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 interaction of the BCR with the intracellular TLRs and on the outcome of these interactions. Over the last year we characterized the effect of CpG on BCR antigen processing and presentation. We now appreciate that key events in T cell-dependent Ab responses in vivo are dependent on antigen-specific T cell-B cell interactions. The initiation of T cell-dependent Ab responses occurs in secondary lymphoid organs at the T cell/B cell border and is dependent on the stable interaction of antigen-primed Th cells that have acquired some of the features of T follicular helper cells (Tfh) with activated antigen-specific B cell through MHC-class II peptide complexes presented on the B cell surface. As a result B cells become fully activated and proliferate and Tfh cells fully differentiate. Dependent, in part, on the quality of the B cell-Tfh cells interaction, B cells will either enter germinal centers (GCs) or differentiate into either short-lived plasma cells (PCs) or GC-independent memory B cells (MBCs). Within the GC B cells proliferate and undergo somatic hypermutation in the GC dark zone prior to entering the GC light zone where antigen-dependent selection occurs. Selection is dependent on the ability of B cells to capture, process and present Ag to Tfh cells an event that ultimately results in the differentiation of GC B cells to long-lived MBCs and PCs. We discovered that the TLR9 agonist, 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 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. Moreover, TLR9 signaling reduced BCR-induced increases in expression of CD86, a key costimulatory receptor for T cells, decreased the expression of IL-6, required for Tfh differentiation and increased the expression of IL-2, an inhibitor of Tfh differentiation. 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
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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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