In one of the earliest events in the initiation of antigen-driven

In one of the earliest events in the initiation of antigen-driven antibody responses na?ve IgM- and IgD-expressing B cells enter germinal centers where they irreversibly isotype switch to the expression of predominately IgG B cell TAK-441 receptors (BCRs). essential for antibody memory responses including enhanced calcium responses in IgG BCR expressing B cells (Wakabayashi et al. 2002; Horikawa et al. 2007; Waisman et al. 2007) and distinct gene expression profiles (Horikawa et al. 2007). Engels (Engels et al. 2009) provided a molecular basis for downstream signaling differences in IgM and IgG BCRs showing that conserved Ig tail tyrosine (ITT) motif in the mIgG cytoplasmic domain was phosphorylated upon BCR crosslinking and recruited the adaptor Grb2 to the IgG BCR resulting in enhanced calcium responses and B cell proliferation. Collectively these studies provide convincing evidence that this IgG tail plays a central role in memory responses and in downstream signaling in B cells (Treanor et al. 2010) provided evidence that BCR crosslinking functions to remodel the actin cytoskeleton allowing BCRs that are confined in the resting state by actin ‘fences’ to diffuse and encounter early BCR signaling components or alternatively to escape TAK-441 inhibitory interactions. Using live cell imaging these authors showed that diffusion of the BCR in resting B cells was restricted by an ezrin-actin network and disruption of the network PTP2C resulted in spontaneous BCR signaling. Subsequent studies showed that BCR-triggered reorganization of the actin cytoskeleton was required for the formation of signaling active BCR clusters (Treanor et al. 2011) and that reorganization may allow for the conversation of the BCR with CD19 to facilitate signaling (Mattila et al. 2013). These studies raise the question: at the molecular level what is meant by the physical crosslinking of BCRs by multivalent Ag? By several experimental criteria including FRET (Tolar et al. 2005; Sohn et al. 2008) single molecule diffusion measurements (Tolar et al. 2009a; Liu et al. 2010a; Liu et al. 2010b) and recently super resolution stochastic optical reconstruction microscopy (STORM) (Mattila et al. 2013); (Lee and Pierce unpublished observation) TAK-441 the majority of BCRs in resting cells do not appear to be in higher order oligomers but form large clusters in response to Ag (Pierce and Liu 2010b). However based on biochemical analyses of immunoprecipitated BCR (Schamel and Reth 2000) and a quantitative bifluorescence complementation assay (Yang and Reth 2010) an alternative model for the effect of multivalent Ag on BCR activation has been proposed in which BCRs TAK-441 on resting B cells exist as auto-inhibited oligomers that multivalent Ags serve to open into signaling active monomers. This de-oligomerization model was attractive as although it required multivalent Ag binding it did not require that antigenic epitopes around the Ag be arrayed in any particular fashion as might be predicted TAK-441 if multivalent Ags were required to bring BCRs into a well-defined oligomeric structure. However both the BCR oligomerization and de-oligomerization models are similar in that in both models multivalent antigens serve the same function namely to actually crosslink BCRs altering their resting state business. The Ag valency requirement for BCR activation is usually important with regard to one of the fundamental functions of the BCR namely to discern the B cells’ affinity for Ag. The affinity of bivalent BCRs for multivalent Ags can be obscured by the avidity of the conversation whereas monovalent engagement of Ag by the BCR would be exquisitely sensitive to the BCR’s affinity for the Ag. Can monovalent Ags activate B cells under any conditions? The answer appears to be yes and that although physical crosslinking of BCRs is sufficient to induce signaling it may not always be necessary. In particular it appears that the context in which B cells encounter Ag may influence the valency requirement. Recently evidence has accumulated that this relevant mode by which B cells encounter Ag may not be in solution but rather on the surfaces of antigen presenting cells (APCs) (Cyster 2010). Batista (Batista et al. 2001) first described B cells responding to Ag around the surfaces of APCs resulting in the formation of immunological synapses. Subsequent high resolution imaging.