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The Journal of Immunology, 2004, 172: 5095-5102.
Copyright © 2004 by The American Association of Immunologists

Identification of Multiple HLA-DR-Restricted Epitopes of the Tumor-Associated Antigen CAMEL by CD4+ Th1/Th2 Lymphocytes

Elisabeth H. Slager, Caroline E. van der Minne, Margreet Krüse, Dilja D. Krueger, Marieke Griffioen and Susanne Osanto1

Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD4+ Th cells play an important role in the induction and maintenance of adequate CD8+ T cell-mediated antitumor responses. Therefore, identification of MHC class II-restricted tumor antigenic epitopes is of major importance for the development of effective immunotherapies with synthetic peptides. CAMEL and NY-ESO-ORF2 are tumor Ags translated in an alternative open reading frame from the highly homologous LAGE-1 and NY-ESO-1 genes, respectively. In this study, we investigated whether CD4+ T cell responses could be induced in vitro by autologous, mature dendritic cells pulsed with recombinant CAMEL protein. The data show efficient induction of CAMEL-specific CD4+ T cells with mixed Th1/Th2 phenotype in two healthy donors. Isolation of CD4+ T cell clones from the T cell cultures of both donors led to the identification of four naturally processed HLA-DR-binding CAMEL epitopes: CAMEL1–20, CAMEL14–33, CAMEL46–65, and CAMEL81–102. Two peptides (CAMEL1–20 and CAMEL14–33) also contain previously identified HLA class I-binding CD8+ T cell epitopes shared by CAMEL and NY-ESO-ORF2 and are therefore interesting tools to explore for immunotherapy. Furthermore, two CD4+ T cell clones that recognized the CAMEL14–33 peptide with similar affinities were shown to differ in recognition of tumor cells. These CD4+ T cell clones recognized the same minimal epitope and expressed similar levels of adhesion, costimulatory, and inhibitory molecules. TCR analysis demonstrated that these clones expressed identical TCR {beta}-chains, but different complementarity-determining region 3 loops of the TCR {alpha}-chains. Introduction of the TCRs into proper recipient cells should reveal whether the different complementarity-determining region 3{alpha} loops are important for tumor cell recognition.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The aim of many immunotherapies for cancer is to stimulate T cell responses against tumor-specific proteins. In the past, CD8+ CTLs have been shown to be the main effector cells in antitumor T cell responses and many MHC class I-binding peptides derived from tumor-specific Ags have now been identified. Although CTLs can act alone in the effector phase of an immune response, it has become clear that priming of naive CD8+ T cells by APCs, like dendritic cells (DCs),2 is dependent on ‘help’ from CD4+ T cells. Upon interaction with Ag-specific CD4+ Th cells, DCs up-regulate their expression of MHC and costimulatory molecules that is required for efficient priming of CD8+ T cells. This interaction between CD4+ T cells and DC has been shown to be mediated by molecules like CD40 and CD40 ligand (1, 2, 3). In addition to DC activation, CD40 and CD40 ligand molecules have also been shown to mediate direct interactions between CD4+ and CD8+ T cells, required for efficient generation of memory CD8+ T cells (4). These and many other studies (5, 6, 7, 8, 9) demonstrate a crucial role of CD4+ Th cells in the induction and maintenance of adequate CD8+ T cell-mediated antitumor responses. Therefore, the identification of MHC class II-restricted tumor antigenic epitopes is indispensable for the development of effective immunotherapies with synthetic peptides.

Recently, CD4+ T cells recognizing MHC class II-restricted epitopes encoded by cancer/testis Ags like NY-ESO-1 (10, 11), MAGE-6 (12), and CAMEL (13) have been detected in peripheral blood of cancer patients, strongly suggesting that these tumor Ags are processed and presented in the MHC class II pathway in vivo. In cancer patients, however, the tumor Ag-specific CD4+ T cells often belong to the Th2 subtype, producing cytokines such as IL-4, IL-5, and IL-13 (12, 13), whereas IL-2- and IFN-{gamma}-producing Th1 cells are believed to be required for proper activation of cytotoxic CD8+ T cells. It has been suggested that tumor Ag-specific CD4+ Th2 cells in cancer patients are induced by malfunctioning DC, expressing low levels of MHC and costimulatory molecules (14, 15, 16, 17), whereas mature DC, expressing high levels of MHC and costimulatory molecules, are required for the induction of Th1 immunity and potent CD8+ T cell-mediated antitumor responses.

