BAFF-R, a Novel TNF Receptor That Specifically Interacts with BAFF

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Science  16 Aug 2001:

DOI: 10.1126/science.1061965

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  • Abstract
    BAFF-R, a Novel TNF Receptor That Specifically Interacts with BAFF
    Jeffrey S. Thompson, Sarah A. Bixler, Fang Qian, Kalpit Vora, Martin L. Scott, Teresa G.Cachero, Catherine Hession, Pascal Schneider, Irene D. Sizing, Colleen Mullen, Kathy Strauch, Mohammad Zafari, Christopher D. Benjamin, Jurg Tschopp, Jeffrey L. Browning, Christine Ambrose

    Supplementary Material

    Methods for Expression Cloning of BAFF-R.

    An oligo-dT primed directionally cloned cDNA library was made from BJAB cell RNA in the expression vector, CH269, a derivative of pCEP4 (Invitrogen). Soluble human BAFF [amino acid (aa) 136-285] was expressed in Pichia pastoris and purified from the supernatant using anion exchange chromatography followed by gel filtration. Biotinylated human BAFF was generated by incubating 0.4 mg of BAFF in PBS with 120 namel of Biotin-XX from a FluoReporter Mini-Biotin-XX Labeling Kit (Molecular Probes) and 61 namel of 1 M sodium carbonate. The protein was stirred for 1 hour at room temperature and then dialyzed against PBS at 4°C. The BJAB cDNA library was transfected into Escherchia coli DH10B cells and seeded in a 96-well format as pools of approximately 2500 independent clones per well. DNA was prepared from these pools using the Qiagen BioRobot 9600 Turbo miniprep kits. The DNA pools were transfected using Lipofectamine (Life Technologies) into 293 containing the EBNA-1 gene (Invitrogen) cells seeded into fibronectin-coated six-well dishes. At 48 hours post-transfection, the medium was removed and the cells were washed with plate assay wash buffer (20 mM HEPES, 0.5 mg/ml bovine serum albumin, 0.1% NaN3). Cell monolayers were overlaid with 100 ng/ml biotinylated human BAFF in binding buffer (PBS, 2% fetal bovine serum, 0.1% NaN3) and incubated at room temperature for 1 hour. The BAFF solution was removed and the cells were washed and fixed by incubation with 1.8% formaldehyde-0.2% glutaraldehyde in PBS for 5 min. Cells were again washed and then incubated for 30 min. with alkaline phosphatase-conjugated streptavidin (SAV-AP) (Jackson ImmunoResearch) at a 1:3000 dilution in binding buffer. Cells were washed and stained with fast red/napthol phosphate (Pierce). Cells binding the biotin-BAFF/SAV-AP complex were identified by the presence of a red precipitate after inspection under low power microscopy. Secondary screening entailed plating out the DH10B glycerol stocks of the BAFF binding pools for single colonies, creating pools of 100, and repeating the BAFF binding assay as described above. Secondary screen positive pools were similarly broken down to individual clones, assayed for BAFF binding and then sequenced. The identified cDNA (JST576) contained an intron sequence, and so the open reading frame of BAFF-R was determined using the Genscan exon prediction software and by PCR analysis. A minor splice variant of human BAFF-R was detected by PCR, which results in the addition of a single alanine residue after amino acid 46.


    Method for cloning of murine BAFF-R.

    Murine BAFF-R was cloned by screening one million lambda phage from the murine A20 cell line cDNA library purchased from Stratagene (La Jolla, CA) using a 425-bp Eco NI fragment from the human BAFF-R cDNA, as the probe. A common splice variant for murine BAFF-R, identified in the library clones, is deleted for the intracellular domain amino acids 117 to 127 (Web fig. 1).


    Method for analysis of BAFF binding to BAFF-R or TACI transfected cells. 293E cells were co-transfected with either full length BAFF-R (JST576), human TACI (CA336) or human LTnameR (CA259) and a GFP expressing plasmid using Lipofectamine 2000 (Life Technologies). At 18 to 20 hours post-transfection, cells were detached from the plates using 5 mM EDTA in PBS. For 1 hour on ice, 5 x 106 cells/ml were incubated with various concentrations of biotinylated BAFF. After the cells were washed, BAFF was detected using a 1:100 phycoerythrin-conjugated streptavidin (Jackson Immuno Research) (Web fig. 2).

