ProTα(100–109), and the tumor antigen epitopes HER-2(9369), tyr(9369) (HLA-A2-restricted) , HER-2(15776) and tyr(15448) (HLA-DR4-restricted) [36, 64] were synthesized by the Fmoc (9-fluorenylmethoxycarbonyl)/tBu chemistry utilizing a multiple peptide synthesizer Syro II (MultiSynTech, Witten, Germany). Crude peptides were purified by HPLC on a reverse phase C18 Nucleosil 100-5C column (HPLC Technologies, UK) to a purity of >95%, using a linear gradient of 5.8% acetonitrile in 0.05% trifluoroacetic acid for 45 min. All peptides were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry and results were in all cases in agreement with the calculated masses. Human recombinant proTα was purchased from Alexis Biochemicals, CA, USA and passed through an Endotoxin removal column (Pierce Biotechnology). Prior to their use, all peptides and proTα were tested for endotoxin levels using the LAL chromogenic Endotoxin Quantitation kit (Pierce Biotechnology, IL, USA) according to the manufacturer’s instructions. They were endotoxin-free.
Cell lines and PBMC isolation
Human T2 cells (HLA-A*0201) were cultured in RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM Hepes, 5 μg/mL Gentamycin, 10 U/mL Penicillin and 10 U/mL Streptomycin (all from Lonza, Cologne, Germany), at 37°C, in a humidified 5% CO2 incubator.
Buffy coats were collected from HLA-A2+ and DR4+ healthy blood donors. Prior to blood draw, individuals gave their informed consent according to the regulations approved by the 2nd Peripheral Blood Transfusion Unit and Haemophilia Centre, ‘Laikon’ General Hospital Institutional Review Board, Athens, Greece. PBMCs were isolated by centrifugation over Ficoll-Histopaque (Lonza) density gradient, resuspended in X-VIVO 15 (Lonza) or cryopreserved in FBS-10% DMSO (Sigma-Aldrich Chemical Co., St Louis, MO, USA) for later use.
DC maturation and T cell stimulation
Highly enriched monocytes (>80% CD14+) were obtained from PBMCs by plastic adherence for 2 h at 37°C . Non-adherent cells were removed and cryopreserved. Monocytes were cultured for 5 days in X-VIVO 15 supplemented with 800 IU/mL recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) and 500 IU/mL recombinant human IL-4 (both from R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany). On day 5, iDCs were treated with LPS (0.5 μg/mL; Sigma-Aldrich), TNF-α (10 ng/mL; R&D Systems), proTα (160 ng/mL) or proαα(100–109) (25 ng/mL) for 1–48 h, concentrations already reported to induce DC maturation . Mature DCs were recovered at various time points for phenotypic and TLR-4 analysis by flow cytometry and immunoblotting, or were used to stimulate autologous T cells. Supernatants from 48 h matured DCs were also collected and the concentrations of TNF-α, IL-10 and IL-12 were quantified using commercially available ELISA kits (all from Life Technologies Corporation, Carlsbad, USA), according to manufacturer’s instructions. For TLR-4 neutralization experiments, iDCs were pre-incubated in the presence of anti-TLR-4 (a-TLR-4) neutralizing monoclonal antibody (mAb; clone W7C11) or an irrelevant mouse IgG1 mAb (both from InvivoGen, San Diego, USA) at a final concentration of 10 μg/mL for 1 h and further stimulated with LPS, proTα or proTα(100–109) for 48 h. TNF-α, IL-10 and IL-12 were determined in culture supernatants.
For T cell stimulation, 48 h matured DCs (1×106/mL) were pulsed with 50 μg/mL HER-2(9369) and HER-2(15776) for 6 h at 37°C, in a humidified 5% CO2 incubator in X-VIVO 15. DCs were washed twice, resuspended in X-VIVO 15 and added to autologous lymphocytes (non-adherent fraction) at a DC:lymphocyte ratio of 1:10. T cells were stimulated thrice at weekly intervals and on days 3 and 5 after each stimulation, 40 IU/mL IL-2 (Proleukin; Novartis Pharmaceuticals Ltd, UK) were added to the cultures. At the third stimulation, Golgi-Plug (1 μL/mL; Becton-Dickinson (BD) Biosciences, Erembodegem, Belgium) was added in the cultures, and 12 h later, T cells were harvested and analyzed for cytokine production by flow cytometry.
Flow cytometry analysis
For DC phenotype analysis, iDCs and mature DCs were stained for the surface molecules HLA-DR, CD80, CD83, CD86, CD11b, CD40 and CD14. Triple staining was performed using appropriate combinations of FITC-, PE- or PE-Cy5-labelled mouse anti-human IgG1 and IgG2 mAbs (BD Biosciences) at saturating concentrations for 30 min on ice. DCs were also stained with irrelevant anti-human IgG1 and IgG2 mAbs (BD Biosciences), as isotype controls. Samples were measured using a FACSCalibur flow cytometer (BD Biosciences) and data were analyzed using CellQuest software. MFI was evaluated for each marker.
