Rabbits were obtained from the allotype-defined pedigreed colony maintained at the National Institute of Allergy and Infectious Diseases. The animal studies described here were reviewed and approved by the animal care and use committees of NIAID/NIH (ASP LI6) and of the Spring Valley Laboratories, Inc. where the NIAID allotype-defined rabbit colony was housed. Normal adult rabbits of known Ig allotypes from our breeding colony, provided whole blood and spleens for flow cytometry, immunohistochemistry, and mRNA detection. In addition, one spleen from a 7-week old rabbit and appendix tissues of young rabbits at 4, 8 and 10 days of age were studied by immunohistochemistry.
Abs and reagents for flow cytometry, cell isolation and ELISA
BAFF was detected using biotin-conjugated goat anti-human BAFF polyclonal Ab (pAb) (Antigenix America Inc.) and rat anti-mouse BAFF monoclonal antibody (mAb) (clone 121809 R&D Systems) that we found to cross-react with rabbit BAFF. BR3 was detected using purified goat anti-human BR3 pAb (R&D Systems) that also cross-reacts with rabbit BR3. Also used were FITC-labeled mouse anti-rabbit CD14 (clone K4), FITC-labeled mouse anti-rabbit CD4 (clone KEN-4), FITC-labeled mouse anti-rabbit CD8 (clone C7) (Antigenix America Inc.), FITC-labeled goat anti-rabbit IgM, FITC-labeled goat anti-rabbit IgG (Southern Biotechnology Associates), biotin-labeled donkey anti-goat IgG, biotin-labeled goat IgG, normal goat IgG (Jackson ImmunoResearch Laboratories, Inc.), and FITC-labeled mouse IgG2a (BD Pharmingen). Biotinylated Abs were visualized by phycoerythrin-(PE)-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc).
Isolation of cell subpopulations
For quantitative analyses of mRNA expression in cell subpopulations, spleen tissues were chopped in small pieces in PBS and pressed through a stainless steel mesh screen with rubber-tipped syringe plunger. Debris and cell clumps were removed by passing the suspension through 70 μm nylon mesh. Freshly isolated spleen cells were stained with mouse anti-rabbit CD14 (Antigenix America) or mouse anti-rabbit CD4 (Serotec) Ab and CD4+ or CD14+ cell subpopulations were isolated with goat anti-mouse IgG covalently bound to Dynabeads (Dynal). To isolate IgM positive B cell subpopulations, cells were stained with biotin-conjugated polyclonal μ-heavy chain specific goat anti-rabbit IgM (Southern Biotechnology Assoc.) followed by Streptavidin-Dynabeads. Viable peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood by density gradient centrifugation on Lympholyte-Mammal (Cedarlane Laboratories Limited). This permitted recovery of lymphocytes and monocytes but eliminated most red cells and granulocytes. IgM positive subpopulations from PBMC were isolated as described above. IgM depleted PBMC (IgM− cells) were then stained with mouse anti-rabbit CD14 mAb and separated into two populations, IgM−CD14+ and IgM−CD14− using anti-mouse IgG-Dynabeads. Upon analyses by flow cytometry, fractions were shown to be at least 90% pure. After isolation, cells were immediately placed into TRIzol™ reagent (Invitrogen) for RNA isolation.
Rabbit BAFF and BR3 cloning
Total RNA was obtained from normal rabbit PBMCs that were immediately placed in TRIzol reagent. PBMC were homogenized and filtered with Qiashredder column (Qiagen). Total RNA was isolated by RNeasy spin column (Qiagen) and precipitated with ethanol. First-stand cDNA was synthesized using the SuperScript first-strand synthesis kit (Invitrogen). The BAFF primers () were designed after searching the rabbit whole genome shotgun (WGS) trace archives database and aligning the sequence with the highly conserved BAFF domains of multiple species. The cDNA was amplified by Platinum® pfx DNA polymerase (Invitrogen). PCR conditions were first melting at 94°C for 2 min, then 30 cycles of amplification: 15 sec at 94°C, 30 sec at 55°C, and 1 min at 68°C. A final extension was at 72°C for 10 min. The rabbit BR3 primers (), were designed based on the ‘working draft’ sequence of genomic DNA in Oryctolagus cuniculus clone LB1-145O11 (AC145540.1) positions 124760 to 123559 on the reverse strand, that appears to contain the sequence of the rabbit homolog of human BR3. Touchdown PCR conditions were melting at 94°C for 30 sec, annealing and elongation at 72°C for 3 min. The annealing temperature was dropped from 72°C at a rate of 2°C for five cycles to 68°C for the remaining 30 cycles. The rabbit BAFF PCR products were cloned into pCR-Blunt II-TOPO (Invitrogen), and sequenced from SP6 and T7 promoter sites with SP6 and T7 primers. The rabbit BR3 PCR products were cloned into pCR 2.1 vector (Invitrogen), and sequenced from the M13 reverse priming and T7 promoter sites with M13 reverse and T7 primers. At least three independent PCR cloning and sequencing experiments were conducted to rule out errors introduced by PCR.