CAMEL (109 aa) is a tumor-specific protein translated from the LAGE-1 gene in an alternative open reading frame (ORF) starting at a second ATG start codon located 40 bp downstream of the first ATG start site (18). LAGE-1 shows 94% homology to tumor Ag NY-ESO-1 and both genes are located on chromosome Xp28 (19, 20). The LAGE-1 and NY-ESO-1 genes are frequently coexpressed in a wide variety of tumors, like melanoma, breast carcinoma, prostate and bladder cancers, but silent in normal tissues except for testis (19, 20, 21). Like LAGE-1, the NY-ESO-1 gene is translated in an alternative ORF, resulting in a 58 aa NY-ESO-ORF2 protein from which the N-terminal 54 aa are identical with CAMEL.

In this study, we investigated whether CD4+ T cell responses could be induced in vitro by mature DC pulsed with recombinant CAMEL protein. The data show efficient induction of CAMEL-specific CD4+ T cells expressing both Th1 and Th2 cytokines in two healthy donors. Because CAMEL and NY-ESO-ORF2 are not expressed in normal tissues, these CAMEL-specific CD4+ T cells resulted from in vitro priming of naive CD4+ T cells. The isolation of CD4+ T cell clones from the T cell cultures of both donors led to the identification of four naturally processed HLA-DR-binding CAMEL epitopes: CAMEL1–20, CAMEL14–33, CAMEL46–65, and CAMEL81–102. Two of these peptides (CAMEL1–20 and CAMEL14–33) also contain previously identified HLA class I-binding CD8+ T cell epitopes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

Melanoma cell lines 518A2, 90.07, 93.08, 93.11, 93.15.2, and 94.03 were established in our laboratory. Line 518A2/IL-2.14 was obtained by transfecting the 518A2 cell line with IL-2 cDNA (22). Melanoma cell lines FM3 and FM6 were kindly provided by J. Zeuthen (Danish Cancer Society, Copenhagen, Denmark). All melanoma cell lines were cultured in DMEM (Invitrogen, Breda, The Netherlands) supplemented with 8% heat-inactivated FCS, 4 mM L-glutamine, 50 µg/ml penicillin and 50 µg/ml streptomycin. EBV-transformed B-lymphoblastic cell lines (EBV-B cells) were generated from PBMC of healthy donors and maintained in RPMI 1640 (Invitrogen) with FCS, L-glutamine and antibiotics. All melanoma cell lines and EBV-B cells were HLA typed using standard serological methods.

Peptides and recombinant protein

Peptides were synthesized by solid-phase methods, using an automated multiple peptide synthesizer (Abimed AMS 422; Abimed Analyes-Technik, Langenfeld, Germany) and Fmoc chemistry. After reversed phase HPLC analysis, peptides were dissolved in DMSO at 50 mg/ml and stored at −70°C. From this stock solution, peptide was diluted in PBS to a final concentration of 1 mg/ml and stored at −20°C.

Recombinant CAMEL protein was produced in a bacterial expression system. Briefly, a 349 bp LAGE-1 cDNA fragment was amplified using a sense primer containing a NdeI site (5'-GAAGAACATATGCTGATGGCCCAGGAGGC-3') and an antisense primer containing a BglII site (5'-TTAAAGATCTCAGAACCGCCCCTGGTCG-3'). The LAGE-1 cDNA fragment only encodes CAMEL due to deletion of the first ATG start site. The cDNA fragment was cloned into the NdeI and BamHI sites of the pET19b vector (Novagen, Madison, WI) in frame with an N-terminal (His)10-tag. Expression of pET19b-CAMEL in Escherichia coli strain BL21(DE3) (Stratagene, Amsterdam, The Netherlands) was induced by addition of 1 mM isopropyl-{beta}-D-thiogalactoside (Sigma-Aldrich, Zwijndrecht, The Netherlands). After 4 h, rCAMEL was purified from bacterial lysate by nickel-chelate affinity chromatography using Ni-NTA agarose according to manufacturer’s instructions (Westburg, Leusden, The Netherlands). Recombinant CAMEL was eluted in 8 M urea, 100 mM NaH2PO4, and 10 mM Tris, pH 4.5 and dialyzed in distilled water. After lyophilization, recombinant CAMEL was dissolved in distilled water at 1 mg/ml and stored at −70°C.

