Materials and methods

Cell lines

COS-1 cells and melanoma cell lines FM6 (gift from J Zeuthen, Copenhagen, Denmark), SK23 (gift from T Wölfel, Mainz, Germany) and 518A2, were cultured in Dulbecco's Modified Eagle Medium (Invitrogen, Breda, The Netherlands) supplemented with 8% heat-inactivated fetal calf serum (FCS), 4 mM L-glutamine, 50 g/ml penicillin and 50 g/ml streptomycin. EBV-transformed B-lymphoblastic cell lines (EBV-B cells) were grown in RPMI-1640 (Invitrogen) with FCS, L-glutamine and antibiotics.

Peptides and recombinant protein

A total of 11 partially overlapping 20- and 22-mer peptides comprising the entire 109-aa CAMEL protein sequence were synthesized by solid-phase methods, using an automated multiple peptide synthesizer (Abimed AMS 422; Abimed Analysen-Technik, Langenfeld, Germany) and Fmoc chemistry. After reverse-phase HPLC analysis, peptides were dissolved in dimethylsulfoxide 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 (rCAMEL) was produced in a bacterial expression system. Briefly, a 349 bp LAGE-1 cDNA fragment was cloned in frame with an N-terminal (His)₁₀-tag into the pET19b vector (Novagen, Madison, WI). This 349 bp fragment contains only the coding sequence for the alternatively translated CAMEL protein due to deletion of the first ATG start site. Escherichia coli strain BL21(DE3) (Stratagene, Amsterdam, The Netherlands) were transformed with pET19b-CAMEL and expression of the vector was induced by addition of 1 mM isopropyl--D-thiogalactoside (IPTG). After 4 hours, rCAMEL was purified from bacterial lysate by nickel-chelate chromatography using nickel–nitrilotriacetic acid agarose according to the manufacturer's instructions (Westburg, Leusden, The Netherlands). rCAMEL was eluted in 8 M urea, 100 mM NaH₂PO₄ and 10 mM Tris, pH 4.5 and dialyzed in distilled water. After lyophilization, rCAMEL was dissolved in distilled water at 1 mg/ml and stored at -70°C.

Adenoviral constructs

Ad5F35-CAMEL is a recombinant Ad5 vector modified to express the fiber shaft and knob of serotype Ad35 containing a 349 bp LAGE-1 cDNA fragment only coding for the alternatively translated CAMEL protein due to deletion of the first ATG start site. Ad5F35-CAMEL was generated and purified as described previously.^26,39 Briefly, PER.C6™ cells were cotransfected with a cosmid (pWE/Ad.AflII-rITR.Pac/Fib35) linearized with PacI and a plasmid (pAdapt) encoding 5 kb of Ad5 sequence of the left side of the Ad5 genome, whereby the E1 region is replaced by an expression cassette. The CAMEL antigen is under the control of the CMV promoter/enhancer. Recombinant virus was purified by cesium chloride density gradient centrifugation and the titer of the virus batch was determined by HPLC as described before.⁴⁰

The Ad5-LAGE-1 and Ad5-NY-ESO-1 vectors are Ad5 vectors containing a 780 bp LAGE-1 and a 950 bp NY-ESO-1 cDNA fragment, respectively. The cDNA fragments were cloned into the Ad5 vector according to the method of He et al.,⁴¹ which is characterized by a recombination step in E. coli. The Ad5 vectors lack the E1 and E3 regions. Both vectors contain a CMV promoter/enhancer to drive expression of the transgene and a separate CMV promoter/enhancer to drive expression of the GFP gene. The Ad5 vectors were propagated on packaging cell line PER.C6™.

