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. Using live cell TIRF microscopy, 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 the ability of the BCR to signal subsequently, revealing clustering to be an intrinsic property of the BCRs. 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. An important prediction from our model is that BCRs exist mostly as monomers on B cell surfaces and that Ag induces their clustering to initiate signaling. However, evidence for an opposing model has emerged, namely that BCRs are organized into clusters on resting cell surfaces and that Ag serve to disassociate these clusters to initiate signaling. Through a collaboration with Dr. Lippincott-Schwartz at the NIH, a leader in the development of methods to quantify the spatial organization of surface receptors by point-localization super resolution imaging using pair correlation analysis, we have now obtained robust evidence that the majority of IgM BCRs expressed by human peripheral blood naive B cells exist as monomers in the resting state. To determine if the organization of the BCRs on the B cell surface varied with the differentiated state of the B cell, we also analyzed the distribution of IgG BCRs on human peripheral blood memory B cells. The results of our analysis provide insights into both the fundamental process of antigen-driven BCR clustering as well as differences in the spatial organization of IgM and IgG BCRs that may contribute to the characteristic differences in the responses of naive and memory B cells to antigen. We provided evidence that although both IgM and IgG BCRs reside in highly heterogeneous protein islands that vary in both size and number of BCR single molecule localizations, both resting and activated B cells intrinsically maintain a high frequency of single isolated BCR localizations, which likely represent BCR monomers. IgG BCRs are more clustered than IgM BCRs on resting cells and form larger protein islands following antigen activation. Small dense BCR clusters likely formed via protein-protein interactions are present on the surface of resting cells and antigen activation induces these to come together to form less dense, larger islands, a process likely governed, at least in part, by protein-lipid interactions. Over the last year we also made considerable progress on a new initiative to characterize BCR signaling and antigen-internalization in human tonsillar germinal center (GC) B cells. GCs are compartments within secondary lymphoid organs in which in response to Ag, B cell clonal expansion, somatic hypermutation and affinity-based selection occur resulting in the production of isotype-switched memory B cells and high affinity antibody secreting plasma cells. In recent years discrete steps in GC reactions have been mapped out in mouse models. However, our understanding of the B cell biology of human GCs lags behind the mouse models and clearly knowledge of the cellular and molecular mechanism by which high affinity memory B cells and plasma cells are generated would aid in vaccine design. We showed that human GC B cells respond to high (KD = 5 10-9) but not low (KD = 3.9 10 -7) affinity membrane-associated antigens by initiating B cell receptor (BCR) signaling and extracting and internalizing antigens for presentation. GC B cells engage antigens through novel actin-rich pod-like structures that concentrate BCRs and exert pulling forces capable of dissociating low affinity BCRs from antigen. Antigen-engaged GC B cells provided with T cell help selectively express interferon-regulatory factor-4, critical for plasma cell differentiation. Our findings have implications for the design and development of human vaccines that induce affinity matured antibody responses.
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