Production of anti-galectin-9 monoclonal antibodies
The recombinant S and M isoforms of human galectin-9 and the M isoform of murine galectin-9 were produced in E. coli as GST-fusion proteins. Tag-free proteins were purified by affinity chromatography on a lactose-agarose column . The c-terminus galectin-9 (residues 191 to 355 of the galectin-9 long isoform) was produced in E. coli as a GST-fusion protein. The tag-free protein was purified by exclusion chromatography. Immunizations were conducted at PX’Therapeutics (Grenoble, France). Five BALB/c female mice (eight weeks old) were immunized with the recombinant c-terminus galectin-9. Immunizations (40 μg of protein) were administered intraperitoneally at days 0, 22, 37 and 54 with complete Freund’s adjuvant for the first immunization, then with incomplete Freund’s adjuvant for subsequent injections. We performed enzyme-linked immunosorbent assay (ELISA) on mice serum to confirm response to galectin-9 immunization, using the same recombinant galectin-9 c-terminus part which was injected into mice. A rabbit polyclonal serum raised against the same portion of galectin-9 was used a positive control. The five immunized mice exhibited a specific and strong immune response against the c-terminus part of galectin-9. Three days after the last boost, the two best responding mice were sacrificed and their splenocytes were collected to use in subsequent liquid or semi-solid fusion with Sp2/0 cells at a ratio of 5:1 and 2:1 respectively. Hybridomas supernatants were assessed in galectin-9 ELISA. The semi-solid fusion was successful and a large collection of monoclonal hybridomas secreting anti-galectin-9 antibodies was obtained.
Galectin-9 ELISA for assessment of mouse sera and selection of hybridomas
Wells of microtiter plates (Greiner Bio-One, Courtaboeuf, France) were coated with 0.05 M carbonate/bicarbonate buffer (Sigma-Aldrich, Saint-Quentin Fallavier, France) pH 9.6 containing 50 ng human c-terminus galectin-9 during 1 H at room temperature. After washing with phosphate buffered saline (PBS) containing 0.1% Tween-20 (Euromedex, Souffelweyersheim, France), the wells were saturated with 3% bovine serum albumine (BSA) (Sigma-Aldrich, St Quentin Fallavier, France) in PBS at room temperature for 1 H. They were then incubated with mouse sera or raw hybridoma supernatants in PBS with 1% BSA at room temperature for 2 H. After a washing step with PBS with 0.1% Tween-20, the anti-galectin-9 antibody level was determined using horseradish peroxidase-coupled (HRP) goat antibodies to mouse IgG (Sigma-Aldrich, St Quentin Fallavier, France) and 3,3’,5,5’ Tetramethylbenzidine (TMB) (Thermo Fisher Scientific, Brebieres, France) as substrate. Microtiter plates were incubated 15 min in the dark under shaking before stopping the reaction with 1 M H2SO4. Then, OD was read at 405 nm and 620 nm using a MultiSkan Ex microplate reader (Thermo Fisher Scientific). Experiments were performed in duplicates.
ELISA for epitope mapping and oligopeptide competition
A panel of 27 oligopeptides representative of the 168 amino-acids from the c-terminus galectin-9 used for mice immunisation was produced. Oligopeptides were 13 amino-acids long, with a 7 amino-acids overlap. For epitope mapping, each peptide was coated in 96-well plate using 100 ng/well during 16 hours at 4 °C. For oligopeptide competition, full length galectin-9 (recombinant S isoform produced in E. Coli) was coated in the same conditions. Wells were saturated in PBS + 0.1% BSA at room temperature during 2 hours, then incubated with purified mouse monoclonal antibodies during 2 hours at room temperature. For oligopeptide competition, 1G3 was tested in parallel with the mab 9S2-3 which targets the n-terminus of galectin-9 . The antibodies were pre-incubated during 2 H at room temperature with control oligopeptide (“ITQTVIHTVQSAP”) or target oligopeptide (“TPAIPPMMYPHPA”) using serial dilutions ranging from 4 μg/ml to 2 ng/ml. Revelation of bound monoclonal antibodies was performed using a peroxidase-conjugated secondary antibody as described in the previous paragraph.
