As we had found that human TACI-S exhibits greatly enhanced activation in both transduced murine and human B cells (13 (link)), we tested if TACI-S might have an enhanced affinity for ligands as compared to TACI-L, which would provide a potential reason for these signaling differences. For this we separately incubated BJAB or HEK-293T cells bearing TACI-L or TACI-S (or both) with increasing amounts of FLAG-tagged multimerized MegaAPRIL (Adipogen) or MegaBAFF (Enzo) (0–800 ng). After this, 1 μg/mL biotin-anti-FLAG monoclonal M2 antibody was added to multimerize the ligands (18 (link)). Cells were washed, streptavidin-phycoerythrin-PE (Becton Dickinson) added and then examined by FACS (LSRII) (10 (link), 13 (link)). To ensure comparable expression of TACI receptors on BJAB cells, GFP expression, integral to both constructs, was determined (13 (link)). For HEK-293T cells, comparable TACI expression was determined by FACS using an anti-TACI antibody which binds both TACI isoforms (mAb174, R&D). As our FRET studies suggested that mixed short and long isoforms may occur on human B cells, we also compared APRIL binding on BJAB and HEK-293T cells transduced with both TACI-S and TACI-L, separately labeled with either mCherry or YFP to validate transduction of both isoforms.
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