The B cell antigen receptor (BCR) serves dual, interrelated functions in B cell activation. The first is to initiate signal cascades that result in the transcription of a variety of genes associated with B cell activation. The second is to traffic bound antigen into the cell to deliver the antigen into intracellular compartments where the antigen is proteolytically cleaved and the resulting peptides are assembled into MHC class II-peptide complexes. The MHC class II-peptide complexes are expressed on the B cell surface for recognition by helper T cells that will as a consequence be activated to provide essential growth and differentiation factors to the B cell. The signaling and antigen transport functions of the BCR are interdependent in that the BCR signaling is necessary for the correct and rapid intracellular targeting of the antigen. Our current evidence indicates that the BCR enters the class II-containing compartment as an intact, signaling competent, receptor. However, at present it is an open question as to whether the BCR signals intracellularly and if so what role intracellular signaling might play in B cell activation. The long term goal of this project is to gain a better understanding of the molecular mechanisms that underlie the correct targeting of the BCR to the class II-containing compartments including determining where in the pathway discrete signaling events occur and to further define the role of intracellular signaling on B cell activation with particular focus on the synergistic interaction of the BCR with the intracellular Toll like receptors (TLRs). Studies to delineate the components of the BCR signaling cascade necessary for correct targeting of the BCR provided evidence that the BCR-induced activation of phospholipase D (PLD) is necessary for the correct targeting of the BCR into MHC class II-containing intracellular compartments. PLD is a signal-transduction-activated enzyme that hydrolyzes phosphatidylcholine to generate the second messenger phosphatic acid and choline. PLD has been implicated in controlling a wide variety of cellular processes including the endocytosis and vesicular trafficking of cellular receptors. We showed that the activity of PLD was not required for endocytosis of the BCR from the plasma membrane but was required for subsequent steps in the BCRs trafficking pathway. We also provided evidence that components of the BCR signaling pathway are activated sequentially and in defined subcellular locations. We observed that pSyk appeared on the plasma membrane immediately following BCR crosslinking, whereas the phosphorylated forms of p38, ERK and JNK were not detected until the BCR had internalized from the plasma membrane and trafficked to the class II-containing compartments. We also investigated the function and regulation of the intracellular signaling of the BCR. BCR signaling is regulated and fine tuned by an array of B cell coreceptors many of which are present on the cell surface where they presumably interact with the BCR. However, certain members of one family of receptors, the TLRs, that have been shown to influence BCR signaling are located in intracellular endosomes and not on the plasma membrane. One endosomal TLR, TLR9, that recognizes unmethylated CpG-DNA motifs has been shown to enhance BCR signaling resulting in hyperactivation of B cells to proliferate and differentiate into antibody secreting plasma cells. Recent evidence indicates that synergistic signaling between the BCR and TLR9 underlies the production of self-reactive antibodies in systemic autoimmune diseases and partly explains the preponderance of auto-antibodies that react with DNA-containing antigens. Using confocal microscopy and antibodies specific for the BCR, TLR9, p38 and pJNK we showed that following the binding and internalization of DNA-containing antigens, 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 through to p38 and JNK occurs. The recruitment of TLR9 to the BCR was by a dynein-mediated, microtubule-network dependent process and was necessary for B cell hyper-responses to DNA-containing antigens. Of considerable interest was the observation that recruitment of TLR9 was dependent on the BCRs activation of PLD. Thus, PLD plays a role in both the correct targeting of the BCR to intracellular compartments and the recruitment of TLR9 to the same compartments. The BCRs PLD-activation dependent recruitment of the TLR9 to the autophagosome represents a novel mechanism underlying the hyper-response of B cells 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. Studies are underway to better define the role of TLR3 phosphorylation on its function and to determine what intracellular substrates might activate the BCR-recruited TLRs.

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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:
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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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