BAFF 3-mer and 60-mer bind BCMA and TACI, but only BAFF 60-mer, BAFF oligomers, and membrane-bound BAFF activate a reporter signaling pathway dependent on ligand oligomerization. (A) Jurkat T cells expressing a BCMA:Fas chimerical receptor were exposed for 16 hours to various concentrations of BAFF 3-mer or 60-mer. Cells were also exposed to Flag-BAFF (3-mer) alone or in the presence of a cross-linking anti-Flag antibody, or to Fc-BAFF (6-mer). Cell viability was measured with the PMS/MTS assay. (B) Jurkat BCMA:Fas cells were incubated with various ratio of 293T cells transfected with full-length BAFF R125A R126A (293T-BAFF uncleaved) or with empty plasmid (293T mock). Myc-BAFF (3-mer) and BAFF A134 (60-mer) were used instead of cells to calibrate the assay. BAFF present in transfected 293T cells was quantified by ELISA. The highest concentration used (40 ng/mL) corresponds to an effector to target ratio of 1:1. Maximal cell death obtained with this assay was approximately 50%. (C) BAFF 3-mer and BAFF 60-mer were immunoprecipitated with TACI-Fc. Inputs and immunoprecipitates (IP) were analyzed by Western blotting using an anti-BAFF antibody (Buffy-2). The same blot was reprobed with an anti-Fc antibody (bottom). The higher band of BAFF carries an N-linked oligosaccharide on asparagine 242, a site that is not used in mammalian cells.⁹  Note that recombinant TACI-Fc is partially processed at Lys¹⁰⁸ and Arg¹¹⁰.²⁸  (D) Recombinant BCMA-Fc, TACI-Fc, and BAFF-R-Fc were immobilized on ELISA plates and exposed to a fixed concentration of Flag-BAFF, whose binding was visualized with anti-Flag secondary reagents. BAFF 3-mer and BAFF 60-mer were added at the indicated concentrations to compete with Flag-BAFF binding and therefore reduce the ELISA signal.