Construction of soluble recombinant HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules for insect cells expression
All the DR α and β constructs were cloned in the pMT/Bip/V5-His vector (Invitrogen, Groningen, the Nederlands) in frame with the Drosophila BiP secretion signal, under the control of the Drosophila metallothionein promoter.
The extracellular region of the HLA-DRα chain, deprived of the leader sequence, was cloned by RT-PCR using the following primers: oligo-1-Up DRα (BglII) 5'GGGAGATCTATCAAAGAAGAACATGTGATCATCCAG3', oligo-2-Dw DRα (BamHI) 5'GGATCCTCCACCTCCGTTCTCTGTAGTCTCTGGG3'. The PCR product was cloned into the PCR2.1 vector (Invitrogen) and sequenced.
To generate the HLA-DRα-Bir chain, a cassette containing the sequence for the Acidic Leucine Zipper-Bir A target peptide was generated by fusion PCR with the following primers, as illustrated in Figure 1:
Oligo-3-Up AZ (BamHI) 5'GGAGGATCCACTACAGCTCCATCAGCTCAG3', Oligo-4-Dw AZ (Bir) 5'ACCACCACCGTCCCCACCCTGAGCCAGTTC3', Oligo-5-Up Bir (AZ) 5'GGTGGGAGCGGTGGTGGTCTGAACGATATTTTTG3' and Oligo-6-Dw Bir (NotI) 5'CTAGCGGCCGCTATTCATGCCATTCGAT3'. The PCR product was cloned into the PCR2.1 vector and sequenced, then subcloned in frame with the HLA-DRα fragment separated by a five-aminoacid linker (GGGGS). The resulting sequence encoding the HLA-DRα-AZ-Bir fusion protein was excised with BglII and NotI restriction enzymes, and cloned into the Drosophila melanogaster expression vector pMT/Bip/V5/His.
To generate the HLA-DRα-Ig chain, a cassette containing the sequence for the Acidic Leucine Zipper-hIgG1 constant region was generated by fusion PCR as illustrated in Figure 1, using the following primers:
oligo-3-upAZ(BamHI) described above, oligo-9-Dw AZ (Ig) AGATTTGGGCTCAGATGCTGCCTGAGCCAGTTC, oligo-10-Up Ig (AZ) GCAGCATCTGACGCCCAAARCTTGTGACAAAACTCAC and oligo-11-Dw IgG1 (NotI) 5'TGGCGGCCGCCGCACTCATTTACCCGGAGA. The PCR product was cloned into the PCR2.1 vector, sequenced and then subcloned in frame with the HLA-DR*1101α chain fragment. The resulting sequence encoding for the HLA-DR*1101α-AZ-IgG1 fusion protein was excised with BglII and NotI, and cloned in the Drosophila melanogaster expression vector pMT/Bip/V5/His.
The HLA-DR*1101β chain, shared by both type of soluble recombinant HLA-DR*1101 molecules, was generated as follows. The extracellular region of the HLA-DR*1101β, chain deprived of its leader sequence, was cloned by RT-PCR using the following primers:
oligo-7-Up DRβ (BglII) 5'GGGAGATCTGGGGACACCAGACCACGTTTC3', oligo-8-Dw DRβ (BamHI) 5'GTGGATCCTCCACCTCCCTTGCTCTGTGCAGATTC3'. The PCR product was cloned into the PCR2.1 vector and sequenced. The HLA-DR*1101β cDNA was subcloned in frame with a Basic Leucine Zipper-His tag cassette derived from the pCO268 plasmid spaced by a five-aminoacid linker (GGGGS) [21]. The resulting coding sequence was then excised with BglII and SalI restriction digestion, and cloned into the Drosophila melanogaster expression vector pMT/Bip/V5/His, digested with BglII and XhoI.