Primers and probes for cloning, expression and quantitation of rabbit BAFF and BR3
Construction, expression, and purification of recombinant rabbit BAFF protein
A construct was designed with a signal sequence of human Ig heavy chain (VH1), a (His)6 tag at the N-terminus, followed by the cDNA sequence of the predicted rabbit BAFF extracellular domain (amino acids 139-290) and a stop codon. The desired product was amplified by two-rounds of PCR from the pCR-Blunt II-TOPO-BAFF construct using primers shown in . After the second-round overlapping PCR, the specific DNA fragment was cloned by KpnI/XhoI ligation into the mammalian cell expression vector pCEP4 (Invitrogen). Following transient transfection, the (His)6-tagged rabbit BAFF protein was expressed and secreted by HEK293F cells (Invitrogen) cultured in free-Style serum-free medium (Invitrogen). Supernatants were collected after 3, 6 and 9 days of culture by centrifugation, filtered through a 0.45-μm membrane, and concentrated tenfold using an ultrafiltration device with a 5-kDa cutoff membrane (Millipore). The concentrate was loaded on a 1-ml HisTrap FF crude column (GE Healthcare) and washed with 40-volumes of washing buffer (PBS containing 500 mM NaCl and 30 mM imidazole, pH 7.4). The purified protein was eluted with elution buffer (PBS containing 500 mM NaCl and 500 mM imidazole, pH 7.4), the imidazole was immediately removed and the protein was concentrated using 5-kDa cut off centrifugal filters (Millipore). The quality and quantity of purified rabbit recombinant BAFF protein ((His)6rBAFF) was monitored by SDS-PAGE, Western blotting and A280 absorbance.
Synthesis of miniBR3
Biotinylated miniBR3 [26
] was synthesized as the C-terminal amide on a 433A peptide synthesizer (Applied Biosystems) using standard Fmoc (fluorenylmethoxy carbonyl) chemistry[26
] (Research Technology Branch, NIAID Peptide Synthesis and Analysis Unit). MiniBR3 was biotinylated at the amino terminus while on the resin using NHS-LC-Biotin reagent (Pierce). The peptide was cleaved from the resin using a mixture of 92% trifluoroacetic acid (TFA; Aldrich), 5% thioanisole (Aldrich), and 3% 3,6-dioxa-1,8-octanedithiol (DODT, Aldrich) for 2.5 hr at room temperature. After removal of TFA by rotary evaporation, the peptide was precipitated by addition of methyl t-butyl ether (MTBA, Burdick & Jackson), then purified by reversed-phase HPLC (acetonitrile/H2O/0.1% TFA). Peptide identity was confirmed by MALDI-TOF mass spectrometry. Spontaneous folding of the fully unprotected peptide in aqueous solution under redox control of reduced and oxidized glutathione was carried out as described [27
]. The progress of the oxidation was monitored by analytical HPLC, and the final product was again purified by HPLC.
Purified rabbit (His)6
rBAFF protein was analyzed by sandwich-ELISA. Briefly, polystyrene 96-well plates (Corning) were coated with 50 μl/well of purified goat anti-human BAFF polyclonal Ab (Antigenix America Inc.) at 2 μg/ml in bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C. All subsequent incubations were done for 1 h at room temperature. Plates were washed six times with 1x TBS (pH 7.4) and blocked with 100 μl of SuperBlock T20 PBS Blocking Buffer (Pierce). Wells were then incubated with serial tenfold-diluted BAFF protein or with serial tenfold-diluted human BAFF recombinant protein as a control. After washing six times with 1x TBS (pH 7.4), wells were further incubated with 50μl/well of 1/1000 diluted rat anti-mouse BAFF mAb (clone 121809, R&D Systems), then detected by horse radish peroxidase-(HRP)-conjugated goat anti-rat IgG secondary Ab (Jackson ImmunoResearch Laboratories). After washing as above, wells were developed with 3,3′, 5,5′-tetramethylbenzidine (TMB) (INOVA Diagnostics) and the resulting OD read at 450 nm. In order to detect the interaction between rabbit (His)6
rBAFF and the synthesized rabbit miniBR3 homolog, rBAFF or control rabbit (His)6
] (600 ng/well) in carbonate buffer (pH 9.6) were coated on plates at 4 °C overnight. After washing as above, the plates were blocked with 100 μl of blocking buffer (Pierce). Wells were incubated with serial twofold dilutions of biotin-labeled miniBR3. After washing as above, wells were incubated with 40 mU/ml of streptavidin-β-peroxidase (POD) conjugate (Roche), developed with TMB and the resulting OD read at 450 nm.