Generation of dendritic cells

Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood obtained from two healthy donors, who were HLA-typed as A1, A^*0201, B7, B35 (donor 1) and A1, A^*0201, B8, B13 (donor 2) using standard serological techniques. PBMC were depleted for T and B cells using CD2- and CD19-dynabeads (Dynal, Oslo, Norway) and cultured in six-well plates (2 10⁶ cells/well) in AIM-V (Invitrogen) with 116 g/ml L-arginine, 36 g/ml L-asparagine and 215 g/ml L-glutamine (AAG) and antibiotics, supplemented with 500 U/ml IL-4 (PeproTech Inc., Rocky Hill, NJ) and 800 U/ml GM-CSF (Behringwerke, Marburg, Germany) for 6 days. Immature DC were harvested and seeded at 10⁶ cells/well in 24-well plate in 1 ml AIM-V containing activating anti-CD40 Ab (1 g/ml; Cymbus Biotechnology, Chandlers Ford, UK), IL-4 (500 U/ml) and GM-CSF (800 U/ml). After overnight incubation with the activating anti–CD40 Ab, Ad5F35-CAMEL vector was added to the mature DC at doses ranging from 2.5 10³ to 6 10³ virus particles (VP)/cell. After 2 hours at 37°C, 1 ml AIM-V supplemented with anti-CD40 Ab, IL-4 and GM-CSF as described above was added. The Ad5F35–CAMEL-infected, mature DC were used as stimulators 24 hours after infection. For loading with rCAMEL protein, immature DC were pulsed with 10 g/ml rCAMEL for 4 hours at 37°C and subsequently matured for 2 days in AIM-V containing the anti-CD40 Ab, IL-4 and GM-CSF as described above. For peptide pulsing, immature DC were incubated for 2 days in AIM-V containing the anti-CD40 Ab, IL-4 and GM-CSF as described above and subsequently pulsed with 10 g/ml peptide for 2 hours at 37°C.

Generation and isolation of CAMEL-specific T cells

To generate CAMEL-specific T-cell lines, 20 10⁶ nonadherent PBMC of healthy donor 1 and 2 were incubated with autologous DC infected with Ad5F35-CAMEL or pulsed with the rCAMEL protein at ratios ranging between 5:1 and 10:1 at 2 10⁶ PBMC/well in 24-well plates containing 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, Zwijndrecht, The Netherlands). T-cell cultures were weekly restimulated as described above in IMDM with 5% HS, AAG and antibiotics, supplemented with 150 U/ml IL-2. At day 36, the T-cell line stimulated with DC infected with Ad5F35-CAMEL of healthy donor 1 was seeded at 1 cell/well in 96-well U-bottom plates to generate T-cell clones. Each well contained 10⁵ irradiated, allogeneic PBMC, 5 10³ irradiated, allogeneic EBV-B cells, 5 10³ irradiated, autologous DC pulsed with 2 g/ml CAMEL_41–62 and CAMEL_46–65 and 1 g/ml leucoagglutinin (Sigma-Aldrich) and 150 U/ml IL-2. Growing clones were weekly restimulated as described above.

Tetramer staining

T cells (2 10⁵) were stained with PE-conjugated HLA-A^*0201/CAMEL_1-11 tetramers (kindly provided by P Romero, Lausanne, Switzerland) in PBS containing 0.5% BSA and 0.02% NaN₃ for 30 minutes at room temperature (RT), followed by an additional incubation of 30 minutes at 4°C with anti-CD8-FITC (Becton Dickinson, Mountain View, CA). Prior to the measurement, propidium iodide was added to a final concentration of 5 g/ml to exclude dead cells during the analysis by flow cytometry (FACSCalibur, Becton Dickinson) using CellQuest software.

IFN- ELISPOT assay

IFN- ELISPOT assays were performed as described previously with small modifications.⁴² Briefly, 96-well nylon Silent Screen plates (Nalge Nunc International Corporation, Rochester, NY) were coated with 100 l of a mouse monoclonal antibody against human IFN- (1-D1 K, Mabtech AB, Nacka, Sweden) diluted to 5 g/ml in PBS overnight at 4°C. Wells were washed several times with PBS and blocked with 50 l/well IMDM/5% HS for 1 hour at 37°C. T cells (1–2 10⁴ cells/well) were incubated with target cells (1–2 10⁴ cells/well) in duplicate. For blocking studies, target cells were preincubated with antibodies against HLA class I (w6/32) or HLA class II (IC-2) for 30 minutes at 37°C. To inhibit proteasomal activity, target cells were pretreated with 10 M lactacystin (Sigma-Aldrich) for 2 hours at 37°C. After 4-hour incubation with 10 g/ml peptide, target cells were washed and fixed with 1% paraformaldehyde to prevent further processing. After overnight incubation at 37°C, the plates were washed several times with PBS/0.05% Tween-20 and subsequently incubated with 100 l of a biotinylated antibody against human IFN- (7-B6-1-biotin, Mabtech) diluted at 0.3 g/ml in PBS for 2 hours at RT. Wells were washed several times and incubated with 100 l streptavidin–alkaline phosphatase (Mabtech) in PBS/0.5% FCS for 1 hour at RT. After several washes, wells were incubated with 100 l substrate solution (AP-conjugate substrate kit; Bio-Rad, Hercules, CA) for 15–30 minutes at RT. The colorimetric reaction was stopped under running tap water and spots were counted using computer-assisted video image analysis with KS ELISPOT software (Carl Zeiss Vision GmbH, Hallbergmoos, Germany).