Expression of HLA-DR*1101 in Drosophila cells
S2 cells were grown in SFX medium (Hy-clone, South Logan, UT, USA) supplemented with 10% heat inactivated foetal calf serum (Euroclone, Milano, Italy), 50 u/ml penicillin, 25 μg/ml streptomycin, 25 μg/ml Kanamycin (Gibco) and 1 μg/ml Amphotericin B (Gibco, Paisley, Scotland, UK) at 27°C to a density of 2–4 × 106 cells/ml. The α- and β-chain expressing vectors (15 μg each) were co-transfected with 0,5 μg of the selection plasmid pCoHYGRO using a calcium phosphate transfection kit (Invitrogen). Three days after the transfection, selection medium containing 300 μg/ml of Hygromicin B (Roche, Indianapolis, IN, USA) was added to cells. Transfected cells were cultured in SFX medium supplemented with 2% of foetal calf serum and 300 μg/ml of Hygromicin B. Cells were kept in the exponential growth phase (7–20 × 106 cells/ml) and the protein production was induced by the addition of 1 mM of CuSO4 when the cells were at a density of 107/ml. Protein production was monitored by dot blot analysis with either mouse anti-human HLA-DR α monoclonal antibody L243 (ATCC, Manassas, VA, USA) or rabbit anti-His tag polyclonal antibody His-probe (Santa Cruz, Santa Cruz, CA, USA), followed by an HRP-labelled goat anti mouse or anti rabbit antibody (Southern Biothecnology, Birmingham, AL, USA). The assay was developed with ECL (Amersham, Uppsala, Sweden). To improve the production of recombinant proteins, transfected cells were cloned by limiting dilution. S2 cells expressing the desired HLA-DR*1101 molecule were diluted to 2.5 cell/ml in a suspension of irradiated (8000 rad) S2 wild type cells at the concentration of 3 × 105 cells/ml in medium+Hygromicin B, and plated in flat bottomed 96 w plates. The best producer clones were selected by dot blot analysis on the culture supernatant as described above, upon induction with 1 mM of CuSO4 of the same number of cells, and used for routine protein production.
Purification of soluble HLA-DR*1101 molecules
Both HLA-DR*1101-Ig and HLA-DR*1101-Bir molecules were purified from the S2 cells supernatant by immunoaffinity chromatography using ProtA-Sepharose (Amersham Bioscience) and HLA-DRα-specific L243 mAb-Sepharose, respectively. The HLA-DR*1101-Ig molecules were stored at -80°C at a concentration of 1 mg/ml in PBS buffer. HLA-DR*1101-Bir molecules were dialyzed against 10 mM Tris buffer, pH 8.0, and concentrated up to 3 mg/ml on Vivaspin2 concentrator (Vivascience, Hannover, Germany). The protein was biotinylated using the BirA enzyme according to the manufacturer's instructions (Avidity, Denver, CO, USA), then was diluted to 1 mg/ml and stored at -80°C. The formation of molecular aggregates was assessed by size exclusion chromatography on a Superdex 200 HR 10/30 column using a ÅKTA FPLC chromatography system (Amersham Bioscience). The calibration of the Superdex 200 column was performed with the Gel Filtration Calibration Kit high molecular weights (Amersham Bioscience), following the manufacturer indications. Partition coefficients were calculated using the relation Kd = (Ve-Vo)/(Vc-Vo), where Ve, Vc and Vo are the elution, column and void volumes, respectively.
Peptide loading into soluble HLA-DR*1101 molecules
Both HLA-DR*1101-Ig and HLA-DR*1101-Bir were loaded with synthetic peptides by incubation at 37°C for 72 hours with 50 fold molar excess of either Tetanus Toxoid peptide residues 829–844 (p2), Hemagglutinin peptide residues 306–318 (HA) or MAGE-3 tumour associated antigen peptide residues 191–205 (p39) in 10 mM Tris, 50 mM Glycine pH 5.0 with 0.2% of n-octyl-β-D-glucopyranoside (Sigma-Aldrich, St. Louis, MO, USA).