Generation of DCs

PBMC were isolated by Ficoll gradient centrifugation from heparinized blood from two healthy donors. Donors were HLA class II-typed as HLA-DR3, -DR7, -DR52, -DR53, -DQ2 (donor 1) and HLA-DR3, -DR4, -DR52, -DR53, -DQ2, -DQ8 (donor 2) using standard serological techniques. PBMC were depleted for T and B cells using CD2- and CD19-dynabeads (Dynal Biotech, Oslo, Norway) and cultured at 2 x 106 cells/well in 6-well plates in AIM-V (Invitrogen) with 116 µg/ml L-arginine, 36 µg/ml L-asparagine, 215 µg/ml L-glutamine (AAG), and antibiotics, supplemented with 500 U/ml IL-4 (PeproTech, Rocky Hill, NJ) and 800 U/ml GM-CSF (Behringwerke, Marburg, Germany) for 6 days. Immature DC were pulsed with 10 µg/ml recombinant CAMEL for 4 h at 37°C and subsequently matured for 2 days in AIM-V containing 1 µg/ml activating anti-CD40 Ab (Cymbus Biotechnology, Chandlers Ford, U.K.), 500 U/ml IL-4 and 800 U/ml GM-CSF. For peptide loading, immature DC were cultured for 2 days in AIM-V containing anti-CD40 Ab, IL-4 and GM-CSF as previously described and subsequently pulsed with 10 µg/ml peptide for 2 h at 37°C.

Generation of CAMEL-specific CD4+ T cells

For the induction of CAMEL-specific CD4+ T cells, 20 x 106 nonadherent PBMC were incubated with autologous, mature DC pulsed with recombinant CAMEL at a ratio of 10:1 at 2 x 106 PBMC/well in 24-well plates in IMDM (Invitrogen) with 5% pooled human AB serum (HS), AAG and antibiotics, supplemented with 10 ng/ml IL-7 (PeproTech) and 100 pg/ml IL-12 (Sigma-Aldrich). T cell cultures were weekly restimulated as previously described in IMDM with 5% HS, AAG and antibiotics, supplemented with 150 U/ml IL-2.

To obtain CAMEL-specific CD4+ T cell clones, T cell cultures were seeded at 1 cell/well in 96-well U-bottom plates, each well containing 5 x 103 irradiated, autologous DC pulsed with recombinant CAMEL, 105 irradiated allogeneic PBMC, 5 x 103 irradiated allogeneic EBV-B cells and 1 µg/ml leucoagglutinin (Sigma-Aldrich) and 150 U/ml IL-2. Because many T cell clones isolated from donor 1 were shown to be specific for bacterial Ags, the T cell clones from donor 2 were stimulated with autologous DC pulsed with CAMEL peptide mix 1 and mix 2 instead of recombinant CAMEL protein. Growing clones were weekly restimulated as previously described.

IFN-{gamma} ELISPOT assay

IFN-{gamma} ELISPOT assays were performed as previously described with small modifications (23). Ninety six-well nylon Silent Screen plates (Nalge Nunc International, Rochester, NY) were coated with 100 µl of a mouse mAb against human IFN-{gamma} (1-D1K; Mabtech, Nacka, Sweden) diluted to 5 µg/ml in PBS overnight at 4°C. Wells were washed with PBS and blocked with 50 µl IMDM with 5% HS for 1 h at 37°C. T cells (1–5 x 104) were incubated together with target cells (1–2 x 104 cells/well) in duplicate. For blocking studies, target cells were preincubated with Abs against HLA class I (w6/32), HLA-DR (B8.11.2), HLA-DQ (SPV-L3), or HLA-DQ (B7.21) (24, 25) for 30 min at 37°C. After overnight incubation at 37°C, wells were washed in PBS/0.05% Tween 20 and incubated with 100 µl of a biotinylated Ab against human IFN-{gamma} (7-B6-1-biotin; Mabtech) diluted at 0.3 µg/ml in PBS for 2 h at room temperature. After several washes, wells were incubated with complexes between avidin and biotinylated HRP (Vectastain ABC system; Vector Laboratories, Burlingame, CA) for 1 h at 37°C and subsequently with 100 µl substrate solution containing 3-amino-9-ethylcarbazole (Sigma-Aldrich). Alternatively, wells were incubated with 100 µl streptavidin-alkaline phosphatase (Mabtech) in PBS/0.5% FCS for 1 h at room temperature and subsequently with substrate solution (alkaline phosphatase-conjugate substrate kit; Bio-Rad, Hercules, CA). Colorimetric reactions were stopped under running tap water and spots were counted by computer-associated video image analysis using KS ELISPOT software release 4.1 (Carl Zeiss Vision, Hallbergmoos, Germany).

