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 succeeded in characterizing the effect of the TLR9 agonist 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 signaling through Toll-like receptor 9 (TLR9) blocked the ability of antigen-specific B cells to capture and present antigen and antagonized the BCR-induced increases in the expression of both MHC class II and important co-receptors, including CD86, resulting in the inability of B cells to activate antigen-specific CD4+ T cells. 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. A principle component analysis of RNA seq data showed little overlap between B cells stimulated through the BCR or TLR9 alone or together indicating that TLR9 antagonism of BCR-dependent antigen presentation is through the activation of a novel transcriptional program. In a mouse model and in a human clinical trial the TLR9 agonist, CpG, enhanced the magnitude of the antibody response to a protein vaccine but failed to promote affinity maturation. Thus, TLR9 signaling may enhance the magnitude of an antibody response at the expense of the ability of B cells to engage in germinal center events that are highly dependent on antigen capture and presentation. We also explored the outcome of immunization with protein antigens adjuvanted with the TLR9 agonist CpG. Of the two major classes of CpG-containing oligonucleotides, CpG-A appears restricted to inducing type 1 IFN in innate immune cells and CpG-B to activating B cells to proliferate and produce antibodies and inflammatory cytokines. Although CpGs are candidates for adjuvants to boost innate and adaptive immunity, our understanding of the effect of CpG-A and CpG-B on B cell responses is incomplete. We showed that both CpG-B and CpG-A activated B cells in vitro to proliferate, secrete antibodies and IL-6, and that neither CpG-B nor CpG-A alone induced type 1 IFN production. However when incorporated into the cationic lipid, DOTAP, CpG-A, but not CpG-B, induced a type 1 IFN response in B cells in vitro and in vivo. We provide evidence that differences in the function of CpG-A and CpG-B may be related to their intracellular trafficking in B cells. These findings fill an important gap in our understanding of the B cell response to CpGs with implications for the use of CpG-A and CpG-B as immunomodulators. We also compared immunization with the T-cell dependent antigen, NP-conjugated to chicken gamma globulin (NP-CGG) adjuvanted with CpG-A or CpG-B, alone or conjugated with the cationic lipid carrier, DOTAP. We provided evidence that only NP-CGG adjuvanted with DOTAP-CpG-B was an effective vaccine in mice resulting in robust germinal center responses, isotype switching and high affinity NP-specific antibodies. The effectiveness of DOTAP-CpG-B as an adjuvant was dependent on the expression of the TLR9 signaling adaptor MyD88 in immunized mice. These results indicate DOTAP-CpG-B but not DOTAP-CpG-A is an effective adjuvant for T cell-dependent protein antigen-based vaccines.

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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
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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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