(569.7) BAFF 60-mer binding to BAFF receptor 3 utilizes the NF-κB1 signaling pathway to hyperactivate B cells

Poster Board Number: E261

Melissa Lempicki (East Carolina University), Vlad Serbulea (University of Virginia), Saikat Paul (East Carolina University), Clint Upchurch (University of Virginia), Srabani Sahu (University of Virginia), Jake Gray (East Carolina University), Gorav Ailawadi (University of Virginia), Brandon Garcia (East Carolina University), Coleen McNamara (University of Virginia), Norbert Leitinger (University of Virginia), Akshaya Meher (East Carolina University)

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.  

Support or Funding Information

This study was supported by National Institutes of Health (NIH) NHLBI R01 HL146685, American Heart Association (AHA) 14SDG20380044, University of Virginia LaunchPad for Diabetes Innovation Grant, and funds from East Carolina University to A.K. Meher and NIH R01 DK096076 to N. Leitinger.