Tetramerization of soluble HLA-DR*1101 molecules and staining of specific T cells
Tetramerization of HLA-DR*1101-Ig and HLA-DR*1101-Bir were achieved by adding FITC-labelled protein A (Molecular Probes, Leiden, The Netherlands) at 5:1 molar ratio, or PE-labelled streptavidin (Molecular Probes) at 5:1 molar ratio, respectively. Tetramer staining was performed for 2 hours at 37°C in complete medium. The cells are then washed twice, transferred on ice and stained for the other surface markers. Immediately before the analysis TOPRO-3 (Molecular Probes) is added following the manufacturer's instructions to exclude dead cells. The analysis is performed on CD3+CD4+ viable cells.
Generation of specific CD4+ T cell lines and clones
20 million of freshly purified PBMCs from a HLA-DR*1101/1104 healthy donor were cultured for 7 days in RPMI 1640 (Gibco) supplemented with 10% of human serum, 2 mM Glutamax I (Gibco), 100 u/ml penicillin and 50 μg/ml streptomycin (Gibco), in the presence of the specific peptide at a concentration ranging from 1 to 10 μg/ml. After seven days, blasts were separated from small T cells on a 30–70% discontinuous Percoll gradient [39], and put in culture in complete medium with 10–20 u/ml of human recombinant IL-2 (Roche). Peptide specific T cells were kept in culture by weekly re-stimulation with the same amount of peptide and irradiated (4000 rad) autologous PBMCs as antigen presenting cells. Peptide-specific T cell clones were generated by limiting dilution of Percoll-purified T cell blasts blasts as follows. Blasts were diluted at 25 cells/ml in a suspension containing 0.5 × 106 cells/ml of a mixture of two different allogeneic PBMCs supplemented with 1 μg/ml of PHA-L (Roche) and 200 u/ml of human recombinant IL-2 (Roche). The cloning mix is dispensed in 25 μl in flat-bottomed Terasaki plates and incubated at 37°C for 10–20 days. Growing cells are then tested for the recognition of peptide pulsed HLA matched EBV cell lines.
T cells functional assays
To test IFN-γ production, 104 T cells were plated in U bottom 96 well plates in RPMI 1640 supplemented with 10% of human serum, 2 mM Glutamax I, 100 u/ml penicillin, 50 μg/ml streptomycin, together with 5 × 104 irradiated (6000 rad) HLA-DR*1101 LCL cells as antigen presenting cells, and the specific peptide at the concentrations detailed in the figure legends. IFN-γ production in the supernatant was measured by ELISA after 48 hours of incubation (IFN-γ detection kit-Endogen).
For the intracellular analysis of IFN-γ production, T cells were stimulated for 6 hours with HLA-DR*1101 LCL of at 1:5 ratio in three conditions: i. without peptide; ii. with 5 μg/ml of peptide; and iii. with 50 ng/ml of PMA (Sigma) plus 500 ng/ml of Ionomycin (Sigma). After the first hour of stimulation, Brefeldin A was added at the final concentration of 10 μg/ml. At the end of the stimulation, the cells were harvested, fixed with PFA1%, permeabilized with 0,5% saponin and stained for intracellular cytokine expression.
For the T cell activation with peptide-loaded soluble recombinant HLA-DR molecules, 1 μg of peptide loaded HLA-DR*1101-Ig or HLA-DR11-Bir molecules in 50 μl of PBS were incubated in U bottomed 96 well plates for 3 hours at 37°C. After washing, 2 × 104 specific T cells were added to the wells and incubated for 48 hours at 37°C in the presence of 1 ng/ml of PMA. Cells incubated either with PMA alone or in the presence of 1 μg of plate coated TR66 anti-CD3 mAb are used as negative and positive control, respectively. IFN-γ production in the supernatant is measured by ELISA (Endogen, Woburn, MA, USA) after 48 hours of culture.
Antibodies and flow cytometry
The following antibodies have been used: FITC- and APC-labelled mouse anti human CD3 (Becton Dickinson, San Jose, CA, USA), Quantum Red-conjugated mouse anti-human CD4 (SIGMA), PE-labelled rat anti-human IFN-γ (Becton Dickinson). Data were collected using a Becton Dickinson FACScalibur flow cytometer and analysed using the Cell Quest software (Becton Dickinson)