For TLR-4 expression, iDCs and DCs matured with LPS, proTα or proTα(100–109) for 15 min, 30 min, 1 h, 18 h and 36 h were harvested and treated with human immunoglobulin (GAMUNEX; Bayer, Leverkusen, Germany) and ethidium monoazide (EMA; Invitrogen, Karlsruhe, Germany) to block Fc receptors and label nonviable cells, respectively. DCs were then stained with TLR-4/Brilliant Violet 421, CD11c/PE-Cy7 (both from BioLegend, San Diego, CA) and Lineage 1 cocktail/FITC (BD Biosciences) mAbs and measured immediately using LSR II or FACSCanto II and FACSDiva software (BD Biosciences). Data were analyzed using FlowJo software (TreeStar, Ashland, OR). Duplicates were excluded using the forward-scatter area versus forward-scatter height plot, TLR-4+ cells were gated within viable DCs (EMA-negative (−), CD11c + and Lineage 1-) and their MFI was determined. For TLR-4 neutralization experiments, a-TLR-4-treated iDCs were stimulated as above and stained with CD14/FITC (BioLegend) and TLR-4/Brilliant Violet 421 or PE (BioLegend) mAbs at saturating concentrations for 30 min on ice. DCs were also stained with irrelevant anti-human IgG2 mAbs (BD Biosciences), as isotype controls. Samples were measured using a FACSCanto II and data were analyzed using FACSDiva software.
For cytokine production analysis, T cells were harvested and treated with GAMUNEX and EMA. They were then stained with the following mAbs: CD3/eFluor 605, IL-10/PE, and IL-17/PerCP-Cy5.5 (eBioscience, San Diego, CA); CD-4/PerCP, CD-8/APC-H7, IL-4/APC, IFN-γ/PE-Cy7 and CD107a/FITC (BD Biosciences); IL-2/Alexa700 and TNF-α/Brilliant Violet 421 (BioLegend). Samples were analysed immediately using an LSR II and FACSDiva software and data were processed using FlowJo software. Duplicates were excluded using the forward-scatter area versus forward-scatter height plot, and CD4+ and CD8+ cells were gated within viable CD3+ lymphocytes and analyzed separately for cytokine production. The percentage of cells producing each cytokine on gated T cells was determined.
The cytotoxic activity of thrice stimulated T cells was determined by standard 51Cr- release assay. T2 cells were incubated for 2 h at 37°C with 10 μg/mL HER-2(9369) or tyr(9369), washed and labeled with sodium chromate, as previously described . Non-loaded T2 were similarly labeled for controls. Effectors (1×106/mL in X-VIVO 15; 100 μL/well) were seeded in 96-well U-bottom plates (Greiner Bio-one, Kirchheim, Germany) and T2 targets were added (5×104/mL; 100 μL/well), at an effector:target (E:T) ratio of 10:1. Where indicated, mAb to MHC class I molecules (W6/32, kindly donated by Prof. S. Stevanovic, University of Tübingen) was added to the cultures at a final concentration of 5 μg/mL for the entire incubation period . After 18 h of coincubation at 37°C, 5% CO2, 100 μL of supernatant were removed from each well and isotope (counts per minute (cpm)) was counted in a γ-counter (1275 Mini-gamma LKB Wallac, Turku, Finland). To determine maximal and spontaneous isotope release, targets were incubated with 3 N HCl and in plain medium, respectively. All cultures were set in triplicate. Percentage of specific cytotoxicity was calculated according to the formula: [(cpm experimental-cpm spontaneous)/(cpm maximal-cpm spontaneous)] ×100.
Stimulated T cells were seeded in 96-well U-bottom plates (1 × 106/mL; 100 μL). Autologous matured DCs pulsed with 50 μg/mL HER-2(15776) or tyr(15448) for 6 h, were added (1 × 105/mL; 100 μL/well) and cocultured for 5 days. T cells incubated with unpulsed matured DCs or in the presence of IL-2 (500 IU/mL) were used as controls. Where indicated, mAb to MHC class II molecules (L243, kindly donated by Prof. S. Stevanovic) was added to the cultures at a concentration of 5 μg/mL for the entire culture period . For the last 18 h of culture, 1 μCi 3H-thymidine (Amersham Pharmacia Biotech, Amersham, Bucks, UK) was added per well and cells were harvested in a semi-automatic cell harvester (Skatron Inc., Tranby, Norway). The amount of incorporated radioactivity, proportional to DNA synthesis, was measured in a liquid scintillation counter (Wallac, Turku, Finland) and expressed as cpm. The S.I. of each experimental group was calculated using the formula: (average cpm of sample in the presence of peptide-pulsed DCs)/(average cpm of sample in the presence of unpulsed DCs).
Total cell extracts from 4–5×105 iDCs and DCs matured with LPS, proTα or proTα(100–109) were extracted as described . Briefly, cells were lysed in NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris pH 8.0) containing protease inhibitors (Protease Inhibitor Cocktail, Sigma-Aldrich) and lysates were cleared by centrifugation for 10 min at 19,000 g (4°C). The protein content of extracts was determined by the Bradford assay, samples were mixed with reducing Laemmli buffer and equal protein amounts (15–25 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using 12% (w/v) polyacrylamide gels. Separated proteins were blotted on nitrocellulose membranes and probed with primary antibodies (goat anti-human αRIF/Novus Biologicals, Ltd, Cambridge, UK; rabbit anti-human MyD88 and rabbit anti-human TIRAP/eBioscience; rabbit anti-human GAPDH/Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit-IgG and anti-goat-IgG/Santa Cruz Biotechnology). Immunoblots were developed using an enhanced chemiluminescence reagent kit (Santa Cruz Biotechnology) and quantified by scanning densitometry (Gel Analyzer v.1.0, Biosure, Athens, Greece).
Data were analyzed by the Student’s t-test and statistical significance was presumed at significance level of 5% (p < 0.05).