Abstract

B cells form a crucial component of humoral immunity within the adaptive immune system, being involved in antigen presentation, cytokine secretion and production of antibodies. B cells signal through the B cell receptor (BCR), a transmembrane receptor composed of an immunoglobulin plus the signal transduction moiety CD79. Stimulation of the BCR invokes a signalling cascade with multiple proteins phosphorylated including spleen tyrosine kinase (SYK), Bruton’s tyrosine kinase (BTK) and phospholipase C gamma 2 (PLCγ2). The cascade plays a key role in B cell survival, proliferation, and function, and is a desirable target for both B cell malignancies and autoimmune diseases including rheumatoid arthritis. BCR-mediated phosphorylation can be studied in detail using flow cytometry (“phosflow”), which offers higher throughput compared to techniques such as Western blotting and allows simultaneous cell phenotyping using cell surface markers. Here, we used phosflow to investigate the impact of anti-IgM stimulation on BTK and PLCγ2 phosphorylation in human B cells. In addition, we investigated the impact of the SYK inhibitor BAY-61-3606 and the BTK inhibitor zanubrutinib on anti-IgM stimulation-induced BTK and PLCγ2 phosphorylation. We show here that anti-IgM stimulation results in a concentration dependent increase in protein phosphorylation, with peak BTK phosphorylation after 30 seconds and PLCγ2 phosphorylation after 2 minutes, highlighting the temporal difference for these proteins in the BCR signalling cascade. BAY-61-3606 inhibited anti-IgM-induced phosphorylation of BTK and PLCγ2 in a concentration-dependent manner in different B cell subsets, including naïve and memory B cells. However, zanubrutinib failed to reduce phosphorylation of either BTK or PLC2γ. Phosflow provides a powerful platform for the kinetic study of protein phosphorylation cascades in immune cell subsets and for high-throughput screening to target these signalling pathways.