The signals regulating human B cell function have not been thoroughly explored. The research has focused on the mechanisms by which signals delivered through the B cell surface molecule, CD40, regulate human B cell function. Specifically, the research of the BCBG investigates the role of interactions between CD154/CD40L/TNFSF5 and CD40/TNFRSF5 as well as other TNF superfamily member receptor-ligand pairs in the activation and differentiation of human B cells to memory cells and plasma cells. Signals delivered through CD154-CD40 interactions regulate essential B cell maturational events such as germinal center formation, immunoglobulin heavy chain class switching, somatic hypermutation and differentiation to memory cells or plasma cells, but the exact details of the signaling cascades involved have not been delineated. The goal of the research of the B Cell Biology Group is to determine signaling events that occur during a normal humoral immune response so that one can understand defects in these pathways that occur in immunodeficiencies such as HyperIgM syndrome and autoimmune diseases such as systemic lupus erythematosus. Work has nvestigated signaling in five component steps: 1) receptor engagement; 2) recruitment of adaptor molecules; 3) proximal kinase activation; 4) transcription factor stimulation; and ultimately, 5) production of a functional outcome. Mechanisms connecting cell surface occupancy with downstream signaling events, specific gene transcription and functional outcomes are examined following engagement of TNF superfamily members on B cells with recombinant ligands. Signaling cascades are dissected using novel biochemical and multi-parameter flow cytometric assays following transfection or transduction of B cells with constructs expressing constitutively active or dominant negative proteins. Gene expression is examined by microarray and confirmed by quantitative PCR, Western blotting, the proteomic technique Immunoaffinity Capillary Electrophoresis (ICE) and flow cytometry. Functional outcomes are examined in vitro, ex vivo immediately following isolation of human blood or tonsillar tissue samples, and following transplantation of activated human secondary lymphoid tissue and primary lymphocyte populations into the RAG KO/NOD/Perforin KO mouse model. Despite the complexity, specific checkpoints within the cascades regulate signaling and provide evidence of novel means to regulate B cell function therapeutically. In summary, the results of the experiments of the B Cell Biology Group have delineated the role of CD40 signaling in abnormal germinal center reactions in patients with systemic lupus erythematosus that lead differentiation of autoimmune plasma cell and memory B cell populations. Moreover, a novel fluorescence resonance transfer (FRET) technique has been developed to examine CD40-induced signaling events by multi-parameter flow cytometry and confocal microscopy. Futhermore, a novel role for the transcription factor, ICSBP/IRF8, in the regulation of the germinal center gene program and plasma cell differentiation has been documented. Further delineation of the signaling cascades and the genes expressed following engagement of CD40 on the surface of human B cells should provide unique targets that could be exploited to regulate B cell activity in autoimmune diseases such as Systemic Lupus Erythematosus.
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