Capture of biotinylated galectins on surface-bound antibodies
For this assay, we used the following recombinant galectins: the S and M isoforms of human galectin-9, the murine M isoform of galectin-9 and human galectin-1, -2, -3, -4, -8 (M isoform) and −10. Preparation of recombinant galectins and their biotinylation have been already described in previous works [15–17]. 1G3 and control mouse IgG1 antibody (MOPC21 clone) were coated in 96-well plates overnight at 4 °C in assay buffer (PBS-Tween, 2% fetal calf serum (FCS), 0.05% NaN3). Plates were blocked with 5% FCS overnight at 4 °C then washed five times with PBS-Tween. Biotinylated galectins (1 nM) were incubated during 1 H at 37 °C in antibody-coated wells; then plates were washed five times with PBS-Tween. Revelation of captured biotynilated galectins was done by addition of SA-HRP for 1 H at 37 °C. After washing, TMB substrate was added during 2.5 min at room temperature. Reaction was stopped with 1 M phosphoric acid and OD was read at 405 nm and 620 nm. Experiments were performed in duplicates.
BL2 and REMB1 cells were grown in RPMI 1640 medium (Gibco-Invitrogen, Carlsbad, CA) supplemented with 10% FCS. BL2 is an EBV-negative B-cell line derived from a Burkitt’s Lymphoma. REMB1 is a lymphoblastoid cell line (LCL) resulting from in vitro EBV-transformation of B lymphocytes from a normal donor. HeLa cells were cultured in DMEM supplemented with 10% FCS. C15 is an EBV-positive NPC xenograft which was propagated by subcutaneous passages into nude mice . C666-1 cells which are EBV-positive NPC cells were grown in vitro in RPMI 1640 medium supplemented with 25 mM HEPES and 7.5% FCS, in plastic flasks coated with collagen I (Biocoat; Becton-Dickinson, Franklin Lakes, NJ) .
Cell pellets were solubilized in RIPA buffer (150 mM NaCl, 25 mM Tris–HCl pH 7.5, 5 mM EDTA, 0.5% sodium deoxycholate, 0.5% NP40, 0.1% SDS) supplemented with the complete protease inhibitor cocktail (Roche Applied Science, Neuilly-sur-Seine, France) and sonicated on ice. Extracts were clarified by centrifugation for 15 minutes at 16 000 g at 4 °C. Protein concentration was assayed by the Bradford method using BioRad protein assay (BioRad, Marnes-la-coquette, France). Cell protein extracts were separated on 12% polyacrylamide gels in standard conditions. Gels were blotted on PVDF membranes (Immobilon-P; Millipore, Molsheim, France) then blocked during one hour with TBS containing 3% non-fat milk powder and 4% glycine. Membranes were incubated overnight with mouse monoclonal antibodies at the concentration of 2 μg/ml in blocking solution. Specific protein bands were visualized using goat anti-mouse HRP–conjugated secondary antibodies and revealed by chemiluminescence using Immobilon Western kit (Millipore, Molsheim, France).
Thirteen samples from head and neck carcinomas were selected from a retrospective collection of biopsies collected for diagnosis purpose at Lariboisière hospital (Paris, France). This set of biopsies included ten nasopharyngeal carcinomas and three carcinomas of the oropharynx and oral cavity. Nine liver samples were selected from a retrospective collection obtained following partial or complete hepatectomy at Beaujon hospital (Paris, France). This set of surgical samples included three specimens infected by HCV, three specimens infected by the hepatitis B virus (HBV) and three specimens of uninfected patients having undergone partial hepatectomy for benign tumors. All these samples were obtained and processed according to guidelines of Lariboisière and Beaujon hospitals institutional review boards.
Biopsies from head and neck carcinomas and surgical liver samples were initially fixed in paraformaldehyde 4% and paraffin-embedded. Prior to galectin-9 staining, tissue sections were dewaxed with xylene and ethanol and rehydrated. Antigens were unmasked using a pH 6.0 citrate solution at 98 °C during 30 min. After adequate washing steps, sections were incubated 10 min with 3% H2O2, washed again and saturated with blocking serum (Biogenex, MM France, Francheville, France) for 1 hour. Sections were then incubated with mouse monoclonal antibodies diluted at 2 μg/ml in blocking serum for 1 hour at room temperature. Visualization was achieved by exposing sections to a goat anti-mouse HRP 1:50 (Southern Biotech, Clinisciences, Nanterre, France) during 30 min at room temperature then adding DAB substrate. The slides were counterstained with Mayer’s hematoxylin diluted at 1:2 during 1 min at room temperature.