Cell lines and culture medium
The CTL clone M134.12, melanoma cell line M117 were established in our laboratory (Godefroy et al 2005). M134.12 was obtained from Tumor Infiltrating Lymphocytes (TIL) of melanoma patient M134 by limiting dilution and selected for it specificity against the antigen MMP-2 in an HLA-A*0201 context. Non-small cell lung cancer cell line (1355) (established by H. Oie) was gift from C. Saï (UMR 892 INSERM/Université de Nantes, France). Mouse fibrosarcoma WEHI 164 clone 13 (established by Rollinghoff and N.L. Warner) and COS-7 cells (established by Y. Gluzman) were gifts from T. Boon (Ludwig Institute for Cancer Research, Brussels, Belgium).
Tumor cell lines and COS-7 cell were maintained in RPMI-1640 and DMEM 1 g glucose/L respectively, supplemented with penicillin-streptomycin (100 U/ml and 10 µg/ml respectively), L-Glutamine (2 mM) and 10% fetal calf serum (all from Sigma-Aldrich). The CTL clone was maintained in RPMI-1640 supplemented with penicillin-streptomycin, L-Glutamine, IL-2 (150 U/ml) and 8% pooled human serum (pHS).
Written consents were obtained from all patients and healthy donors. The local ethics committee “Comité de Protection des Personnes Ouest IV- Nantes” and the “Agence française de sécurité sanitaire des produits de santé” approved all these studies.
Cloning of cDNA encoding modified MMP-2
NA-134A corresponding to the end of the MMP-2 sequence and containing the epitope was obtained from a cDNA library of melanoma cell line M134. Truncated MMP-2 Δ96MMP-2 was obtained by PCR. ΔpreMMP-2 and ΔproMMP-2 were obtained by deletion by PCR of the nucleotide sequence coding for the signal peptide (Δpre) or the propeptide (Δpro). Point mutations were introduced into the cDNA coding for MMP-2 using directional mutagenesis: site-directed mutagenesis was performed using primers with nucleotide substitutions in order to change cystein in alanine at amino acid 60 and/or 65 and 233.
Each of the cDNA constructs was digested with EcoRI and XbaI, inserted into pcDNA3.1 vector (Invitrogen) and plasmids were electroporated into a bacterial strain (Escherichia coli TOP 10 F').
Transient transfection of COS-7 cells
We used the DEAE-dextran-chloroquine method as described (Brichard et al. 1993, Coolie et al. 1994).
Briefly: 1,5.10 4/well COS-7 cells were plated in flat-bottom 96 well plates. After 24 h cells were cotransfected with 125 ng of plasmids containing the cDNA coding for the different constructs of MMP-2 and with 125 ng of plasmids containing the cDNA coding for HLA-A*0201. Each transfection condition was performed in duplicate. After 48 h, cells were tested in a CTL stimulation assay. Transfection efficiency was controlled by transfected COS-7 cells with plasmid coding for the GFP and measured by flow cytometry analysis.
Transfection of COS-7 cells using the Cell Line Nucleofector™ kit V from Amaxa
1.106 COS-7 cells were transfected with 5 µg of plasmid (coding for mutated MMP-2) according to the manufacturer's instructions.
For pulse-chase experiments, 7.106 COS-7 transfected with the same plasmid were pooled in 100 mm dishes and cultured 48 h at 37°C before metabolic labeling.
For zymography and Western blotting assays, 1.106 transfected COS-7 were plated in a 6-well plate. 1 day later, cells were washed and cultured for 48 h in medium without FCS.
Transient transfection of tumor cell lines using lipofectamine reagent
2.104/well tumor cell lines were plated in flat-bottom 96 wells plates 24 h before transfection. Cells were washed with OPTI-MEM medium (Invitrogen) and were transfected using lipofectamine and Plus reagent (Invitrogen) and 100 ng of plasmid containing the cDNA coding for the mutated MMP-2, in OPTI-MEM. All transfection conditions were performed in duplicate. After 48 h, cells were tested in a CTL stimulation assay. Transfection efficiency was controlled by transfected tumor cells with plasmid coding for the GFP and measured by flow cytometry analysis.
