Amplification of Toll-like receptor-mediated signaling through spleen tyrosine kinase in human B-cell activation

Background: B cells are activated by combined signals through the B-cell receptor (BCR) and CD40. However, the underlying mechanisms by which BCR signals synergize with Toll-like receptor (TLR) signaling in human B cells remain unclear. Objective: We sought to elucidate a role of spleen tyrosine kinase (Syk), a key molecule of BCR signaling, in TLR-mediated activation of human B cells. Methods: Human naive and memory B cells were stimulated with combinations of anti-BCR, soluble CD40 ligand, and CpG. Effects of the Syk inhibitors on several B-cell functions and expression of TLR9, TNF receptor-associated factors (TRAFs), and phospho-nuclear factor κB in B cells were assessed. Results: Activation of BCR synergized with CD40- and TLR9-mediated signals in driving robust proliferation, cell-cycle progression, expression of costimulatory molecules, cytokine production, and immunoglobulin production of human B-cell subsets, especially memory B cells. However, the Syk inhibitors remarkably abrogated these B-cell functions. Notably, after stimulation through all 3 receptors, B-cell subsets induced marked expression of TLR9, TRAF6, and phospho-nuclear factor κB, which was again significantly abrogated by the Syk inhibitors. Conclusion: Syk-mediated BCR signaling is a prerequisite for optimal induction of TLR9 and TRAF6, allowing efficient propagation of TLR9-mediated signaling in memory B cells. These results also underscore the role of Syk in aberrant B-cell activation in patients with autoimmune diseases.