IL-13 ELISPOT assay

The IL-13 ELISPOT assay was performed as previously described (13). Briefly, 96-well ELISA plates (Greiner, Alphen aan den Rijn, The Netherlands) were coated with 100 µl of an Ab against human IL-13 (QS-13; U-CyTech, Utrecht, The Netherlands) diluted to 10 µg/ml in PBS overnight at 4°C. Plates were washed several times with PBS/0.05% Tween 20 and blocked with 50 µl/well PBS/1% BSA for 1 h at 37°C. T cells (1–5 x 104 cells/well) were seeded together with target cells (1–2 x 104 cells/well) in duplicate. For blocking studies, target cells were preincubated with Abs against HLA class I (w6/32), HLA-DR (B8.11.2), HLA-DQ (SPV-L3), or HLA-DP (B7.21) (24, 25). After 5 h at 37°C, wells were incubated with 200 µl ice-cold deionized water on melting ice for 10 min, washed several times and subsequently incubated with 100 µl of a biotinylated polyclonal Ab against human IL-13 (U-CyTech) diluted in PBS/1% BSA overnight at 4°C. After several washes, wells were incubated with 50 µl of a gold-labeled anti-biotin Ab for 1 h at 37°C. Wells were washed several times and incubated with 30 µl activator mix (U-CyTech) until silver salt precipitates were formed at the site of gold clusters. Plates were washed and spots were counted using light microscopy.

Cloning of TCR {alpha}- and {beta}-chain

TCR {alpha}-chain and {beta}-chain were identified as described before (26). The complete coding sequences of the TCR {alpha}- and {beta}-chains were amplified using the following specific primers containing the ATG start site: V{alpha}3 ATG, 5'-CCACCATGGAAACTCTCCTGGGAG-3' in combination with C{alpha}, 5'-GATGGCGGAGGCAGTCTCTG-3' for the TCR {alpha}-chain and V{beta}5 ATG, 5'-TGCCATGGGCTCCAGGCTGC-3' in combination with C{beta}1, 5'-TCCAGGGCTGCCTTCAGAAATCC-3' for the TCR {beta}-chains. PCR products were cloned into TA-cloning vector pCR2.1-topo (Invitrogen) and sequenced (BaseClear, Leiden, The Netherlands).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
For the induction of CAMEL-specific CD4+ Th cells, monocyte-derived DC from two healthy donors were pulsed with bacterially expressed recombinant CAMEL protein. T cell cultures were weekly restimulated with autologous, mature DC pulsed with recombinant CAMEL and tested at day 14 against three mixes of overlapping peptides covering the entire CAMEL protein sequence in IFN-{gamma} (Th1) and IL-13 (Th2) ELISPOT assays. Peptide mix 1 consisted of three 20-mer peptides (CAMEL1–20, CAMEL14–33, and CAMEL46–65) with predicted binding motifs for several HLA-DR alleles (data not shown), whereas peptide mixes 2 and 3 contained four 22-mer peptides covering aa 21–72 and aa 61–109 of the CAMEL protein sequence, respectively. As shown in Fig. 1, the T cell culture of donor 1 recognized autologous DC pulsed with CAMEL peptide mix 3, whereas autologous DC pulsed with CAMEL peptide mix 1 and to a lesser extent CAMEL peptide mix 2 were recognized by the T cell culture of donor 2. Fig. 1 shows the number of spots produced in IFN-{gamma} ELISPOT assays, but similar results were obtained in IL-13 ELISPOT assays (data not shown). For further analysis of the recognized CAMEL epitope(s), several CD4+ T cell clones were isolated from the T cell cultures of donor 1 and donor 2 by limiting dilution. Two CD4+ T cell clones (clones 1/21 and 1/53) of donor 1 and four CD4+ T cell clones (clones 2/12, 2/33, 2/52, and 2/54) of donor 2 were isolated.



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  		FIGURE 1. Induction of CAMEL-specific T cells by DC pulsed with recombinant CAMEL protein. T cell cultures of donor 1 (A) and donor 2 (B) stimulated with autologous, mature DC pulsed with recombinant CAMEL protein were tested at day 14 against autologous DC, autologous DC pulsed with 10 µg/ml recombinant CAMEL protein for 2 h at 37°C, and autologous DC pulsed with CAMEL peptide mix 1 (CAMEL1–20, CAMEL14–33, and CAMEL46–65), mix 2 (CAMEL21–42, CAMEL31–52, CAMEL41–62, and CAMEL51–72), or mix 3 (CAMEL61–82, CAMEL71–92, CAMEL81–102, and CAMEL88–109) for 2 h at 37°C (10 µg/ml each peptide). T cells (104 cells/well) were incubated with the target cells (104 cells/well) in duplicate in IFN-{gamma} ELISPOT assays.

