B cell activation is initiated by the binding of the antigen to the B cell receptor (BCR), triggering signal cascades that result in the transcription of a variety of genes associated with B cell activation. Following the initiation of signaling the antigen-bound BCR enters the cell and trafficks to specialized MHC class II-containing intracellular compartments where the antigen is proteolytically cleaved and the resulting peptides bound to MHC class II molecules that are ultimately expressed on the B cell surface allowing for interaction with antigen-specific helper T cells. We recently determined that BCR signaling also triggers reorganization of the endocytic compartments, recruiting endosomes containing toll-like receptors to autophagosome compartments into which the BCR trafficks. We now understand that the BCR continues to signal as it enters the cell and that the correct intracellular trafficking of the BCR and its recruitment of the TLRs depend on these signals. The goal of this project is to understand where discrete steps in the BCR signaling cascade occur and how the spatial and temporal organization of signaling regulates the outcome of antigen binding to the BCR. Particular focus will be on the synergistic interaction of the BCR with the intracellular TLRs and on the outcome of these interactions in terms of cytokine production. Our recent studies provided evidence that components of the BCR signaling pathway are activated sequentially and in defined subcellular locations. We observed that the phosphorylated form of Syk kinase, pSyk, appeared on the plasma membrane immediately following BCR crosslinking, whereas the phosphorylated forms of the MAPKinases p38, ERK and JNK were not detected until the BCR had internalized from the plasma membrane and trafficked to autophagosome-like class II-containing compartments. Using a highly selective inhibitor of endocytosis we showed that blocking BCR internalization resulted in the recruitment of both proximal and downstream kinases to the plasma membrane where MAP kinases were hyper-phosphorylated and Akt and its downstream target Foxo were hypo-phosphorylated leading to the dysregulation of gene transcription controlled through these pathways. These studies are important in demonstrating that the cellular location of the BCR serves to compartmentalize kinase activation to regulate the outcome of signaling. Future studies aimed at defining the molecular composition of the intracellular BCR signaling sites may provide new targets for therapeutics to block BCR signaling in autoimmune disease and in BCR-dependent B cell tumors. During the last year we collaborated with Dr. Joshua Milner, LAD, NIAID, to characterize the effect of mutant phosphatase Cgamma2 on the trafficking of the BCR in human B cells. Dr. Milner recently described PLCgamma2-exon deletions in individuals in three families with dominantly inherited complex of cold-induced urticarial, antibody deficiencies and susceptibility to infection and autoimmune disease. We would like to know if the antibody deficiencies in these individuals are due to alterations in BCR signaling and in trafficking. The syndrome is termed phospholipase Cgamma2-associated antibody deficiency and immune dysregulation (PLAID). The cSH2 domain of PLCgamma2 is auto-inhibitory and the PLAID mutant PLCgamma2 has increased phospholipase activity. Because PLCgamma2 is recruited to the early BCR signaling complex, it was anticipated that BCR signaling in PLAID B cells would be enhanced. Contrary to this prediction, B cells from PLAID patients show reduced responses to BCR cross-linking compared to B cells from healthy controls as measured by either calcium flux or phosphorylation of ERK. We showed that in response to BCR-crosslinking, B cells from PLAID patients, as well as human peripheral B cells transiently transfected with a PLAID mutant PLCgamma2 plasmid, have reduced phosphorylation of Syk as compared to healthy controls. In addition, PLAID B cells showed reduced phosphorylation of the early BCR signaling components, BTK and BLNK but not of PI3K. PLAID B cells also showed reduced phosphorylation of the MAP kinases ERK and p38, but normal JNK phosphorylation and enhanced NF-kappaB phosphorylation. Early BCR signaling also regulates intracellular trafficking of the BCR necessary for MHC class II antigen processing. Even though the rate of internalization of the BCR from the B cell surface upon BCR-XL was normal in PLAID B cells, by confocal microscopy, the BCR in PLAID B cells failed to properly traffic into the lamp1-positive late endosomal/lysosome class II processing compartments. These data suggest that PLAID mutant PLCgamma2 in the heterozygous state alters early BCR signaling by destabilizing Syk/BLNK/BTK complexes resulting in decreased signaling to the MAP kinases ERK and p38 and failure of the BCR to properly traffic to the class II processing compartments. However, mutant PLCgamma2 does not appear to affect the Syk/PI3K pathway or NF-kB activation, possibly due to the amplification of PI3K activity through interaction of the BCR with CD19. We are interested in understanding how the intracellular trafficking of the BCR facilitates interaction with the intracellular TLRs. We showed that following the antigen binding and internalization the BCR signals for the recruitment of TLR9 from multiple small endosomes to an LC3-positive autophagosome into which the BCR trafficks antigen and where synergistic signaling to p38 and JNK activation occurs. The recruitment of TLR9 to the BCR was by a dynein-mediated, microtubule-network dependent process. TLR9 is responsive to DNA and the recruitment of TLR9 to the autophagosome-like compartment was necessary for B cell hyper-responses to DNA-containing antigens. We have now determined that BCR signaling also results in the recruitment of the intracellular TLRs, TLR7 and TLR3, to autophagosomes. Thus, the recruitment of TLRs to the autophagosomes into which the BCR trafficks appears to be a general feature of BCR-TLR interactions. One important output of TLR9 signaling in other cell types is the production of both gamma-interferon (gamma-IFN) and type 1 IFNs. Recent evidence indicates that the subcellular location in which TLRs signal dictates the outcome of signaling with type 1 IFN production resulting from TLR9 signaling in early endosomes and gamma-IFN production requiring signaling from late endosomes. In monocytes the location of signaling is dictated by the delivery of TLR9 agonist-containing immune complexes to early versus late endosomes by activating FcRs. Over the last year we initiated experiments to determine if the TLR-dependent production of cytokines by B cells is similarly regulated by the BCR. These studies have the potential to provide therapeutic targets to regulate cytokine production by B cells in autoimmune disease.
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