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 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. 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. 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. We also characterized the effect of CpG on BCR antigen processing and presentation. To our surprise we discovered that CpG ablated the ability of B cells to process and present antigen to antigen-specific helper T cells. When Ag was provided to B cells in the presence of CpG the BCR receptor initiated early signaling events involving phosphorylation of several early kinases and adaptors and the BCR was endocytosed into the cell similarly to the response of B cells to Ag alone. However, in the presence of CpG, BCR trafficking was dysregulated, the BCR did not reach Ag processing compartments and peptide-MHC complexes, detected by complex-specific antibodies, did not appear on the B cell surface. When placed in culture with Ag-specific CD4+ T cells Ag-specific B cells were unable to activate T cells in response to Ag in the presence of CpG. Using a new methodology that allowed us to view and quantify B cells pulling antigen from membrane sheets we determined that CpG greatly diminished the amount of antigen captured by B cells through the BCR. We conclude from these results that the presence of CpG results in a switch in the outcome of B cell encounter with Ag, strongly promoting proliferation and differentiation of the B cells at the expense of Ag-dependent interactions with helper T cells.

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Akkaya, Munir; Akkaya, Billur; Kim, Ann S et al. (2018) Toll-like receptor 9 antagonizes antibody affinity maturation. Nat Immunol 19:255-266
Akkaya, Munir; Akkaya, Billur; Sheehan, Patrick W et al. (2017) T cell-dependent antigen adjuvanted with DOTAP-CpG-B but not DOTAP-CpG-A induces robust germinal center responses and high affinity antibodies in mice. Eur J Immunol 47:1890-1899
Akkaya, Munir; Akkaya, Billur; Miozzo, Pietro et al. (2017) B Cells Produce Type 1 IFNs in Response to the TLR9 Agonist CpG-A Conjugated to Cationic Lipids. J Immunol 199:931-940
Portugal, Silvia; Obeng-Adjei, Nyamekye; Moir, Susan et al. (2017) Atypical memory B cells in human chronic infectious diseases: An interim report. Cell Immunol 321:18-25
Akkaya, Billur; Miozzo, Pietro; Holstein, Amanda H et al. (2016) A Simple, Versatile Antibody-Based Barcoding Method for Flow Cytometry. J Immunol 197:2027-38
Portugal, Silvia; Tipton, Christopher M; Sohn, Haewon et al. (2015) Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function. Elife 4:
Liu, Wanli; Chen, Elizabeth; Zhao, Xing Wang et al. (2012) The scaffolding protein synapse-associated protein 97 is required for enhanced signaling through isotype-switched IgG memory B cell receptors. Sci Signal 5:ra54
Simone, Olivia; Bejarano, Maria Teresa; Pierce, Susan K et al. (2011) TLRs innate immunereceptors and Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) CIDR1?-driven human polyclonal B-cell activation. Acta Trop 119:144-50
Chaturvedi, Akanksha; Martz, Rebecca; Dorward, David et al. (2011) Endocytosed BCRs sequentially regulate MAPK and Akt signaling pathways from intracellular compartments. Nat Immunol 12:1119-26
Pierce, Susan K; Liu, Wanli (2010) The tipping points in the initiation of B cell signalling: how small changes make big differences. Nat Rev Immunol 10:767-77

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