Protective antibody responses to vaccination or infection depend on affinity maturation, a process by which high-affinity germinal center (GC) B cells are selected based on their ability to bind, gather and present antigen to T follicular helper (Tfh) cells. Here we show that human GC B cells have intrinsically higher affinity thresholds for both B cell antigen receptor (BCR) signaling and antigen gathering as compared to naïve B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to antigen-affinity- and Tfh cell-dependent differentiation to plasma cells. GC B cells bound antigen through highly dynamic, actin- and ezrin-rich pod-like structures that concentrated BCRs. The behavior of these structure was dictated by the intrinsic antigen-affinity thresholds of GC B cells. Low affinity antigens triggered continuous engagement and dis-engagement of membrane associated antigens whereas high affinity antigens induced stable synapse formation. The pod-like structures also mediated affinity-dependent antigen internalization by unconventional pathways distinct from those of naïve B cells. Thus, intrinsic properties of human GC B cells set thresholds for affinity selection.

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B lymphocytes use B cell receptors (BCRs) to sense the chemical and physical features of antigens. The activation of isotype-switched IgG-BCR by mechanical force exhibits a distinct sensitivity and threshold in comparison with IgM-BCR. However, molecular mechanisms governing these differences remain to be identified. In this study, we report that the low threshold of IgG-BCR activation by mechanical force is highly dependent on tethering of the cytoplasmic tail of the IgG-BCR heavy chain (IgG-tail) to the plasma membrane. Mechanistically, we show that the positively charged residues in the IgG-tail play a crucial role by highly enriching phosphatidylinositol (4,5)-biphosphate (PI(4,5)P2) into the membrane microdomains of IgG-BCRs. Indeed, manipulating the amounts of PI(4,5)P2 within IgG-BCR membrane microdomains significantly altered the threshold and sensitivity of IgG-BCR activation. Our results reveal a lipid-dependent mechanism for determining the threshold of IgG-BCR activation by mechanical force.

B cell activation is regulated by the stimulatory or inhibitory co-receptors of B cell receptors (BCRs). Here, we investigated the signaling mechanism of Fc receptor-like 1 (FcRL1), a newly identified BCR co-receptor. FcRL1 was passively recruited into B cell immunological synapses upon BCR engagement in the absence of FcRL1 cross-linking, suggesting that FcRL1 may intrinsically regulate B cell activation and function. BCR cross-linking alone led to the phosphorylation of the intracellular Y281ENV motif of FcRL1 to provide a docking site for c-Abl, an SH2 domain-containing kinase. The FcRL1 and c-Abl signaling module, in turn, potently augmented B cell activation and proliferation. FcRL1-deficient mice exhibited markedly impaired formation of extrafollicular plasmablasts and germinal centers, along with decreased antibody production upon antigen stimulation. These findings reveal a critical BCR signal-enhancing function of FcRL1 through its intrinsic recruitment to B cell immunological synapses and subsequent recruitment of c-Abl upon BCR cross-linking.

Lymphocytes have evolved sophisticated signaling amplification mechanisms to efficiently activate downstream signaling after detection of rare ligands in their microenvironment. B cell receptor microscopic clusters (BCR microclusters) are assembled on the plasma membrane and recruit signaling molecules for the initiation of lymphocyte signaling after antigen binding. We identified a signaling amplification loop derived from phosphatidylinositol 4,5-biphosphate (PIP) for the sustained B cell activation. Upon antigen recognition, PIP was depleted by phospholipase C-γ2 (PLC-γ2) within the BCR microclusters and was regenerated by phosphatidic acid-dependent type I phosphatidylinositol 4-phosphate 5-kinase outside the BCR microclusters. The hydrolysis of PIP inside the BCR microclusters induced a positive feedback mechanism for its synthesis outside the BCR microclusters. The falling gradient of PIP across the boundary of BCR microclusters was important for the efficient formation of BCR microclusters. Our results identified a PIP-derived amplification loop that fuels the sustained initiation of B cell activation.