Culture materials Ag and Ab reagents
The culture medium used was RPMI-l640 (HyClone, Logan, UT), supplemented with 1 × non-essential amino acids (Life Technologies, Gaithersburg, MD), 10% fetal bovine serum (FBS; Life Technologies) and 50 μg/ml of gentamycin and kanamycin (Sinton Chemical & Pharmaceutical, Hsinchu, Taiwan). Purified and biotinylated human CD152-murine Ig fusion protein (CD152-muIg), CD80-muIg and CD86-muIg (Ancell, Bayport, MN) were used in Ag-specific and competing enzyme-linked immunosorbent assay (ELISA), together with peroxidase-labeled goat antibodies against human IgG and IgM (Zymed Laboratories, South San Francisco, CA) or avidin horseradish peroxidase (eBioscience, San Diego, CA) as the reporting system. The fluorochrome-conjugated mouse mAb against human IgGs and human CD3 (UCHT1; mouse IgG1), together with rat mAb against mouse IgG2a were commercially available from Becton Dickinson Immunocytometry Systems (San Jose, CA) and Abcam (Cambridge, UK). The anti-CD3 (OKT3; mouse IgG2a) used for T cell activation and the antagonistic anti-CD152 (BNI3; mouse IgG2a) were purchased from eBioscience and Abcam, respectively.
Preparation of human PBMC
Plasma and buffy coat samples from healthy routine blood donors, screened negative for HIV-1/2, HTLV-I/II, HCV, HBsAg and containing normal levels of alanine transferase (ALT), were obtained from the Tainan and Hualien Blood Centers, Taiwan Blood Services Foundation. Written informed consents were obtained from five repeatedly-healthy regular blood donors after an explanation of the nature, purpose, and potential risks of the study and then 230 ml of whole blood was used for the purpose of site-directed in vitro immunization. PBMC's were isolated by density centrifugation on Ficoll-Paque (GE Healthcare Bio-Sciences, Uppsala, Sweden) as described elsewhere.
Magnetic cell purification and depletion
PBMC's were magnetically labeled with CD45RO MACS® microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) then separated by a VarioMACS™ (Miltenyi) instrument according to the manufacturer's instructions. The purified CD45RO+ T cells were cultured at a density of 2 × 106 cells/ml in the culture medium supplemented with 50 μM 2-mercaptoethanol and 10 μg/ml pokeweed mitogen (PWM; Sigma, St. Louis, MO). After 24 h, cells were removed by 400 × g centrifugation to collect CD45RO+ T cell replacing factor. Removal of cytotoxic cell populations, which inhibit in vitro immunization , was similarly performed by using colloidal super-paramagnetic microbeads conjugated to monoclonal anti-human CD8 and anti-CD56 antibodies (Miltenyi). Removal of IL-10-producing cells was achieved by using rat anti-human IL-10 (SouthernBiotech, Birmingham, AL) and goat anti-rat IgG microbeads (Miltenyi).
Site-directed in vitroimmunization
Cytotoxic cell-depleted PBMCs were immunized in vitro based on a previously described two-step principle . Primary immunization was performed by incubating the cells for 6 days in a medium containing 10 nM of the heterotopic peptide Ag (QYIKANSKFIGITELAATYMMGNELTFLDDSICT; Fine Research Biochem, Taoyuan, Taiwan), 50 μM 2-mercaptoethanol, 10% heat-inactivated human serum, 0.05 ng/ml recombinant human (rh) IL-2 (eBioscience), and 25% (v/v) CD45RO+ T cell replacing factor. For secondary immunization, 3 × 107 primary-immunized cells were mixed with the peptide in a flask that had been immobilized overnight with 5 mg/ml of CD40L (CD154; eBioscience) together with 1 × 107 QYIKANSKFIGITEL (Fine Research Biochem)-stimulated CD4+ T cells and 5 ng/ml rh IL-15 (eBioscience). The cells were cultured for 3–5 days in a medium supplemented with 5% human serum, 50 mM 2-mercaptoethanol and 10 nM heterotopic peptide Ag. The significance of differences between treated and control cultures was established by using Student's t test. A P value of less than 0.05 was considered statistically significant.
Epstein-Barr virus (EBV) infection, ELISA and somatic cell hybridization
The in vitro immunized cells were infected with EBV by virus-containing supernatant derived from the EBV-producing marmoset cell line B95-8 (American Type Culture Collection, ATCC CRL 1612; kindly provided by Dr. L.-F. Sheu, Tri-Service General Hospital, Taipei). The infected cells were seeded at 105/well in 96-well plates together with mytomycin (Kyowa Hakko Kogyo, Tokyo, Japan)-treated PBMC as feeder cells (104/well) for the establishment of lymphoblastoid cells and screened for Abs by ELISA.
Ag-specific ELISA was performed by coating 0.25 μg/ml purified rhCD152-muIg, 0.5 μg/ml monoclonal mouse IgG2a (mIgG2a; Ancell), 1 μg/ml bovine serum albumin (BSA; Sigma) or 1 μg/ml tetanus toxoid (TT; ADImmune, Taichung, Taiwan) onto microtitre plates overnight at 4°C. Culture supernatants were diluted to the desired level in 10 mM sodium phosphate buffer (pH 8.0), containing 0 5 M sodium chloride and 0.1% Tween-20. Coated plates were incubated with diluted culture supernatants, washed, incubated with peroxidase-labeled goat antibodies against human IgG and IgM and developed (15 min) by addition of 100 μl of the chromogenic substrate o-phenylaenediamine (OPD) (Sigma). The reaction was stopped after 30 min by adding 1 M sulphuric acid, and the absorbances were read at 490 nm.