Purified expressed rabbit (His)6rBAFF and control human (hBAFF) (Antigenix) protein were first analyzed using SDS-PAGE 4–15% Tris-HCl Ready gels (Bio-Rad, Hercules, CA) and stained with Coomassie blue. Additional separate sets of SDS gels were electrophoretically transferred onto nitrocellulose membranes and analyzed using cross-reacting biotin-conjugated polyclonal goat anti-human BAFF Ab (Antigenix) and BM Chemiluminescence Blotting kit according to the manufacturer’s instructions (Roche).
Detection of expression levels of BAFF and BR3 mRNA
Quantitative PCR to determine the relative expression levels of BAFF and BR3 mRNA was performed on a 9700HT Sequence Detection System (Applied Biosystems). Synthesized cDNA from isolated PBMCs, and subpopulations of PBMCs and splenocytes was directly used as template for real time PCR using TaqMan 2x PCR Master Mix Reagents Kit (Applied Biosystems). The total volume of the PCR was 25 μl and the PCR conditions were: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C, 15 s for denaturation and 60°C, 1 min for annealing and extension. The primers and probes for BAFF and BR3 and the primers for the control housekeeping gene Peptidylprolyl isomerase A (PPIA) are shown in . Each sample from three independent experiments was run in duplicate. The unit number showing relative mRNA levels in each sample was determined as a value of mRNA normalized against PPIA. Previous studies in our laboratory (Rai G et al., ms in preparation), showed that there was very uniform expression of PPIA in PBMCs collected from rabbits at different time points prior to and after immunization.
Purified cells were stained using standard flow cytometric methodology. Briefly, cells were incubated on ice for 40 min with primary Ab before washing twice with cold PBS containing 1% FCS, followed by incubation with various secondary reagents or secondary Ab. A biotinylated donkey anti-goat IgG Ab (Jackson ImmunoResearch Laboratories, Inc) was used as secondary Ab to detect BR3 on cells stained with purified goat anti-human BR3 Ab (R&D Systems) that cross-reacts with rabbit BR3. Biotinylated Abs were visualized by PE-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc). After washing, cells were analyzed using a FACS-Calibur flow cytometer (BD Pharmingen) and FlowJo analytical software (Tree Star). In order to test whether recombinant rabbit BAFF (rBAFF) would influence staining by anti-BAFF, a fixed quantity of biotin-labeled goat anti-human BAFF polyclonal Ab (Antigenix America Inc.) was incubated with different dosages of rBAFF protein on ice for 40 min, then transferred to isolated PBMC for another 30 min on ice. In additional experiments, different dosages of rBAFF were incubated with isolated rabbit PBMC on ice for 40 min. After washing, a fixed amount of biotin-labeled goat anti-human BAFF pAb was added. Biotinylated Ab was visualized by PE-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Inc).
Cryostat serial sections (7 μm) of rabbit appendix tissues were cut and stained with primary reagents, mouse anti-human CD79a specific for the cytoplasmic domain that cross reacts with rabbit (BD Pharmingen), mouse anti-rabbit CD5 mAb (clone 2B10) [28
] followed by biotin-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) or with cross-reacting biotin-conjugated goat anti-human BAFF pAb (Antigenix). The sections were then incubated with ABC-Alkaline phosphatase kit, labeled cells visualized by Vector blue alkaline phosphatase substrate kit III, and counterstained with Nuclear Fast Red (Vector, Laboratories, Inc.). In other experiments, spleen sections were first stained with mouse anti-rabbit CD14 (Antigenix America) or mouse anti-rabbit CD4 (Serotec) Ab followed by peroxidase-conjugated goat anti-mouse IgG Ab (Jackson ImmunoResearch Laboratories, Inc.) and ABC-Peroxidase kit. Labeled cells were visualized by ABC-DAB substrate kit III. Sections were then stained with biotin-conjugated polyclonal goat anti-human BAFF Ab followed by ABC-Alkaline phosphatase kit, labeled cells were visualized by Vector blue alkaline phosphatase substrate kit III and counterstained with Nuclear Fast Red (Vector, Laboratories, Inc.).