Raji (human Burkitt lymphoma, ATCC: CCL-86) and Jurkat (human acute T-cell leukemia, ATCC: TIB-152) cells were grown in RPMI 1640 supplemented with 10% fetal calf serum (FCS, Gibco BRL, LIFE TECNOLOGIES™) in plastic flasks with gentle agitation at 37°C and 5% CO2, as source of poly-A mRNA. Murine T-cells CTLL-2 (cytotoxic T cell, ATCC: TIB-214), human HEp-2 (laryngeal carcinoma, ATCC: CCL23) and Colo 205 (human colorectal adenocarcinoma, ATCC: CLL-222) cell lines were used to evaluate the biological activity of the antagonist. Cells were cultured in RPMI 1640 (CTLL-2, Colo 205) and MEMCANE (HEp-2) containing gentamycin (50 μg/mL) and 10% FCS at 37°C in a humidified 5% CO2 environment. E. coli cells W3110 P3 were used for pHu (AnTH1) plasmid expression.
Isolation of cDNA coding for IL-2 N-terminal and IFNGR1 extracellular region
Poly-A mRNA purified from 2 × 108 Jurkat and Raji cells, respectively, using the MessageMaker® mRNA Isolation System (Invitrogen Life Technologies) was employed for cDNA RT-PCR amplification. 1–2 μg poly-A mRNAs were reverse transcribed with GeneAmp RNA PCR kit (Perkin Elmer Cetus, Norwalk, Conn.) using random hexamers. The specific primer pairs were the following (overlapping nucleotides are underlined): 5'CCATGACCTACTTTCAAGTTCTACAAAG3' and 5'CATCATATGGGTCTAGACACTGAAGATGTTTC3' for the amplification of IL-2 N-terminal, and 5'CCCATATGATGAGCAGGGCTGAGATGGGC3' and 5'GATCCTTATTTTATACTGCTATTGAAAATG3' for the IFNGR1 extracellular region. Primers for the second round PCR were: 5'CCATGGCACC TACTTTCAAGTTCTACAAAG3' and 5'GGATCCTTATTTTATACTGC TATTGAAAATG 3'.
In brief, first-strand DNA was synthesized in a final volume of 20 μL from 1 μg poly-A mRNA in DEPC-H2O using 5 mM MgCL2, 1 mM dNTP, 1 U/μL Rnase inhibitor, 2.5 U/μL MuLV reverse transcriptase, 2.5 μM random hexamers, 0.75 μM primers, and 1X PCR buffer II. The mixture was incubated in a DNA Thermal Cycler (MJ Research, Inn.) at 25°C for 10 minutes, then 1 hour at 42°C followed by 5 minutes at 99°C and then frozen until use. Complementary DNA (10 μl) were mixed as recommended by providers with the appropriately specific primers and reagents (2 mM MgCl2, 2.5 U/100 μL AmpliTaq DNA polymerase, 0.15 mM specific primers and 1X PCR Buffer II). Amplification started with 5 minutes denaturation at 94°C, followed by 30 PCR cycles. Each cycle consisted of 60 seconds at 94°C for denaturation, 40 seconds for annealing (temperature depended on the primers were used), and 40 seconds at 72°C for extension. Final extension lasted 5 minutes at 72°C. Twenty five percent of the RT-PCR reaction (5 μl) was transferred to a second round reaction mix with the second round primers and reagents in the same concentrations as described for PCR. The second round PCR (25 cycles) was done using the same cycle schedule.
Construction of the TH1 antagonist expression vector
The pTPV-1 plasmid, supplied by the Biomedical Research Division at CIGB, was employed as expression vector. It carries the E. coli tryptophan promoter, the bacteriophage T4 terminator and the ampicillin resistance gene. PTPV-1 vector was digested stepwise with the NcoI and BamHI enzymes. Second PCR amplified cDNA was digested with the BamHI enzyme and processed to eliminate the enzyme and the buffer. Then PCR product was digested with NcoI, and processed as described before. Finally, the vector and the amplified gene fragment were ligated using the T4 ligase enzyme.
Sequencing of the pHu (AnTH1) plasmid
The sequencing of pHu (AnTH1) plasmid was done using the Taq Dye Deoxy terminator cycle sequencing kit (Applied Biosystems). One μg of pHu (AnTH1) plasmid was used as template and annealed with 10 ng forward or reverse primers, which hybridize with a promoter region of expression vector. After the separation of unincorporated dye terminators and primers, the products were dried in SpeedVac centrifuge, resuspended in loading buffer, heat denatured and immediately loaded on acrylamide gel in an automated DNA sequencer.