 
To determine the CAMEL epitope(s) recognized, the CD4+ T cell clones 1/21 and 1/53 of donor 1 were tested against autologous EBV-B cells pulsed with the individual peptides of mix 3. Both clones recognized the CAMEL81–102 peptide and produced IFN-{gamma} as well as IL-13, as illustrated for clone 1/21 in Fig. 2A. CAMEL81–102 is a naturally processed epitope because the CD4+ T cell clones 1/21 and 1/53 also recognized DC pulsed with recombinant CAMEL protein. Recognition of the CAMEL81–102 peptide could be blocked by Abs against HLA-DR, whereas Abs against HLA-DQ, HLA-DP, and HLA class I had no effect (Fig. 2B). Pulsing CAMEL81–102 on EBV-B cells sharing one or more HLA-DR alleles with donor 1 demonstrated that the CD4+ T cell clones 1/21 and 1/53 both recognize the CAMEL81–102 epitope in the context of HLA-DR7 (illustrated for clone 1/21 in Fig. 3). Whether the CAMEL81–102 epitope is also presented by tumor cells could not be investigated because HLA-DR7-positive, CAMEL-expressing tumor cells were not available.



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  		FIGURE 2. CD4+ T cell clone 1/21 of donor 1 recognizes a naturally processed CAMEL81–102 epitope presented in HLA-DR. A, CD4+ T cell clone 1/21 was tested against autologous EBV-B cells, autologous EBV-B cells pulsed with single CAMEL peptides of mix 3 (10 µg/ml) for 2 h at 37°C, autologous DC, and autologous DC pulsed with recombinant CAMEL protein as described in Materials and Methods. Target cells (104 cells/well) were seeded together with CD4+ T cell clone 1/21 (104 cells/well) in duplicate in IFN-{gamma} and IL-13 ELISPOT assays. B, CD4+ T cell clone 1/21 was tested against autologous EBV-B cells and autologous EBV-B cells pulsed with CAMEL81–102 for 2 h at 37°C, preincubated with Abs against HLA class I, HLA-DR, HLA-DQ, and HLA-DP for 30 min at 37°C. Target cells (104 cells/well) were incubated together with CD4+ T cell clone 1/21 (104 cells/well) in duplicate in IFN-{gamma} ELISPOT assays.

 


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  		FIGURE 3. CD4+ T cell clone 1/21 recognizes the CAMEL81–102 epitope in the context of HLA-DR7. EBV-B cells expressing one or more HLA-DR alleles in common with donor 1 (DR3, DR7, DR52, and/or DR53) were pulsed with 10 µg/ml CAMEL81–102 for 2 h at 37°C. Target cells (104 cells/well) were seeded together with CD4+ T cell clone 1/21 (104 cells/well) in duplicate in IFN-{gamma} ELISPOT assays. HLA-DR alleles that are shared with those of donor 1 are depicted in bold.

 
The CD4+ T cell clones 2/12, 2/33, 2/52, and 2/54 of donor 2 were tested against autologous EBV-B cells pulsed with single CAMEL peptides of mix 1 and 2 (Fig. 4A). Clones 2/12 and 2/33 were shown to recognize the CAMEL46–65 and CAMEL1–20 peptides, respectively, whereas clones 2/52 and 2/54 both recognized the CAMEL14–33 peptide. All four CD4+ T clones recognized autologous DC pulsed with recombinant CAMEL protein, demonstrating that CAMEL1–20, CAMEL14–33, and CAMEL46–65 are naturally processed epitopes. CD4+ T cell clones 2/12, 2/52, and 2/54 released IFN-{gamma} as well as IL-13, whereas CD4+ T cell clone 2/33 only produced IL-13 and not or hardly IFN-{gamma} (Fig. 4A). For all four CD4+ T cell clones, peptide recognition was inhibited by Abs against HLA-DR, but not by Abs against HLA-DQ, HLA-DP, or HLA class I (Fig. 4B), indicating that the CAMEL epitopes are presented in HLA-DR. For further analysis of the restriction elements, CD4+ T cell clones 2/52 and 2/54 were tested against a panel of CAMEL14–33-pulsed EBV-B cells sharing one or more HLA-DR alleles with donor 2. Fig. 5 shows that CD4+ T cell clone 2/52 as well as clone 2/54 specifically produced IFN-{gamma} upon stimulation with the CAMEL14–33 peptide in the context of HLA-DR3. The CD4+ T cell clones 2/12 and 2/33 failed to grow and thus could not be further analyzed for their restriction elements.