Human Th1/Th2 cytokine cytometric bead array assay

Target cells were seeded at 5 10⁴ cells/well together with 5 10⁴ T cells in a 96-well flat bottom plate overnight at 37°C in the presence of 150 U/ml IL-2. To inhibit proteasomal activity, target cells were pretreated with 10 M lactacystin (Sigma-Aldrich) for 2 hours at 37°C. After 4-hour incubation with 10 g/ml peptide, target cells were washed and fixed with 1% paraformaldehyde to prevent further processing. The release of IFN, TNF, IL-2, IL-4, IL-5 and IL-10 by the T cells in the supernatant was analyzed using the Human Th1/Th2 Cytokine Cytometric Bead Array (CBA) assay according to the manufacturer's instructions (BD Biosciences, Franklin Lakes, NJ).

Granzyme B ELISA

The release of granzyme B in the culture supernatant was measured by ELISA, according to the manufacturer's instructions (CLB, Amsterdam, The Netherlands). Briefly, 96-well ELISA plates were coated overnight at RT with monoclonal antibodies directed against human granzyme B. Wells were washed several times with PBS/0.02% Tween-20 and subsequently blocked with kit buffer for 1 hour at RT. Wells were incubated with serial dilutions of culture supernatants for 1 hour at RT. After several washes, 100 l biotinylated antibody against human granzyme B was added for 1 hour at RT. Wells were washed several times and incubated with streptavidin–poly-HRP conjugate for 30 minutes at RT. After washing, wells were incubated with 100 l substrate solution. The reaction was stopped by addition of 100 l 0.18 M sulfuric acid solution and OD450 was measured using a microplate reader (Wallac, Turku, Finland).

Results

Induction of HLA-A^*0201-restricted CAMEL/NY-ESO-ORF2_1–11-specific CD8⁺ T cells by DC infected with Ad5F35-CAMEL

Nonadherent PBMC from two HLA-A^*0201-positive healthy donors (donors 1 and 2) were incubated with autologous DC infected with Ad5F35-CAMEL or pulsed with recombinant CAMEL protein (rCAMEL). T-cell cultures were weekly restimulated with autologous DC infected with Ad5F35-CAMEL or pulsed with rCAMEL protein and tested for the presence of CD8⁺ T cells specific for the previously described HLA-A^*0201-binding epitope CAMEL/NY-ESO-ORF2_1–11³³ by tetramer staining. In both donor 1 (Fig 1) and donor 2 (Fig 2), CAMEL/NY-ESO-ORF2_1–11-specific CD8⁺ T cells could be obtained when DC infected with Ad5F35-CAMEL were used for stimulation, but not when DC pulsed with rCAMEL protein were used. CAMEL/NY-ESO-ORF2_1-11-specific CD8⁺ T cells were not detectable in nonstimulated PBMC, raised to maximum numbers after 14 days of stimulation (donor 1: 0.45%, donor 2: 0.13%) and gradually decreased at day 21 to undetectable levels at day 28.

Figure 1.

Detection of HLA-A^*0201/CAMEL_1–11-specific CD8⁺ T cells in T-cell cultures of donor 1. T cells of donor 1 stimulated with DC infected with Ad5F35-CAMEL or pulsed with rCAMEL protein were double-stained with PE-labeled HLA-A^*0201/CAMEL_1–11-tetramers and CD8-FITC and analyzed by flow cytometry. The percentages of CD8⁺ T cells that are tetramer-positive are indicated.

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Figure 2.