Expression of the recombinant protein
E. coli cells, strain W3110P3, containing pHu (AnTH1) plasmid were grown at 37°C in LB medium with 50 μg/mL tryptophan and 100 μg/mL ampicillin until the cells reached an optical density (OD) of 2.0 at 620 nm. Then cells were inoculated in M9 medium with 4% glucose without tryptophan and 50 μg/mL ampicillin to a final OD of 0.25 and grown during 8 hours at 37°C with agitation. Cells were harvested and kept frozen (-20°C) for future processing.
Extraction of E. coli cells and refolding
To suspend the cells, 10 mL of 10 mM Tris buffer pH 7.2, 1 mM EDTA was added per g of E. coli cells. The cells were digested with lysozyme and the pellet from the digestion was washed in a Polytron homogenizer (IKA, Germany) in 50 mM Tris pH 7.2, 1 mM EDTA containing increasing urea concentrations (from 1 to 8 mol/L). Cell suspensions were centrifuged at 12000 rpm at 4°C, 5 min. and supernatants discarded. The pellet from 4 M urea wash was finally extracted in a Polytron homogenizer with 10 mM Tris buffer pH 7.2, 1 mM EDTA containing 8 mol/L urea (extraction buffer). Cell homogenate was centrifuged at 12000 rpm 5 min. 4°C and supernatant decanted and stored at -20°C until refolding. Supernatant from the 8 M urea extraction (150 mL) was loaded on a Sephadex G-100 column (K9/60 (Pharmacia, Uppsala), equilibrated with 50 mM Tris HCL pH 7.4 containing 4 M urea. The elution was performed in the same buffer at 3 mL/minute. Eluted fractions containing the recombinant protein were pooled and dialyzed against a buffer containing 0.1 Tris-HCl, pH 9.0. The dialysis was then continued against phosphate buffered saline (PBS), pH 7.4.
Ligand-bloting, SDS-PAGE, immuno-blot
For ligand blot, 5-μL fractions from gel filtration chromatography containing folded AnTH1 recombinant protein, were directly applied to nitrocellulose strips and incubated with 10% defatted milk during 2 h at room temperature. After washing with PBS containing 0.05% Tween 20 (PBS-T), the strips were incubated with 125-I labed recombinant IFN-γ, without or in the presence unlabed recombinant IFN-γ. Finally, the strips were thrice washed with PBS-T and exposed for autoradiography. For SDS-PAGE the samples were loaded in sample buffer with or without reducing agents. Bands were visualized by Coomassie Blue R-250 (Sigma) staining.
For immunoblotting samples were loaded as for SDS-PAGE. After blotting, nitrocellulose strips were incubated with 10% defatted milk during 2 h at room temperature, washed (as for ligand blot) and incubated with mouse anti-IFNGR1 monoclonal antibody R99 (9 μg/mL) or rabbit polyclonal antibody against human IL-2 protein, washed, incubated with anti-mouse or anti-rabbit (IgG)-peroxidase conjugate in 1% defatted milk and finally washed. Then, the strips were incubated with developing solution (H2O2, 5 mg/mL, o-phenylendiamine, and 15%).
Aminoacid sequence. In-gel digestion
An aliquot (0.5 μg) of purified protein was analyzed by SDS-PAGE and reversed-stained with Zn-imidazol . The band was excised and incubated with a 1% citric acid solution during 5 minutes until complete destaining and incubated another 10 minutes in water to remove the excess of chelating agent. The transparent band was additionally cut in approximately one mm3 cubes dehydrated in a 90 % acetonitrile aqueous solution without TFA, and completely dried in a SpeedVac centrifuge. The gels pieces were rehydrated in 20 μL of 50 mM NH4HCO3 solution containing 12.5 ng of trypsin, sequencing grade (Promega, USA). The in-gel digestion was incubated overnight at 37°C in a thermomixer (Eppendorf, USA). Additionally, 20 μL of 50 mM NH4HCO3 solution were added and incubated further for 45 min. Tryptic peptides were extracted using ZipTips C18 (Millipore, USA) previously activated and equilibrated as recommended by the manufacturer. Twenty loading cycles were carried out for extracting tryptic peptides. The digest was acidified with formic acid, incubated 45 minutes at room temperature and another twenty loading cycles were achieved. The Ziptips were washed extensively using a 5 % formic acid solution and proteolytic peptides eluted in 2 μL of 60 % acetonitrile containing 1 % formic acid.