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  		FIGURE 4. CD4+ T cell clones 2/12, 2/33, 2/52, and 2/54 of donor 2 recognize naturally processed CAMEL epitopes in HLA-DR. A, CD4+ T cell clones 2/12, 2/33, 2/52, and 2/54 were tested against autologous EBV-B cells, autologous EBV-B cells pulsed with single CAMEL peptides (10 µg/ml) for 2 h at 37°C, autologous DC, and autologous DC pulsed with recombinant CAMEL protein as described in Materials and Methods. Target cells (104 cells/well) were seeded together with the CD4+ T cell clones (104 cells/well) in duplicate in IFN-{gamma} and IL-13 ELISPOT assays. B, CD4+ T cell clones were tested against autologous EBV-B cells and autologous EBV-B cells pulsed with CAMEL46–65 (clone 2/12), CAMEL1–20 (clone 2/33), or CAMEL14–33 (clones 2/52 and 2/54) for 2 h at 37°C, preincubated with Abs against HLA class I, HLA-DR, HLA-DQ, and HLA-DP for 30 min at 37°C. Target cells (104 cells/well) were incubated together with the CD4+ T cell clones (104 cells/well) in duplicate in IFN-{gamma} (clones 2/12, 2/52 and 2/54) and IL-13 (clone 2/33) ELISPOT assays.

 


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  		FIGURE 5. CD4+ T cell clones 2/52 and 2/54 recognize the CAMEL14–33 epitope in the context of HLA-DR3. EBV-B cells expressing one or more HLA-DR alleles in common with donor 2 (DR3, DR4, DR52, and/or DR53) were pulsed with 10 µg/ml CAMEL14–33 for 2 h at 37°C. Target cells (104 cells/well) were seeded together with the CD4+ T cell clones 2/52 or 2/54 (104 cells/well) in duplicate in IFN-{gamma} ELISPOT assays. HLA-DR alleles that are shared with those of donor 2 are depicted in bold.

 
To investigate whether the CAMEL14–33 epitope is presented on the surface of tumor cells, a panel of melanoma cell lines was tested for recognition by CD4+ T cell clones 2/52 and 2/54. Fig. 6 shows that CD4+ T cell clone 2/54 recognized all HLA-DR3-positive, CAMEL-expressing melanoma cell lines but not HLA-DR3-negative and/or CAMEL-negative melanoma cell lines, further confirming recognition of an endogenously processed HLA-DR3-binding CAMEL epitope. In contrast to clone 2/54, CD4+ T cell clone 2/52 did not produce IFN-{gamma} upon stimulation with melanoma cell lines that expressed HLA-DR3 and CAMEL (Fig. 6), nor granzyme B, TNF-{alpha}, IL-4, and IL-13 (data not shown).



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  		FIGURE 6. Recognition of HLA-DR3-positive, CAMEL-expressing melanoma cell lines by CD4+ T cell clones 2/54 and 2/52. The CD4+ T cell clones 2/52 and 2/54 were tested against a panel of melanoma cell lines and autologous EBV-B cells pulsed with 10 µg/ml CAMEL14–33 for 2 h at 37°C. The HLA-DR surface expression, as determined by FACS analysis, and the CAMEL mRNA expression, as determined by RT-PCR, are indicated. Target cells (104 cells/well) were seeded together with CD4+ T cell clones 2/52 or 2/54 (104 cells/well) in duplicate in IFN-{gamma} ELISPOT assays.

 
To determine whether the observed difference in tumor cell recognition between CD4+ T cell clones 2/52 and 2/54 is caused by a different affinity for the HLA-DR3-peptide complex, HLA-DR3-expressing EBV-B and tumor cells were pulsed with titrated concentrations of the CAMEL14–33 peptide and tested for recognition by CD4+ T cell clones 2/52 and 2/54. The peptide titration analysis, as shown in Fig. 7A, did not reveal any difference in affinity between the two CD4+ T cell clones. To investigate whether CD4+ T cell clones 2/52 and 2/54 recognize different minimal epitopes derived from the CAMEL14–33 peptide, N- and C-terminal truncated variants of the CAMEL14–33 peptide were synthesized and tested for their ability to stimulate IFN-{gamma} release by CD4+ T cell clones 2/52 and 2/54. As shown in Fig. 7B, CD4+ T cell clones 2/52 and 2/54 recognize the same minimal CAMEL epitope: AAQERRVPR (aa 19–27). Moreover, substitution of amino acids in the minimal epitope with alanine residues had similar effects on the recognition by CD4+ T cell clones 2/52 and 2/54 (Fig. 7C), indicating that the same amino acids are contacted by the TCRs of both clones. Analysis of surface expression of CD3 and other molecules involved in T cell activation (CD2, CD4, CD25, CD28, LFA-1) did not reveal any difference between CD4+ T cell clones 2/52 and 2/54 (data not shown). In addition, expression of CTLA-4, CD94, and killer cell Ig-like receptors could not be detected (data not shown).



