B cell antibody responses are triggered by the binding of antigen to the clonally distributed B cell antigen receptors (BCRs). Over the last several years a great deal has been learned about the biochemistry of the complex signal cascades triggered by BCR antigen engagement. However, what remains relatively poorly understood are the molecular events that trigger the initiation of signaling. We understand that the BCR, like other members of the multichain immune recognition receptor family, is composed of a ligand binding chain, for the BCR a membrane form of Ig (mIg) the most common forms of which, mIgM and mIgD, have short cytoplasmic domains with no capacity to interact directly with the components of the signaling cascades. To do so the mIg associates with two additional chains, Ig alpha and Ig beta, that contain within their cytoplasmic domains immunoregulatory tyrosine activation motifs (ITAMs). The BCR has no inherent kinase activity but following antigen binding one of the first events observed is the phosphorylation of the BCR ITAMs by the Src-family kinase, Lyn. This project represents an approach to gain an understanding of the changes that occur in the BCR following antigen binding that allow Lyn to discriminate antigen-bound BCRs from the unbound BCRs. Based on the crystal structure of antigens bound to Fab of antibodies there is at present no evidence for an antigen-induced structural change in the BCR that could propagate the information that the BCR has bound antigen from the BCR ectodomains, across the membrane, to the cytoplasmic domains. In the absence of such an allosteric effect of antigen binding one is left with antigen-induced clustering of the BCR as the trigger for the initiation of signaling. Thus, the goal of this project is to gain an understanding of how antigen induces BCRs to cluster and how BCR clustering leads to the initiation of signaling. The critical events that trigger signaling are likely to occur within seconds of antigen binding to the BCR and to be highly dynamic, involving many weak protein-protein and protein-lipid interactions. In general, the biochemical approaches that have been used so effectively to describe the BCR signaling pathways are inadequate to capture events that occur as rapidly and as transiently as those predicted to initiate BCR signaling. In addition, antigen binding to the B cell involves a dramatic spatial change in the BCRs resulting in their patching and capping and the formation of an immune synapse. All this potentially important spatial information is lost with the addition of detergents to cells for biochemical analyses. Consequently we have taken advantage of new live cell imaging technologies that allow analyses of the BCR and components of the BCR signaling pathway with the temporal and spatial resolution necessary to view the earliest events in B cell activation at the single molecule level without the complications that the addition of detergents introduce. Using live cell imaging, we showed that in B cells responding to antigen presented on a planar lipid bilayer, simulating an antigen presenting cell, BCRs form signaling active microclusters by a mechanism that does not require physical crosslinking of BCRs by multivalent antigens. We observed that in response to either monovalent or multivalent antigens the BCRs accumulate and form microclusters at the initial points of contact of the B cell membrane with the bilayer. Clustering did not depend on signaling active BCRs revealing an intrinsic tendency of the BCRs to cluster. The microclusters grew by trapping mobile BCRs and the larger clusters were actively organized into an immune synapse. The kinetics of these events and signaling were identical for monovalent and multivalent antigens. We determined that the membrane-proximal ecto-domain of the BCR mIg was both necessary and sufficient for BCR oligomerization and signaling. BCRs that contained a mIg in which Cmu4 was deleted failed to cluster and to signal when engaging monovalent antigen. Conversely, Cmu4 expressed alone on the B cell surface spontaneously clustered and activated B cells. These findings lead us to propose a novel mechanism by which BCRs form signaling active microclusters that we termed the conformation induced oligomerization model for the initiation of BCR signaling. According to our model BCR in the resting state are not in an oligomerization receptive conformation such that random bumping has no repercussion. The binding of antigen on an opposing membrane exerts a force on the BCR to bring it into an oligomerization receptive form so that when two antigen-bound BCRs bump they oligomerize. We also approached a fundamental question in B cell biology namely what advantage is conferred on B cell in activation by high affinity, isotype switched BCRs such as those expressed by memory B cells. We demonstrated that high affinity BCRs, as compared to low affinity BCR form immobile, signaling active BCR clusters more efficiently providing evidence that affinity discrimination is a BCR intrinsic function. We also determined that as compared to mIgM-containing BCRs, mIgG BCRs more efficiently form signaling active BCR oligomers by a mechanism that depends on the cytoplasmic domain of the mIgG. These results are important in providing a molecular understanding of the functional advantage of high affinity, isotype-switched BCRs. Over the last year we mapped the functional difference between IgG and IgM BCRs to the 15 membrane proximal region of the mIgG cytoplasmic tail. We showed that this highly conserved region of the cytoplasmic tail associated with synapse associated protein 97 (SAP97), a member of the membrane associated guanylate kinase family of scaffolding molecules that play a role in controlling receptor density and signal strength at neuronal synapse. SAP97 accumulated and bound to IgG BCRs in the immune synapses that formed in B cells in response to antigen engagement. Deletion of SAP97 in IgG-expressing B cells by RNA interference or by targeted gene knocking out in SAP97-KO mice impaired immune synapse formation and the initiation of BCR signaling. This finding is important in showing that the enhanced response of memory B cells is encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immune synapse. Despite their importance, little is known about how antigens trigger human memory B cells, even though our understanding of the molecular basis of antigen activation of B cells in model systems has advanced considerably. We used quantitative, high-resolution, live cell imaging at the single cell and single molecule levels to describe the earliest antigen-driven events in human isotype-switched, IgG-expressing memory B cells and compare these to those in IgM-expressing nave B cells. We showed that human memory B cells are more robust than nave B cells at each step in the initiation of BCR signaling, including interrogation of Ag-containing membranes, formation of sub-microscopic BCR oligomers and recruitment and activation of signaling-associated kinases. Despite their robust response to antigen, MBCs remained highly sensitive to FcγRIIB-mediated inhibition. We also demonstrated that in the absence of antigen a portion of memory B cells BCRs spontaneously oligomerized and phosphorylated kinases accumulated at the membrane and speculate that heightened constitutive signaling may play a role in maintaining memory B cell longevity.
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