Detection of HLA-A^*0201/CAMEL_1–11-specific CD8⁺ T cells in T-cell cultures of donor 2. T cells of donor 2 stimulated with DC infected with Ad5F35-CAMEL or pulsed with rCAMEL protein were double-stained with PE-labeled HLA-A^*0201/CAMEL_1–11-tetramers and CD8-FITC and analyzed by flow cytometry. The percentages of CD8⁺ T cells that are tetramer-positive are indicated.

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Generation of a CAMEL_46–54-specific CD8⁺ T-cell line by DC infected with Ad5F35-CAMEL

The T-cell cultures at days 14 and 21 that were shown to contain CAMEL/NY-ESO-ORF2_1–11-specific T cells after stimulation with DC infected with Ad5F35-CAMEL were also screened for T cells specific for unknown CAMEL epitopes in IFN- ELISPOT assays. The Ad5F35-CAMEL-stimulated T-cell cultures were tested against autologous DC infected with Ad5F35-CAMEL and, as a control, empty Ad5F35-vector and autologous DC pulsed with mixes of overlapping peptides covering the entire CAMEL protein sequence. Peptide mix 1 contained three 20-mer peptides (CAMEL_1–20, CAMEL_14–33 and CAMEL_46–65) with predicted binding motifs for various HLA alleles (data not shown), whereas peptide mixes 2 and 3 each consisted of four overlapping 22-mer peptides covering aa 21–72 and aa 61–109 of the CAMEL protein sequence, respectively. At day 14, T-cell cultures of donor 1 (Fig 3a) and donor 2 (Fig 3b) both produced IFN- upon stimulation with DC infected with Ad5F35-CAMEL as well as Ad5F35-empty, demonstrating the presence of adenovirus-specific T cells. T cells specific for CAMEL-derived peptides could not be detected in both donors at day 14. At day 21, the adenovirus-specific T cells were hardly detectable in the T-cell cultures of both donors, as demonstrated by low numbers of IFN- spots produced upon stimulation with DC infected with Ad5F35-empty. The T-cell culture of donor 1, however, specifically released high amounts of IFN- upon stimulation with DC infected with Ad5F35-CAMEL (Fig 3a), strongly suggesting the presence of CAMEL-specific T cells. The release of IFN- was also observed upon stimulation with DC pulsed with mixes of CAMEL-derived peptides, further supporting the presence of CAMEL-specific T cells in the Ad5F35-CAMEL-stimulated T-cell culture of donor 1.

Figure 3.

Detection of CAMEL-specific T cells in Ad5F35-CAMEL-stimulated T-cell cultures of donors 1 and 2 by the IFN- ELISPOT assay. T-cell cultures of donor 1 (a) and donor 2 (b) stimulated with DC infected with Ad5F35-CAMEL were tested at day 14 and 21 against autologous DC infected with 4 10³ VP/cell Ad5F35-CAMEL or Ad5F35-empty, as described in Materials and Methods, and autologous DC pulsed with CAMEL peptide mix 1 (CAMEL_1–20, CAMEL_14–33 and CAMEL_46–65), mix 2 (CAMEL_21–42, CAMEL_31–52, CAMEL_41–62 and CAMEL_51–72) or mix 3 (CAMEL_61–82, CAMEL_71–92, CAMEL_81–102 and CAMEL_88–109) for 2 hours at 37°C (10 g/ml of each peptide). The Ad5F35-CAMEL-stimulated T cells of donor 1 (10⁴ cells/well) and donor 2 (2 10⁴ cells/well) were incubated with the target cells (10⁴ cells/well) in duplicate in IFN- ELISPOT assays.

Full figure and legend (36K)

To determine the epitope(s) recognized by the Ad5F35-CAMEL-stimulated T-cell culture of donor 1, recognition of DC pulsed with the single CAMEL peptides was measured in an IFN- ELISPOT assay. As shown in Figure 4a, two partially overlapping peptides, CAMEL_41–62 and CAMEL_46–65, stimulated IFN- production by the Ad5F35-CAMEL-stimulated T-cell line. Recognition of these 22- and 20-mer peptides was inhibited by anti-HLA class I, but not by anti-HLA class II, antibodies strongly suggesting the presence of CD8⁺ T cells specific for a previously unknown HLA-class I-binding CAMEL epitope. Screening the CAMEL_41–62 and CAMEL_46–65 peptides for binding motifs for the HLA class I alleles of donor 1 (HLA-A1, A^*0201, B7, B35) revealed the presence of two predicted HLA-B7-binding motifs: APRGVRMAV at aa 46–54 and GVRMAVPLL at aa 49–57. Two 9-mer peptides containing the predicted HLA-B7-binding motifs (CAMEL_46–54 and CAMEL_49–57) were synthesized and tested for recognition by the Ad5F35-CAMEL-stimulated T-cell line of donor 1. Figure 4b demonstrates the specific release of IFN- upon stimulation with DC pulsed with CAMEL_46–54, but not CAMEL_49–57, indicating that CD8⁺ T cells specific for a new HLA-B7-binding CAMEL_46–54 epitope were induced by DC infected with Ad5F35-CAMEL.