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  		FIGURE 7. CD4+ T cell clones 2/52 and 2/54 recognize the minimal CAMEL19–27 epitope (AAQERRVPR) with similar affinities. A, Autologous EBV-B cells (filled symbols) and 93.08 melanoma cells (open symbols) were pulsed with titrated concentrations of the CAMEL14–33 peptide for 2 h at 37°C. Target cells (104 cells/well) were seeded together with CD4+ T cell clones 2/52 (circles) and 2/54 (triangles) at 104 cells/well in duplicate in IFN-{gamma} ELISPOT assays. B, Autologous EBV-B cells were pulsed with N- and C-terminal truncation variants of the CAMEL14–33 peptide at 10 µg/ml for 2 h at 37°C and seeded at 104 cells/well together with the CD4+ T cell clones 2/52 or 2/54 (104 cells/well) in duplicate in IFN-{gamma} ELISPOT assays. C, Autologous EBV-B cells were pulsed with variants of the CAMEL15–31 peptide containing single amino acids of the minimal CAMEL19–27 (AAQERRVPR) epitope substituted for alanine residues at 10 µg/ml for 2 h at 37°C. Peptide-pulsed EBV-B cells (104 cells/well) were seeded together with the CD4+ T cell clones 2/52 and 2/54 (104 cells/well) in duplicate in IFN-{gamma} ELISPOT assays.

 
To investigate whether CD4+ T cell clones 2/52 and 2/54 express different TCRs, TCRV{beta} expression was analyzed using a panel of Abs recognizing 24 different TCRV{beta}. FACS analysis demonstrated that CD4+ T cell clones 2/52 and 2/54 both expressed TCRV{beta}5.1 (data not shown). Sequencing of the TCR {alpha}- and {beta}-chains demonstrated that the CD4+ T cell clones 2/52 and 2/54 express identical TCR {beta}-chain, but revealed expression of different TCR {alpha}-chain (Table I). In the TCR {alpha}-chain of clone 2/52, the AV3S1 gene is combined with the AJ39 gene, whereas in the TCR {alpha}-chain of clone 2/54 the AJ-48 gene is incorporated, indicating that CD4+ T cell clones 2/52 and 2/54 express TCRs that differ in 10 aa in the complementarity-determining region (CDR)3{alpha} loop of the TCR {alpha}-chain.


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  		Table I. CDR3 regions of the TCR expressed by clones 2/52 and 2/54a

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have shown that Ag-specific CD4+ Th1 cells producing IFN-{gamma} and IL-2 are required for the efficient activation and maintenance of CD8+ T cell-mediated immune responses (1, 2, 3, 4, 5, 6, 7, 8, 9). Therefore, induction of tumor-specific CD4+ Th1 cells should be addressed in the development of immunotherapy for cancer. In this study, CD4+ T cells specific for the alternatively translated tumor Ag CAMEL were induced in vitro by mature DC pulsed with bacterially expressed recombinant CAMEL protein in two healthy donors. Because CAMEL is not expressed in normal tissues, these CAMEL-specific CD4+ T cells could be considered as induced by in vitro priming of naive CD4+ T cells by DC pulsed with recombinant CAMEL protein.

It has been reported by Chaux et al. (27) that stimulation with DC pulsed with bacterially expressed recombinant MAGE-3 protein resulted in strong activation of T cells specific for bacterial contaminants. In our experiments, T cells specific for bacterial components were also stimulated by DC pulsed with recombinant CAMEL protein, as demonstrated by recognition of a bacterially expressed control protein (data not shown). However, the T cell cultures of both donors were also shown to contain T cells specific for CAMEL peptides, demonstrating that CAMEL-specific T cells can still be generated even in the presence of T cells reactive with bacterial Ags.

The isolation of CD4+ T cell clones from the T cell cultures stimulated with DC pulsed with recombinant CAMEL protein led to the identification of four naturally processed, HLA-DR-binding CAMEL epitopes: CAMEL1–20, CAMEL14–33, CAMEL46–65, and CAMEL81–102. The HLA-DR restriction elements of the CAMEL14–33- and CAMEL81–102-specific CD4+ T cell clones were HLA-DR3 and HLA-DR7, respectively. These HLA-DR alleles are expressed in 15–25% (HLA-DR3) and 25–30% (HLA-DR7) of the study population. We recently demonstrated that the CAMEL81–102 peptide is recognized in the context of HLA-DR11 and HLA-DR12 by CD4+ T cell clones isolated from peripheral blood of a melanoma patient (13). These HLA-DR11 and HLA-DR12-restricted CD4+ T cell clones and the HLA-DR7-restricted CD4+ T cell clone described in this study, both recognize the same minimal CAMEL86–93 (PWKRSWSA) epitope (Ref. 13 and data not shown), indicating that CD4+ T cells can be stimulated by the CAMEL86–93 epitope in the context of various HLA-DR alleles.