The Electrospray ionization (ESI-MS) mass spectra were acquired using a hybrid quadrupole orthogonal acceleration tandem mass spectrometer QTOF from Micromass (Manchester, UK) fitted with a Z-spray nanoflow electrospray ion source. The mass spectrometer was operated with a source at 80°C and a drying gas flow of 50 L/h. Two μL of the tryptic peptides were loaded onto the borosilicate nanoflow tip and 900 V and 35 V potentials were applied to nanoflow tip and entrance cone, respectively. To obtain information on peptide sequence, the ESI-MS/MS spectra were acquired as described previously . Data acquisition and processing were performed using the MassLynx system (v 3.5) from Micromass. The most intense signals observed in the ESI-MS spectra were sequenced by MS/MS, sequence tags were manually extracted and used to identify the proteins by Peptide Search program . Peptide mass tolerance was 2 Da in order to identify peptides containing deamidated asparagines residues.
Inhibition of recombinant rhu IL-2 biological activity
The biological activity of rhu IL-2 was assessed as described  using IL-2-dependent murine T lymphocyte cell line CTLL-2 . Cells were grown in RPMI-1640 medium containing 1 mM pyruvate, 2 mM L-glutamine, 40 mM HEPES, 100 U/mL penicillin, 50 μg/mL streptomycin, 50 μM 2- mercaptoethanol and 10% FCS supplemented with 8 U/mL rhu IL-2 (Heber Biotec, Havana; 1.2 × 107 IU/mg). Before use, cells were washed thrice, resuspended in complete culture medium without IL-2 and incubated during 1 h at 37°C in a humidified 5% CO2 atmosphere. Cells were suspended at a density of 4 × 105 cells/mL, and distributed into 96-well microtiter plates (100 μL per well) already containing 100 μL of two-fold serial dilutions of rhu IL-2 or samples, in complete medium. Samples consisted of a constant amount of rhu IL-2 containing serial dilutions of the antagonist. Following 36 h of incubation at 37°C, 20 μL of 5 mg/mL of MTT were added and plates incubated for 4 h in the same environment. Finally, 50 μL of 10% SDS/0.1 N HCl/50% isopropylalcohol solution were added per well, the plates agitated for 1 h at 37°C, and the absorbance readed at 570 nm using a microplate reader (Tecnosuma, Havana). Results were expressed as hu IL-2 units.
Inhibition of rhu IFN-γ antiproliferative action
HEp-2 at 2.5 × 103 cells/mL were seeded in 96-well microtiter plates (COSTAR, Cambridge), cultured in MEMCANE containing 50 μg/ml gentamycin and 10% FCS, at 37°C in a humidified 5% CO2 environment. Serial dilutions in MEM-CANE medium with 10 % FCS of chimeric recombinant protein samples were tested. Samples were mixed with an equal volume of medium containing appropriated amounts (see figures notes) of rhu IFN-γ (Heber Biotec, Havana; 1.0 × 107 IU/mg). After adding the samples, monolayers were incubated during 72 h at 37°C, 5 % CO2. The amount of growing cells in triplicate cultures at each point was determined by Crystal Violet staining, and absorbance measured at 490 nm using the microplate reader. The result is defined as % of growth as follows:
% of growth = (AT72h-AC0h/AC72h-AC0h) × 100.
AT72h = Absorbance from treated culture at 72 hr.
AC72h = Absorbance from control culture at 72 hr.
AC0h = Absorbance from culture just prior to the addition of IFN.
Inhibition of HLA expression
The biological activity of TH1 antagonist was assessed further by testing its ability to prevent IFN-γ from inducing expression of HLA-DR. A bio-ELISA assay according to the method of Gibson and colleagues  was carried out. Colo 205 cells were grown to confluence in RPMI 1640 containing 10% FCS, trypsinized and seeded in 96-well tissue culture plates at a density of 2.5 × 105 cells/well in 0.1 mL of RPMI 1640 containing 10% FCS and finally incubated for 12 h at 37°C in 5% CO2. Culture media containing the rhu IFN-γ samples and mixtures of rhu IFN-γ and antagonist protein were added in a 0.1 mL volume to the wells containing Colo 205 cells, and then incubated for 1 h at 37°C. Following incubation, the media was removed and wells washed three times with culture media. Fresh culture media (0.2 ml/well) was added and plates incubated for 48 h at 37°C to allow for induction of HLA/DR antigen. Then, the wells were washed with PBS and fixed for 2 min with ice-cold anhydrous ethyl alcohol. After the alcohol was removed, the wells were washed with PBS and incubated for 1 h at room temperature with mouse monoclonal anti-HLA/DR (DAKO, California) antibody diluted in PBS containing 0.5% bovine serum albumin. PBS was used to wash the wells, and peroxidase-labeled goat anti-mouse IgG was added to each well for 1 h at room temperature. The wells were washed three times with PBS and then developed as described for the ELISA plates.