Figure 4.

The Ad5F35-CAMEL-stimulated T-cell line of donor 1 contains CD8⁺ T cells specific for a new HLA-B7-binding CAMEL_46-54 epitope. (a) Ad5F35-CAMEL-stimulated T cells of donor 1 (5 10³ cells/well) were incubated with autologous DC infected with Ad5F35-CAMEL as described in Materials and methods or autologous DC pulsed with single CAMEL peptides (10 g/ml) in duplicate in an IFN- ELISPOT assay. (b) Ad5F35-CAMEL-stimulated T cells of donor 1 (5 10³ cells/well) were incubated with autologous DC infected with Ad5F35-CAMEL or autologous DC pulsed with CAMEL peptides (10 g/ml) in duplicate in an IFN- ELISPOT assay.

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Crosspresentation of CAMEL_46–54 into HLA-B7 by pulsing antigen-presenting cells with long synthetic peptides

Pulsing DC with the long 22- and 20-mer CAMEL_41–62 and CAMEL_46–65 peptides clearly leads to processing and presentation of the epitope CAMEL_46–54 into HLA-B7 (Fig 4a). We next tested whether crosspresentation in HLA class I also occurs in nonprofessional antigen-presenting EBV–B cells. HLA-B7-expressing DC and EBV-B cells were pulsed with rCAMEL protein, the long 22- and 20-mer CAMEL_41–62 and CAMEL_46–65 peptides or the minimal CAMEL_46–54 peptide and were subsequently tested for recognition by the Ad5F35-CAMEL-stimulated T-cell line of donor 1. To minimize the possibility of degradation of the 20- and 22-mer peptides by serum proteases, experiments were performed in serum-free conditions.^43,44 Both DC and EBV-B cells pulsed with the long CAMEL_41–62 and CAMEL_46–65 peptides were clearly recognized by the Ad5F35-CAMEL-stimulated T-cell line (Fig 5a). Crosspresentation of the long synthetic peptides in HLA class I proved very efficient, since DC and EBV-B cells pulsed with these peptides for 4 hours were able to stimulate the Ad5F35-CAMEL-stimulated T-cell line to similar levels as DC and EBV-B cells exogenously pulsed with the minimal CAMEL_46–54 peptide. These data also show that cross-presentation of long synthetic peptides in HLA class I is more efficient than crosspresentation of the full-length protein, since DC and EBV-B cells pulsed with the rCAMEL protein were not recognized by the Ad5F35-CAMEL-stimulated T-cell line. DC and EBV-B cells pulsed with rCAMEL protein, however, were clearly recognized by an HLA-DR3-restricted, CAMEL_14–33-specific CD4⁺ T-cell clone (Fig 5b), demonstrating efficient processing and presentation of rCAMEL protein in HLA class II. As shown in Figure 6a, recognition of EBV–B cells pulsed with the long CAMEL_41–62 peptide by the Ad5F35-CAMEL-stimulated T-cell line was not reduced upon treatment with lactacystin, whereas recognition of EBV-B cells pulsed with a long PRAME_98–124 peptide by an HLA-A^*0201-restricted, PRAME_100–108-specific CD8⁺ T-cell clone was strongly inhibited. These experiments strongly suggest proteasome-independent processing of the CAMEL_46–54 epitope. This is supported by in vitro experiments, demonstrating disruption of the CAMEL_46–54 epitope due to the presence of major proteasome cleavage sites at positions 50 and 53 (data not shown).

Figure 5.

