CD4+ T cells against both NY-ESO-1 87–111 and NY-ESO-1 157–170 epitopes (Fig. 5C). The two HLA-DP4+ long-term survivors with NY-ESO-1-expressing tumors had only NY-ESO-1 157–170-specific CD4+ T cells, not NY-ESO-1 87–111-specific CD4+ T cells.
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Analysis of NY-ESO-1-specific Ab responses in conjunction with NY-ESO-1-specific CD4+ T cells
We then analyzed sera from melanoma patients for the presence of NY-ESO-1-specific Abs. ELISAs with the NY-ESO-1 protein and sera from patients were performed as previously reported (17). Among the six HLA-DP4+ patients with NY-ESO-1-expressing tumors and NY-ESO-1 87–111-specific CD4+ T cells, two had high titers of NY-ESO-1-specific Abs, whereas four had no detectable level of NY-ESO-1-specific Abs (Fig. 5D). Additionally, three patients had low to high titers of Abs, but no evidence of NY-ESO-1 87–111-specific CD4+ T cells.
Among the five patients with NY-ESO-1-expressing tumors and NY-ESO-1 157–170-specific CD4+ T cells, two had high titers of NY-ESO-1-specific Abs, and three had no NY-ESO-1-specific Abs (Fig. 5E). Additionally, three melanoma patients had low to high titers of Abs, but no evidence of NY-ESO-1 157–170-specific CD4+ T cells.
We also observed that two of the three HLA-DP4+ long-term survivors with NY-ESO-1-expressing tumors displayed high titers of NY-ESO-1-specific Abs.
Of note, the presence of NY-ESO-1-specific Abs was not always correlated with the expression of HLA-DPB1*0401/0402 molecules, as previously reported by others (4); one melanoma patient and one long-term survivor with NY-ESO-1-specific Abs did not express HLA-DP4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a series of in vitro experiments using DCs and PBLs isolated from melanoma patients and normal donors, we have confirmed the implications of the binding data and demonstrated the immunogenicity of peptide NY-ESO-1 87–111 in the context of several HLA-DR and HLA-DP4 molecules. Moreover, our data demonstrate the ability of peptide NY-ESO-1 87–111 to stimulate both HLA-DR- and HLA-DP4-restricted CD4+ T cells from the PBLs of the same patient.
We also demonstrated that the CD4+ T cells and clones were capable of recognizing either autologous melanoma cell lines or autologous DCs previously loaded with NY-ESO-1 protein or transfected with NY-ESO-1 cDNA. This indicates that peptides NY-ESO-1 87–111 and 87–101 contain epitopes that are processed and presented at the surface of multiple HLA-DR and HLA-DP4 molecules.
Gnjatic et al. (7) have recently reported the existence of circulating CD4+ T cells against peptide 80–109 in HLA-DR7+ patients with NY-ESO-1-expressing tumors. These authors have noted NY-ESO-1 80–109-specific CD4+ T cells in three of five HLA-DP4+ patients with NY-ESO-1-expressing tumors. Our data confirm and expand these observations, demonstrating precisely at the molecular level the capability of peptide NY-ESO-1 87–111 to bind to multiple HLA-DR and HLA-DP4 molecules and to stimulate HLA-DR- and HLA-DP4-restricted CD4+ T cell lines and clones.
We have also demonstrated the ability of the NY-ESO-1 87–111 peptide to stimulate both Th1/Th0- and Th2-type CD4+ T cells. We have observed that the phenotype of cloned CD4+ T cells may vary with the type of APCs used in the assay, in particular when using mature DCs vs the L.DP4 cell line as APCs. One category of clones, such as clone 40/1, appears capable of producing both IFN- and IL-5 only in the presence of autologous DCs, not L.DP4 cells. Another category, such as clone 40/10, appears to exhibit a well-defined and fixed Th phenotype that does not change based on the type of APCs added to stimulate these T cells. This may illustrate the flexibility of cytokine gene expression by certain tumor Ag-specific human CD4+ T cell subsets (18). This observation represents one major issue for the phenotype study of Ag-specific CD4+ T cells after in vitro stimulation, in particular any potential disease or therapy-associated Th1/Th2 bias from the blood of patients with cancer (19), because cytokine production may depend on the type of APCs used in the assay.
We also observed that the peptide concentration may modulate the level of IL-5 secretion by the CD4+ T cells, such as clone 40/10. In particular, high doses of peptide (i.e., >500 nM/ml) were better at stimulating IFN- production than IL-5 production by the Th0-type CD4+ T cell clone. In contrast, low peptide doses (i.e., 5–10 nM) better stimulated IL-5 production. These results are in agreement with several studies suggesting that low doses of soluble Ag may optimally stimulate Th2-type CD4+ T cells (20, 21).
We have shown that patients with active melanoma may develop naturally occurring HLA-DR4- and HLA-DP4-restricted CD4+ T cell responses against NY-ESO-1 87–111. Nearly 67% of the HLA-DP4+ patients (8 of 12) with NY-ESO-1-expressing melanomas displayed either NY-ESO-1 87–111- or NY-ESO-1 157–170-specific CD4+ T cells, and nearly 25% displayed both (3 of 12). Patients with active melanoma had mainly Th1-type (seven of eight) and less often Th0-type (one of eight) NY-ESO-1-specific CD4+ T cells. We did not measure any Th1/Th2-associated bias in CD4+ T cell responses to tumor Ag, in contrast to reports by others (19).
No correlation of CD4+ T cell responses against NY-ESO-1 87–111 or NY-ESO-1 157–170 with NY-ESO-1-specific Ab responses was observed; three of eight patients with NY-ESO-1-specific CD4+ T cells did not exhibit NY-ESO-1-specific Abs.
Surprisingly, we detected NY-ESO-1-specific Abs in the sera of two of three long-term survivors. These patients have been disease free for >10 years and are clinically re-evaluated on a yearly basis. Although there was no obvious evidence of active disease in these patients, we cannot exclude that they may still be tumor-bearing, because the presence of NY-ESO-1-specific Abs has been previously correlated with the presence of NY-ESO-1-expressing tumors in the host (8).
Both the immunogenicity and the pan-MHC class II promiscuity of peptide NY-ESO-1 87–111 support its relevance in cancer vaccines designed to treat patients with NY-ESO-1+ tumors. Because NY-ESO-1 is expressed by 30% of metastatic melanoma, breast and prostate carcinomas, non-small cell lung cancer, and bladder, head, and neck tumors (22), peptide vaccines including NY-ESO-1 87–111 may be applicable to a large number of patients with tumors of different histological types.
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Acknowledgments |
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Footnotes |
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1 This work was supported by National Institutes of Health Grant RO1CA90360 and a Clinical Trial Grant from the Cancer Research Institute (to H.M.Z.), grants from l’Association pour la Recherche sur le Cancer et l’Etablissement Francais des Greffes (to B.M.), and a fellowship from the Cancer Foundation for Melanoma Research (to M.M.). 
2 Address correspondence and reprint requests to Dr. Hassane M. Zarour, University of Pittsburgh Cancer Institute, Hillman Cancer Center, Research Pavilion, Suite 1.32, 5117 Centre Avenue, Pittsburgh, PA 15213-2582. E-mail address: zarourhm{at}upmc.edu
3 Abbreviations used in this paper: ORF, open reading frame; DC, dendritic cell.
Received for publication June 11, 2004. Accepted for publication November 18, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in response to HLA-A2.1-binding melanoma and viral peptide antigens. J. Immunol. Methods 191:131.[Medline]
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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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