Treatment of transfected COS-7 cells
COS-7 cells were transiently cotransfected with plasmids coding for mutated MMP-2 and for HLA-A*0201 using DEAE-dextran-chloroquine method. 24 h later, COS-7 cells were treated by addition to the culture medium of 0,38 µM proteasome inhibitor MG-132 (Calbiochem) or 3 µg/ml sec61 inhibitor Exotoxin A (Sigma-Aldrich). After overnight incubation, cells were washed, counted and tested in CTL stimulation assay.
CTL stimulation assay
Transfected cells were tested for their capability to present a MMP-2 derived HLA-A*0201 and consequently to stimulate the production of Tumor Necrosis Factor (TNF) by the specific CTL M134.12.
1.104 CTL were added to 3.104 stimulator cells (transfected tumoral or COS-7 cells) for 6 h at 37°C. Then, culture supernatants were collected and TNF release by CTL was measured by testing TNF cytotoxicity on WEHI 164 clone 13 cells in a MTT colorimetric assay (Espevik et al, 1986).
Gelatine substrate gel zymography
Secretion of MMP-2 was evaluated by gelatin zymography, as previously described (Barille et al. 1999).
Supernatants were obtained from transfected COS-7 cells (Amaxa) cultured in DMEM 1 g glucose/L without FCS during 48 h and total proteins concentrations were measured (BC Assay, Interchim). 22,5 µg of total protein were mixed with sample buffer without reducing agent and proteins were separated by SDS-PAGE (7,5% acrylamide gels containing 0,2% gelatin). After electrophoresis, SDS was removed from the gel by an incubation in 2,5% triton X-100 for 1 h at room temperature. Gels were then incubated in a buffer containing 50 mmol/L Tris-HCl, 5 mmol/L CaCl2, pH 7,6 for 20 h at 37°C and stained with Coomassie blue R250 for 30 min. Proteolytic activity of MMP-2 was evidenced as a clear band against the blue background of stained gelatin.
Supernatants were obtained from transfected COS-7 cells (Amaxa) cultured in DMEM 1 g glucose/L without FCS during 48 h and total proteins concentrations were measured (BC Assay, Interchim). 50 µg of total protein were mixed with sample buffer containing reducing agent and proteins were separated by SDS-PAGE (7,5% acrylamide), electrotransferred to polyvinylidene difluoride (PVDF) membrane and blocked 1 h in 1% Western Blocking Reagent (Roche Diagnostic) TBS 1X. Blots were probed overnight at 4°C with a rabbit polyclonal antibody anti-MMP-2 hinge region (Biomol, Tebu-bio SAS, France), washed in TBS 1X, 0,1% Tween-20 and probed with HRP-conjugated anti-rabbit, anti-mouse antibody (Roche). The signal was detected by enhanced chemiluminescence detection (Perbio).
Pulse-chase experiments, immunoprecipitation and SDS-PAGE
48 h after transfection using the Cell Line Nucleofector™ kit V (Amaxa), 7.106 transfected COS-7 were incubated at 37°C for 1 h30 in cystein and methionine-free DMEM 4,5 g glucose/L (Invitrogen). Cells were then pulsed for 15 min with 350 µCi of [35S] methionine/cystein (GE Healthcare), plus Brefeldin A (BFA) 10 µg/ml (Sigma-Aldrich) in 5 ml of DMEM 4,5 g glucose/L without cystein and methionine. Cells were then recovered and redistributed: 1.106 cells in 2 ml of DMEM containing FCS plus BFA, and chased from 0 to 24 h. After chase, cells were pelleted, resuspended in lysis buffer (10 mM Tris-HCl pH 7,6, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 2 µg/ml aprotinin, 1% Triton X-100) for 45 min on ice and centrifugated at 10000 g for 30 min at 4°C. Protein concentration in supernatants was measured using bicinchoninic acid (BCA Protein Assay, Pierce, Rockford, IL). 70 µg of lysates were precleared with protein G Agarose (Perbio) and then immunoprecipitated overnight at 4°C with a rabbit polyclonal antibody anti-MMP-2 (H-76, Santa-Cruz, Tebu-bio SAS, France) and protein G agarose. Beads were pelleted, washed and boiled in SDS sample buffer. Proteins in immunoprecipitated samples were separated by SDS-PAGE (7,5% acrylamide) and analyzed by autoradiography.
Statistical analysis was done with InStat 2.01. Data were analyzed using ANOVA test. p<0.005 was considered significant.