Megaapril

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.

IL-10-GFP mice were injected IV on D0, D2, and D5 with 20 × 10⁶ apoptotic thymocytes. On D7 mice were sacrificed and blood, peritoneal lavage and spleens harvested. Splenic CD19^+ve B cells were FACs sorted into IL-10-GFP^{+ve or −ve} fractions, phenotyped and cultured for 10 days in the presence of MegaAPRIL [Adipogen (200 ng/ml)], CpGB [ODN1826 Eurofins MWG Operon (1 μg/ml)], and IL-4 [R&D System (50 ng/ml)], after which culture supernatants were checked for IgM and IgG levels (Ready-SET-Go ELISA kit eBioScience).

As TACI-S isoform transfected cells had a greater affinity for ligands than TACI-L cells in binding assays, we then turned to a cell free system to quantify these differences. For this, FLAG-TACI isoform proteins were expressed in HEK-293T cells and purified with anti-FLAG beads. Highly purified recombinant TACI-L and TACI-S were then fluorescently labeled at their N-termini for microscale thermophoresis (MST) using the Monolith NT Protein Labeling Kit RED-NHS (NanoTemper Technologies, München, Germany). Briefly, proteins at concentrations of 20 μM were incubated with 4X dye at a ratio of 1:1 in labeling buffer. To determine the Kd values of TACI-L and TACI-S for BAFF or APRIL, 100 nM of labeled FLAG-tagged TACI-L and TACI-S proteins were incubated with increasing concentrations (from 0 to 10⁴ nM) of BAFF or Mega-APRIL (Adipogen), for 30 min at room temperature in binding buffer (PBS) (17 (link)). The samples were then centrifuged, loaded into premium-coated capillaries (NanoTemper Technologies) and fluorescence values from the binding reactions determined using the Monolith NT.115 (Nano Temper Technologies). Binding data was analyzed using Affinity analysis software (NanoTemper) to determine the Kd values for each TACI isoform for BAFF and APRIL.