    Method for surface staining B cell lines with BAFF and anti-receptor antibodies. Of each B cell line, 5 x 105 cells/sample were incubated with 100 nameg/ml heat-aggregated human IgG for 15 min on ice. One microgram per milliliter biotinylated human BAFF or 10 nameg/ml of the antibodies as follows were then added and incubated on ice for 30 min: mouse anti-TACI monoclonal (C4.D7.4), hamster anti-BCMA monoclonal (C4.E2.1), or rabbit anti-BAFF-R polyclonal antibody (Rb 97). Dilutions were made in FACS buffer. The cells were washed with FACS buffer and then incubated for 30 min on ice with streptavidin conjugated with phycoerythrin, PE-conjugated anti-mouse IgG, PE conjugated anti-rabbit IgG (Jackson ImmunoResearch) or PE conjugated anti-hamster IgG for anti-BCMA antibody detection (BD PharMingen), all at a 1:100 dilution. The cells were again washed with FACS buffer and resuspended FACS buffer containing 20 namel/sample Via Probe (7-amino-actinomycin D) (BD PharMingen). The live cells were analyzed by FACS for PE fluorescence (Web figs. 3 and 4). Data is shown in Web table 1.


    Supplemental Figure 1. Homology between the human BAFF-R and human BCMA intracellular domains.


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    Supplemental Figure 2. BAFF binding to BAFF-R and TACI transfected cells. The relative ability of BAFF to bind to full-length BAFF-R (name), TACI (name) or LTnameR (name) transfected 293EBNA cells was compared using FACS analysis. Various dilutions of biotinylated BAFF were added to a constant number of transfected cells.


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    Supplemental Figure 3. BAFF-R:Fc blocks the ability of BAFF to bind to BJAB cells. The ability of BAFF-R:Fc (name), TACI:Fc (name) or a nonspecific fusion protein, LTnameR:Fc (name), to block the binding of BAFF to the receptor expressing BJAB cells was tested using FACS analysis. Various concentrations of receptor Fc fusion protein BAFF-R:Fc, TACI:Fc (aa 1-115 downstream of murine IgG-(-signal sequence and 5' of the Fc region of hIgG1) or hLTnameR:Fc [P. Crowe et al., Science264, 707 (1994)] were preincubated with 200 ng/ml biotinylated BAFF and then added to the BJAB cells. BAFF binding was detected using 1:00 dilution of PE conjugated streptavidin (Jackson ImmunoResearch).


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    Supplemental Figure 4. Southern blot analysis on A/J and A/WySnJ genomic DNA. DNA was prepared from the thymus [(B. Schiemann et al., Science, in press (this issue)] and digested with various restriction enzymes overnight. The DNA was run on an agarose gel, processed and then blotted to a nylon membrane. The membrane was hybridized with a Sac I + Bgl 2 fragment from the murine BAFF-R cDNA. P, the A/J parental strain; M, the A/WySnJ strain.


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    Supplemental Table 1. BAFF binding profile and surface receptor expression on various B cell lines. BAFF binding to the cell lines was determined by FACS analysis using biotinylated BAFF. Monoclonal (BCMA and TACI) or polyclonal (BAFF-R) antibodies were used to determine the level of surface receptor by FACS. The level of receptor is ranked according to the highest expressing cell line for each antibody.
    Human B Cell lineTypeBAFF BindingBCMATACIBAFF-R
    BJABBurkitt Lymphoma++++--++++
    RamosBurkitt Lymphoma++--+
    NamalwaBurkitt Lymphoma+--+
    RajiBurkitt Lymphoma+++-++++
    SKW6.4B cell, IgM secreting+++-+++++
    RPMI 1788Peripheral blood, IgM secreting+++++/-+++++++
    IM-9Lymphoblast, Ig secreting++++-++++++
    NC-37Lymphoblast+++-+++++
    RPMI 8226Myeloma+/--+/--
    U266Myeloma-+/---
    DaudiBurkitt lymphoma----