High-throughput primary B cell cytokines assay for drug screening in multiple sclerosis

BACKGROUND: Remarkable success of anti-CD20 B cell depleting therapies in multiple sclerosis (MS) highlights antibody-independent role of B cells in driving inflammatory relapsing-remitting stage of the disease. Yet, long-term B cell depletion leads to increased infections, pointing to the need for more targeted treatments. OBJECTIVES: To establish a high-throughput translational assay of primary B cells for drug screening. RESULTS: We present an assay of primary B cells isolated from MS patients and healthy individuals. We tested various methods of B cell isolation, compared impact of combinations of different stimuli, including 2-signal and 3-signal mode of activation at multiple seeding densities on production of proinflammatory cytokines Interleukin 6 (IL-6), Tumor Necrosis Factor (TNF) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), using 384-well microplate with semi-automation. Ibrutinib, a small molecule that suppresses B cell proliferation and survival by inhibiting protein kinases, including Bruton's tyrosine kinase (BTK), was used to validate the miniaturized B cell assay for drug screening. Seven BTK inhibitors, Ibrutinib and six novel compounds currently under later stage clinical development for autoimmune disease, were evaluated with concentration-response as proof-of-concept validation of the assay. CONCLUSIONS: An optimized primary B cells assay in 384-well microplate format was established and validated by Ibrutinib. Moreover, we demonstrated that six clinical stage BTK inhibitors considerably impact B cell activation and secretion of IL-6, TNF and GM-CSF.