BAFF 60-mer binding to BAFF receptor 3 utilizes the NF-kappa B1 signaling pathway to hyperactivate B cells

B cell activating factor (BAFF) is a critical cytokine for survival, differentiation, proliferation, and antibody production of B cells. Abnormal B cell activation by BAFF has been implicated in diseases such as Systemic Lupus Erythematous (SLE) and rheumatoid arthritis. BAFF is expressed as a membrane bound 3-mer and then cleaved to its soluble form where it can form a 60-mer. It is not completely understood how the 3-mer versus 60-mer form can differentially activate B cells. Using surface plasmon resonance (SPR), we determined that human BAFF 60-mer bound strongly to soluble murine BAFF receptor 3 (BR3), Transmembrane activator and CAML interactor (TACI), and B-cell maturation antigen (BCMA), while human BAFF 3-mer only bound to soluble murine BR3. We treated purified mouse splenic B cells with BAFF 3-mer, BAFF 60-mer, or left untreated in the presence of a control antibody or mBAFFR-Fc, which only blocks BAFF binding to BR3. A global transcriptomics study revealed that BAFF 60-mer upregulated genes involved in B cell activation and NF-κB signaling, compared to BAFF 3-mer treatment. mBAFFR-Fc treatment reduced the expression of many of the genes involved in B cell activation and NF-κB signaling. We next assessed B cell energy metabolism utilizing the Seahorse mitochondrial stress test and glycolysis stress test. B cells treated with BAFF 60-mer had significantly increased glucose oxidation by oxidative phosphorylation (OXPHOS) and aerobic glycolysis compared to the 3-mer treated B cells, and this result was attenuated by the addition of mBAFFR-Fc. Treatment with inhibitors that block the NF-κB pathway (using BMS) or only the NF-κB1 pathway (using BI 605906) attenuated OXPHOS and aerobic glycolysis. BAFF 60-mer treatment increased mitochondrial density and mitochondrial membrane potential (MMP), while decreasing cellular reactive oxygen species (ROS) production indicating generation of healthier mitochondria. Treatment with mBAFFR-Fc or BMS impaired the BAFF 60-mer mediated changes to the mitochondrial density, MPP, and ROS. Interestingly, a mitochondrial substrate metabolism assay found that glycerol 3-PO4 and succinate were significantly utilized by the B cells. While the addition of BAFF, mBAFFR-Fc, or BMS did not significantly affect glycerol 3-PO4 usage, succinate utilization significantly increased after BAFF 60-mer treatment. BAFF 60-mer treatment increased glucose uptake which was reduced after mBAFFR-Fc treatment. In contrast, BMS attenuated both the basal and BAFF 60-mer induced glucose uptake. Altogether, these results show that BAFF 60-mer binding, via the BR3, hyperactivates B cells by increasing metabolic activity, mitochondrial health, succinate utilization, and glucose uptake as a result of NF-κB1 activation. Furthermore, our research suggests that BAFF 60-mer formation may be a potential target for future anti-BAFF biologics for diseases such as SLE.