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 molecular events that initiate the complex signal cascades triggered by BCR antigen engagement. Most studies to date have focused on signaling in naive B cells in mice and have described the B cells initial encounter with antigen that in general lead to relatively low affinity IgM antibody responses in vivo. In order to contribute to the development of vaccines for which we currently have none including malaria and HIV-AIDS, it will be necessary to more fully understand the cellular and molecular mechanisms that underlie immunological memory and the affinity maturation of long-lived memory B cells (MBCs). We now understand that naive B cells first encounter antigens in vivo on the surfaces of macrophages, dendritic cells (DCs), or follicular dendritic cells in specialized microenvironments within secondary lymphoid organs termed B cell follicles. B cell engagement of the cell-surface associated antigen triggers BCR signaling and antigen extraction, internalization, processing and presentation of the antigen on MHC class II molecules. B cells then migrate to the border of the follicles with the T cell zone where they present antigen to T cells that have been recently primed by antigen presented on DCs to differentiate into T follicular helper (Tfh) cells. The resulting B cell-Tfh cell interaction drives B cells to enter germinal centers (GCs). GC B cells first enter GC dark zones where they proliferate and undergo somatic hypermutation prior to entering the GC light zones where antigen-affinity based selection occurs. Selection is a competitive process dependent in large part on the amount of antigen B cells are able to gather, process and present to Tfh cells. Thus, there appear to be at least two key checkpoints in the process of affinity maturation, one for naive B cells and one for GC B cells. At these checkpoints, the affinity of B cell for its antigen is tested by the ability of the BCR to differentially signal in response to and internalize, process and present antigen to T cells. We carried out an extensive comparative characterization of the ability of naive B cells and GC B cells to discriminate antigen affinity and to process and present antigen to T cells. We showed that human GC B cells have intrinsically higher affinity thresholds for both antigen binding and gathering as compared to naive B cells and that these functions are mediated by distinct cellular structures and pathways that ultimately lead to Tfh cell-dependent differentiation to plasma cells. GC B cells bind antigen and exert pulling forces on the BCR-antigen complexes through highly dynamic, actin- and ezrin-rich pod-like structures, the behavior of which is dictated by the cells affinity for antigen. During antigen gathering the GC cells endocytic machinery is not polarized with the antigen-bound BCRs and antigen is trafficked to distal sites for internalization resulting in temporally staged antigen presentation. These findings provide a structural framework for human GC B cell affinity-dependent antigen selection that may guide the development of vaccines that produce high affinity antibody. We also collaborated with Dr. Susan Moir (NIAID) to elucidate the cellular and molecular mechanisms underlying the IgG3-mediated suppression of BCR signaling in atypical MBCs from HIV-high viremic individuals. Dr. Moir observed that atypical MBCs that expand by persistent HIV viremia, bound large amounts of IgG3. Using a variety of imaging techniques, we determined that IgG3 induced clustering of BCRs on the IgM+ B cells, which was mediated by direct interactions between soluble IgG3 and membrane IgM of the BCR. The inhibitory IgG receptor CD32b (FcgammaRIIb), complement component C1q and inflammatory biomarker CRP each contributed to the binding of secreted IgG3. Thus, taken together our results demonstrated that IgG3 can regulate B cells during chronic activation of the immune system.
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