The HLA-DR11- and HLA-DR12-restricted, CAMEL81–102-specific CD4+ T cell clones isolated from a melanoma patient have been reported to belong to the Th2 subtype, producing high levels of IL-4, IL-5, and IL-13 (13). Tumor Ag-specific CD4+ Th2 cells are probably not sufficient for tumor eradication because Th2-type immunity has been described to correlate with active disease in melanoma and renal cell carcinoma patients, whereas Th1 or mixed Th1/Th2 immunity was displayed in disease-free patients (12). It can be speculated that in cancer patients tumor Ag-specific Th2 cells are induced by DC impaired in their maturation, whereas fully mature DC are required for Th1 immunity and CD8+ T cell-mediated antitumor responses. In our study, five of the six CD4+ T cell clones produce Th1 as well as Th2 cytokines. The generation of CD4+ T cell clones that produce Th1 cytokines might be attributed to the fully mature DC that were used for induction.

Because melanomas have been described to express MHC class II molecules (28), CD4+ T cells may directly recognize epitopes processed and presented in the MHC class II pathway of tumor cells. Therefore, the CAMEL14–33-specific CD4+ T cell clones 2/52 and 2/54 were both tested for recognition of HLA-DR3-positive, CAMEL-expressing tumor cells. Whereas CD4+ T cell clones 2/52 and 2/54 were both shown to recognize the exogenously pulsed CAMEL14–33 peptide with similar affinities, only CD4+ T cell clone 2/54, and not clone 2/52, recognized HLA-DR3-positive CAMEL-expressing tumor cells. Lack of tumor cell recognition by CD4+ T cell clone 2/52 could not be explained by expression of inhibitory NK receptors as described by others (29). A difference in cell surface expression of costimulatory and adhesion molecules between the CD4+ T cell clones could not be demonstrated. However, despite similar expression, differential engagement of these molecules in tumor cell recognition by CD4+ T cell clones 2/52 and 2/54 cannot be excluded. Interestingly, like our CD4+ T cell clones 2/52 and 2/54, CD4+ T cells specific for the immunodominant epitope of hen-egg white lysozyme (HEL) have been described to differ in recognition of endogenously processed HEL epitopes, whereas exogenously pulsed HEL epitopes were similarly recognized (30). In this study, however, the difference in recognition of endogenously processed epitopes could be explained by TCRs that make different contacts with residues of the HEL epitope (31), whereas the TCRs of CD4+ T cell clones 2/52 and 2/54 were shown to contact similar residues of the minimal CAMEL19–27 epitope.

TCR analysis demonstrated that CD4+ T cell clones 2/52 and 2/54 express identical TCR {beta}-chains, but different TCR {alpha}-chains with different incorporated J gene segments. The junction of the V gene and J gene segment encodes the CDR3 of the TCR {alpha}-chain (32). Crystal structures of TCR-MHC peptide complexes revealed that CDR3 loops play a role in the interaction of the TCR with antigenic peptide bound to the MHC molecule (33, 34, 35). Therefore, the difference in tumor cell recognition between CD4+ T cell clones 2/52 and 2/54 might be explained by the 10 different amino acids in the CDR3 loop of the TCR {alpha}-chain. Introduction of the TCRs of CD4+ T cell clones 2/52 and 2/54 into a proper recipient cell line should reveal whether the different CDR3{alpha} loops are important for tumor cell recognition.

Thus far, two HLA class I-restricted epitopes that are shared by the alternatively translated CAMEL and NY-ESO-ORF2 proteins have been identified, i.e., the HLA-A*0201-restricted CAMEL1–11 epitope (18) and the HLA-A31-restricted CAMEL18–27 epitope (36). Therefore, the CAMEL1–20 and CAMEL14–33 peptides not only contain HLA-DR-binding CD4+ T cell epitopes, as described in this study, but also HLA class I-binding CD8+ T cell epitopes. Because several studies have shown that synthetic peptides comprising overlapping Th and CTL epitopes efficiently induce tumor Ag-specific CD4+ T cell responses and powerful, long-lasting tumor Ag-specific CD8+ T cell responses (37, 38, 39), the CAMEL/NY-ESO-ORF2 peptides harboring CD4+ and CD8+ T cell epitopes are interesting peptides to investigate for future immunotherapy of cancer.


   Acknowledgments
 
We thank Monique ten Dam and Arend Mulder for providing Abs against costimulatory and adhesion molecules, and Jeroen van Bergen for performing the FACS analysis for expression of NK receptors.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Susanne Osanto, Department of Clinical Oncology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail address: s.osanto{at}lumc.nl Back

2 Abbreviations used in this paper: DC, dendritic cell; ORF, open reading frame; HS, human serum; HEL, hen-egg white lysozyme; CDR, complementarity-determining region. Back

Received for publication July 14, 2003. Accepted for publication February 6, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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