Efficient presentation of the CAMEL_46–54 epitope in HLA-B7 after processing of long synthetic peptides by both professional and nonprofessional antigen-presenting cells. (a) HLA-B7-expressing DC and EBV-B cells were pulsed with the 22-mer CAMEL_41–62 and 20-mer CAMEL_46–65 peptides (10 g/ml) and rCAMEL protein (10 g/ml) for 4 and 20 hours at 37°C or pulsed with the 9–mer CAMEL_46–54 peptide (10 g/ml) for 4 h at 37°C. Target cells (10⁴ cells/well) were seeded together with Ad5F35-CAMEL-stimulated T cells of donor 1 (10⁴ cells/well) in duplicate in an IFN- ELISPOT assay. (b) HLA-DR3-expressing DC and EBV-B cells were pulsed with the 20-mer CAMEL_14–33 peptide (10 g/ml) and rCAMEL protein (10 g/ml) for 4 and 20 hours at 37°C. Target cells (10⁴ cells/well) were seeded together with an HLA-DR3-restricted CAMEL_14–33-specific CD4⁺ T-cell clone (10⁴ cells/well) in duplicate in an IFN- ELISPOT assay.

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Figure 6.

Processing of the CAMEL_46–54 epitope is independent of the proteasome. (a) HLA-B7-expressing EBV-B cells were pretreated with 10 M lactacystin for 2 hours at 37°C and subsequently pulsed with the 9-mer CAMEL_46–54 and 22-mer CAMEL_41–62 peptides (10 g/ml) for 4 hours at 37°C. Target cells (10⁴ cells/well) were seeded together with Ad5F35-CAMEL-stimulated T cells of donor 1 (10⁴ cells/well) in duplicate in an IFN- ELISPOT assay. (b) HLA-A^*0201-expressing EBV-B cells were pretreated with 10 M lactacystin for 2 hours at 37°C and pulsed with the 9-mer PRAME_100–108 and 27-mer PRAME_98–124 peptides (10 g/ml) for 4 h at 37°C. Target cells (2 10⁴ cells/well) were incubated together with PRAME_100–108-specific CD8⁺ T cells (2 10⁴ cells/well) in a 96-well plate overnight at 37°C. Release of IL-4 in the culture supernatant was analyzed by ELISA.

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Isolation of HLA-B7-restricted CAMEL/NY-ESO-ORF2_46–54-specific CD8⁺ T-cell clones

To obtain CAMEL_46–54-specific CD8⁺ T-cell clones, the Ad5F35-CAMEL-stimulated T-cell line of donor 1 was seeded at 1 cell/well by limiting dilution. Several growing clones were isolated that recognized not only HLA-B7-positive EBV-B cells pulsed with the CAMEL_46–54 peptide but also COS-1 cells transiently transfected with the HLA-B7 and CAMEL cDNA (data not shown). The CAMEL_46–54-specific CD8⁺ T-cell clones also recognized melanoma cell line SK23 (HLA-B7-pos, LAGE/NY-ESO-1-neg) upon infection with Ad5 vectors encoding LAGE-1 and NY-ESO-1 (Fig 7) as well as melanoma cell line FM6 (HLA-B7-pos, LAGE/NY-ESO-1-pos, Fig 8), indicating that the CAMEL/NY-ESO-ORF2_46–54 epitope is a naturally processed HLA-B7-binding epitope shared by the alternatively translated CAMEL and NY-ESO-ORF2 proteins.

Figure 7.

The HLA-B7-binding CAMEL_46–54 epitope is shared by the alternatively translated CAMEL and NY-ESO-ORF2 proteins. The CAMEL_46–54-specific CD8⁺ T-cell clone 47 was tested against the HLA-B7-positive EBV–JY cell line pulsed with 10 g/ml CAMEL_46–54 peptide for 2 hours at 37°C and melanoma cell line SK23 (HLA-B7-pos, LAGE/NY-ESO-1-neg) infected with Ad5 vectors encoding the LAGE-1 and NY-ESO-1 genes, as described in Materials and methods. Melanoma cell line SK23 was treated with IFN- for 48 hours. Target cells (10⁴ cells/well) were seeded together with the CAMEL_46–54-specific CD8⁺ T-cell clone 47 (10⁴ cells/well) in duplicate in an IFN– ELISPOT assay.

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Figure 8.