Somatic cell hybridization was generated by electrofusion. Briefly, Ag-specific EBV-infected lymphoblastoid cells were fused with heteromyeloma cells  in an isotonic medium (280 mM sorbitol, 0.5 mM magnesium acetate, 0.1 mM calcium acetate and 1 mg/ml BSA; pH6.9–7.1). Cell fusion was induced by high-voltage pulses using a BTX Electro Cell Manipulator ECM 2001 (Harvard Apparatus, Holliston, MA). Ag-specific hybrids were selected and cloned by limiting dilution.
To define the specific epitope of human CD152 recognized by the mAb, we used peptide arrays (Genesis Biotech, Taipei, Taiwan and Fine Research Biochem,) containing in-situ synthesized peptides immobilized on special membrane. In brief, 1 μg/mL of protein A (Proteus MIDI kit, Pro-Chem, Littleton, MA)-purified mAb was incubated by shaking in room temperature for 2 h. After washing, the membrane-bound mAb was then visualized by diluted anti-human IgG conjugated with peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA) and FAST™ DAB (Sigma). The amount of bound mAb was calculated by Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD) on the scanned images.
Flow cytometry analyses
The surface expression of the CD152 epitope recognized by the present mAb was analyzed using two-color flow cytometry on mitogen-stimulated PBMC's by a FACSCaliber™ flow cytometer and CellQuest™ software (Becton Dickinson Immunocytometry Systems). 2 × 106 isolated PBMC's were resuspended in supplemented culture medium and treated with either anti-human CD3 (OKT3; final concentrations in culture 10 μg/ml, eBioscience), concanavalin A (Con A; final concentrations in culture 10 μg/ml, Sigma), 10 μg/ml phytohemagglutinin (PHA; final concentrations in culture 1 μg/ml, GE Healthcare Bio-Sciences), a combination of phorbol 12-myristate acetate (PMA; 50 ng/ml, Sigma) + ionomycin (1 μM, Sigma) or a combination of PHA (1 μg/ml) + PMA (50 ng/ml). Logarithmically amplified fluorescence data were collected on 10,000 CD3+ cells. All flow cytometry staining procedures were performed at 4°C in cytometry buffer. For extracellular detection of CD152, activated cells were first surface stained with the mAb or isotype control at 4°C, followed by anti-human IgG-FITC and anti-CD3-PE (Becton Dickinson) staining.
RT-PCR assays for deduction of Ab primary structures
The Ab primary structures were deduced by cDNA sequencing from cloned trioma cells. Briefly, poly(A)+ RNA was isolated from by using Dynabeads® mRNA DIRECT™ Kit (Invitrogen, Carlsbad, CA). Purified mRNA was then employed as the reaction template in reverse transcription polymerase chain reactions (RT-PCR). The RT-PCR was carried out with Titan One Tube RT-PCR System™ (Roche Diagnostics Corporation, Indianapolis, IN). PCR primers (1 μM) used to amplify human VH and VL were the HuVH-JH set (forward: 5'-caggt caact taagg gagtc tgg-3' and reverse: 5'-tgaga gacgg tgacc gtggt ccc-3') and the HuVλ set (forward: 5'-tccta tgtgc tgact cagcc acc-3' and reverse: 5'-accta ggacg gtgac cttgg tccc-3'), respectively. The 37 temperature cycles include: one 2-min denature cycle of 94°C; 35 cycles of 3-min denaturation at 94°C, 1-min annealing and extension at 68°C; and a final 10-min extension cycle of 68°C. Single banded PCR fragments were seperated by 2% agarose gel electrophoresis. The DNA fragments were purified from gel by Wizard PCR Preps DNA purification system (Promega, Madison, WI). The purified products were subjected to nucleotide sequencing. Sequences were verified (Molecular Clinical Diagnostic Laboratory, Dr. Chip Biotechnology, Inc., Taipei, Taiwan) and converted to corresponding amino acids.
Isoelectric point electrophoresis and affinity analyses
The isoelectric point of secreted IgG was examined by Novex IEF Gels (Invitrogen). Desalted and dialyzed protein samples were mixed with IEF sample buffer at 1:1 (v/v) ratio. Electrophoresis was performed at following condition: 100 V for 1 h, 200 V for 1 h and 500 V for 30 mins. After electrophoresis, gels were removed from gel cassette and fixed in 10% trichloroacetic acid for 30 mins. Fixed gel was developed by Coomassie brilliant blue staining or silver staining. The broad-range calibration kit for pI determinations (#17-0471-01, pH 3–10; GE Healthcare Bio-Sciences) was included as the standard. The isoelectric point of interested proteins was calculated by Phoretix 2D Elite software (Nonlinear Dynamics, Durham, NC).
The affinity of the mAb was determined against CD152-muIg with an IAsys optical biosensor (Affinity Sensors, Cambridge, UK) according to the manufacturer's instructions. Briefly, 200 μg/ml dialyzed and diluted CD152-muIg was immobilized on the activated surface of carboxymethyl dextran cuvettes in 10 mM of sodium acetate buffer at pH 3.8. After conditioning with 10 mM HCl, immobilization of 2 mg/mL CD152-muIg resulted in a response of 1100 arc sec. This represents the highest immobilization response for CD152 and gives a ligate binding capacity (Rmax) of 300 arc sec. Serial dilutions of the mAb in PBS, i.e. 1.34 × 10-9 M, 6.70 × 10-9 M, 1.34 × 10-8 M, 2.68 × 10-8 M and 5.36 × 10-8 M, were added to the CD152-coated cuvettes (final volume, 50 μl). Affinity constants (Kd) were calculated from these measurements as kdiss/kass by using the FASTFIT® program provided by the manufacturer.