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. 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 propose that as the BCR moves into the cell from the plasma membrane and trafficks intracellularly, the local microenvironments of the receptor changes and that in each microenvironment BCR signaling cascades are propagated and regulated differently. 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 recently 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. Although a general feature, the molecular mechanisms by which recruitment is achieved does not appear universal. For example, TLR9 and 7 signaling is not required for the BCR-induced movement of these receptors to the autophagosomes as evidenced by the observation that BCR-induced recruitment of TLR9 and 7 occurs in MyD88-deficient B cells. For TLR3 the case appears different in that BCR-induced phosphorylation of the cytoplasmic domain of TLR3 by Lyn kinase and thus the activation of TLR3 appears to play a role in its recruitment. Over the last year we initiated studies to use atomic force microscopy to describe the morphological changes that occur in B cells following activation through the BCR or TLRs. Our initial results provided evidence for remarkably dynamic rapid changes in morphology within seconds of B cell activation involving large extension of the plasma membrane. These changes are distinctively different for B cells activated through the BCR versus through TLRs. TLRs induce blebbing of the plasma and BCRs induce membrane ruffling. Our future work in this area will focus on understanding the function of the observed morphological changes and use the electron tomography to characterize the molecular structure of these membrane extensions.

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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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