CAMEL_46–54-specific CD8⁺ T cells recognize a naturally processed epitope presented by tumor cells. The CAMEL_46–54-specific CD8⁺ T-cell clone 47 was tested against the HLA-B7-positive EBV-JY cell line pulsed with the CAMEL_46–54 peptide (10 g/ml) for 2 hours at 37°C and melanoma cell lines 518A2 (HLA-B7-neg, LAGE/NY-ESO-1-pos), FM6 (HLA-B7-pos, LAGE/NY-ESO-1-pos) and SK23 (HLA-B7-pos, LAGE/NY-ESO-1-neg). The melanoma cell lines were treated with IFN- (100 U/ml) for 48 hours. Target cells (5 10⁴ cells/well) were seeded together with the CAMEL_46–54-specific CD8⁺ T-cell clone 47 (5 10⁴ cells/well) in a 96-well plate overnight at 37°C. Culture supernatant was analyzed for the presence of cytokines using the Th1/Th2 cytokine CBA assay (a) and granzyme B by ELISA (b). Background production of IFN-, IL-4 and granzyme B by CD8⁺ T-cell clone 47 has been subtracted. Specific release of IL-5 could not be measured due to high background levels. CD8⁺ T-cell clone 47 did not release TNF-, IL-10 and IL-2 upon stimulation with CAMEL_46–54.

Full figure and legend (16K)

Discussion

Since previous studies have shown that CD4⁺ T helper cells contribute to the efficient induction of CD8⁺ T-cell-mediated antitumor reponses,^17,18,19,45 delivery of the full-length tumor antigen might be more successful in immunotherapy than delivery of HLA class I-binding antigenic peptides. Moreover, in contrast to HLA class I-binding peptides, delivery of the full-length tumor antigen is broadly applicable to patients with different HLA types. Although several studies have demonstrated generation of tumor-specific CD8⁺ T cells by DC infected with Ad5 vectors encoding tumor antigens,^20,21,22 infection of DC with Ad5 vectors is rather inefficient due to lack of CAR, the high-affinity ligand for the fiber of Ad5. The chimeric Ad5F35 vector is a modified Ad5 vector, containing the fiber shaft and knob of Ad35, which binds another, yet unknown, surface receptor. The Ad5F35 vector has been shown to infect DC efficiently and therefore, higher expression levels of the transgene are achieved.^26,27 In this study, we tested the in vitro induction of CAMEL/NY-ESO-ORF2-specific T cells by DC infected with a modified Ad5 vector containing a chimeric Ad5/35 fiber.

Upon stimulation with DC infected with the chimeric Ad5F35-CAMEL vector, CD8⁺ T cells specific for the previously identified CAMEL/NY-ESO-ORF2_1–11 epitope were induced in two healthy donors. In one healthy donor, also CD8⁺ T cells specific for a new HLA-B7 binding CAMEL_46–54 epitope were raised. The CAMEL_46–54 epitope is a naturally processed epitope shared by the alternatively translated CAMEL and NY-ESO-ORF2 proteins, as demonstrated by recognition of melanoma cell line SK23 (HLA-B7-pos, LAGE/NY-ESO-1-neg) infected with Ad5 vectors encoding the LAGE-1 and NY-ESO-1 genes and melanoma cell line FM6 (HLA-B7-pos, LAGE/NY-ESO-1-pos). CAMEL-specific CD8⁺ T cells were not induced by DC pulsed with the rCAMEL protein. In previous experiments, DC pulsed with rCAMEL have been shown to induce CAMEL-specific CD4⁺ T cells (manuscript in preparation), suggesting that pulsing DC with rCAMEL protein leads to more efficient processing and presentation of CAMEL epitopes into the HLA class II than HLA class I pathway.

The numbers of CAMEL/NY-ESO-ORF2_1–11-specific CD8⁺ T cells as induced by DC infected with Ad5F35-CAMEL, reached peak levels at day 14 and gradually decreased at day 21 to undetectable levels at day 28. The CAMEL_1–11-specific CD8⁺ T cells could not be detected in IFN- ELISPOT assays (data not shown) due to frequencies that are too low to exceed the background number of spots. Despite several efforts, we have been unable to select the HLA-A^*0201/CAMEL_1–11-tetramer-positive CD8⁺ T cells by FACS sorting and therefore, lysis of tumor cells by the CAMEL/NY-ESO-ORF2_1–11-specific CD8⁺ T cells could not be investigated. Previous experiments, however, have clearly shown lysis of HLA-A^*0201- and CAMEL/NY-ESO-ORF2-expressing melanoma cell lines by an HLA-A^*0201-restricted CAMEL/NY-ESO-ORF2_1–11-specific CTL clone.³³ The reason for the gradual decrease in numbers of CAMEL/NY-ESO-ORF2_1–11-specific CD8⁺ T cells upon repeated exposure to mature DC infected with Ad5F35-CAMEL is unknown, but might be explained by activation-induced cell death due to high levels of expression of the transgene.

Like the CAMEL/NY-ESO-ORF2_1–11-specific CD8⁺ T cells, adenovirus-specific T cells were transiently stimulated by DC infected with Ad5F35-CAMEL, reaching maximum numbers at day 14 in both healthy donors. The adenovirus-specific T cells not only recognized Ad5F35-infected cells, but also cells infected with the regular Ad5 vector (data not shown), indicating that some or all adenovirus-specific T cells were directed against Ad5-derived proteins. The gradual decrease in adenovirus-specific T cells upon repeated exposure to Ad5F35-infected DC is in contrast with other reports, showing that transgene-specific T cells were not induced or overgrown by adenovirus-specific T cells.^46,47 In these studies, however, Ad2 and Ad5 constructs were used for gene transfer into DC. Therefore, as speculated for the CAMEL/NY-ESO-ORF2_1–11-specific CD8⁺ T cells, the adenovirus-specific T cells might disappear due to activation-induced cell death, mediated by mature DC expressing high levels of adenoviral proteins.

Pulsing of nonprofessional (EBV-B) and professional (DC) antigen-presenting cells with long synthetic 22- and 20-mer CAMEL_41–62 and CAMEL_46–65 peptides led to efficient processing and presentation of the 9-mer CAMEL/NY-ESO-ORF2_46–54 epitope into HLA-B7, which could not be inhibited by treatment with lactacystin. This strongly suggests proteasome-independent processing and presentation of the CAMEL/NY-ESO-ORF2_46–54 epitope in HLA-B7. Crosspresentation of CAMEL/NY-ESO-ORF2_46–54 into HLA-B7 could not be measured after pulsing EBV-B cells and DC with the rCAMEL protein, whereas these cells were clearly recognized by HLA-DR3-restricted CAMEL_14–33-specific CD4⁺ T cells. These data demonstrate that delivery of the CAMEL antigen by infection with the Ad5F35 vector or by pulsing with long synthetic peptides leads to efficient processing and presentation of CAMEL epitopes into the HLA class I pathway, whereas delivery of the full-length rCAMEL protein leads to efficient processing and presentation into the HLA class II pathway. Since CD8⁺ T cells have been suggested to be the main effector cells in tumor cell lysis, DC infected with the Ad5F35 vector encoding the tumor antigen or DC pulsed with long synthetic tumor antigenic peptides might be more successful in future T-cell-based immunotherapies than DC pulsed with the full-length tumor protein.

Our data show successful induction of CAMEL/NY-ESO-ORF2-specific CD8⁺ T cells in healthy donors by stimulation with DC infected with Ad5F35-CAMEL. CAMEL and NY-ESO-ORF2 are so-called cancer-testis antigens that are expressed in various tumor types, but silent in normal tissues, except for testis. The lack of expression of CAMEL and NY-ESO-ORF2 in normal tissues suggests that in healthy donors, primary CD8⁺ T-cell-mediated immune responses were induced by DC infected with Ad5F35-CAMEL. Since such immune responses could not be generated by DC pulsed with rCAMEL protein, our data support further investigation of the Ad5F35 vector as a vehicle for gene transfer into DC for the efficient induction of tumor antigen-specific CD8⁺ T cells in vivo.

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Acknowledgements

We thank A Zakhartchouk for cloning the measles antigen into Ad vectors and subsequent generation and in vitro testing of recombinant viruses carrying CAMEL antigen. We also thank B Vogelstein for providing the plasmids required for generation of the Ad5 vectors and RC Hoeben for propagating the Ad5 vectors encoding LAGE-1 and NY-ESO-1 on PER.C6^TM. CAM van Bergen and JHF Falkenburg are gratefully acknowledged for in vitro experiments with purified proteasomes.