B cells express both a clonally rearranged cell-surface expressed antigen-specific receptor, the B cell receptor (BCR), as well as germline encoded Toll-like receptors (TLRs) that respond to microbial products of potential pathogens. The dual expression of the BCR and TLRs allows B cells to modulate the outcome of antigen binding depending on the presence of pathogens. The BCR plays two roles in T-cell dependent antigen-driven antibody (Ab) responses. The first is to signal for the activation of a variety of genes associated with B cell activation. The second is to transport bound antigen to specialized intracellular compartments in which the antigen is degraded and the resulting peptides are bound to MHC class II molecules that are subsequently trafficked to the cell surface for recognition by antigen-specific helper T cell (Th cells). Several studies have provided evidence that TLR activation in B cells can alter the outcome of BCR signaling. We showed earlier that upon binding of CpG-containing antigens the BCRs signal to recruit endosomes containing TLR9 to intracellular compartments into which the BCR is internalized resulting in hyperactivation of MAPK on the surface of those vesicles. Over the last year we focused on the impact of TLR9 signaling on three outcomes of antigen-dependent BCR signaling, namely: 1) the production of cytokines, in particular type 1 interferons; 2) the antibody response to T cell dependent antigens in vivo and 3) metabolic changes that accompany B cells proliferation and differentiation to plasma cells. 1) The impact of TLR9 signaling on B cell cytokine production B cells express the innate receptor, TLR9, which signals in response to unmethylated CpG sequences in microbial DNA. Of the two major classes of CpG-containing oligonucleotides, CpG-A appears restricted to inducing type 1 interferon (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 provided 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. 2) The impact of TLR9 signaling on the antibody response to T-cell dependent antigens The development of vaccines for infectious diseases for which we currently have none, including HIV, will likely require the use of adjuvants that strongly promote germinal center responses and somatic hypermutation to produce broadly neutralizing antibodies. We compared the outcome of immunization with the T-cell dependent antigen, NP-conjugated to chicken gamma globulin (NP-CGG) adjuvanted with TLR9 ligands, 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. 3) The effect of TLR9 signaling on the metabolic changes that accompany antigen-driven B cell proliferation and differentiation to PCs Activation of B cells to proliferate and differentiate into antibody secreting cells is regulated to avoid unwanted antibody responses by two temporally distinct signals, the first provided by antigen binding to the B cell receptor (BCR) and the second by T helper cells. Despite the central role of these two signals in driving antibody responses, our understanding of the consequences of each signal is incomplete. We investigated the metabolic changes that accompany B cell activation and showed that antigen binding to the BCR signals for rapid increases in both oxidative phosphorylation and glycolysis, preparing the cells to respond to a second signal. However, in the absence of a second signal B cells undergo progressive loss of mitochondria function and glycolytic capacity ultimately leading to apoptosis. Mitochondria dysfunction is a result of the gradual accumulation of intracellular calcium which leads to inefficient oxidative phosphorylation and increased ROS production. This process is avoidable for approximately nine hours after B cell antigen binding by either T helper cells or by signaling through Toll-like receptor 9. Thus, antigen binding appears to activate a metabolic program that imposes a short time window in which B cells either receive a second signal and survive or alternatively face elimination.

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Akkaya, Munir; Traba, Javier; Roesler, Alexander S et al. (2018) Second signals rescue B cells from activation-induced mitochondrial dysfunction and death. Nat Immunol 19:871-884
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
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
Traba, Javier; Miozzo, Pietro; Akkaya, Billur et al. (2016) An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes. J Vis Exp :
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Sohn, Hae Won; Krueger, Peter D; Davis, Randall S et al. (2011) FcRL4 acts as an adaptive to innate molecular switch dampening BCR signaling and enhancing TLR signaling. Blood 118:6332-41
Kardava, Lela; Moir, Susan; Wang, Wei et al. (2011) Attenuation of HIV-associated human B cell exhaustion by siRNA downregulation of inhibitory receptors. J Clin Invest 121:2614-24
Liu, Wanli; Won Sohn, Hae; Tolar, Pavel et al. (2010) Antigen-induced oligomerization of the B cell receptor is an early target of Fc gamma RIIB inhibition. J Immunol 184:1977-89
Pierce, Susan K (2009) Understanding B cell activation: from single molecule tracking, through Tolls, to stalking memory in malaria. Immunol Res 43:85-97
Sohn, Hae Won; Pierce, Susan K; Tzeng, Shiang-Jong (2008) Live cell imaging reveals that the inhibitory FcgammaRIIB destabilizes B cell receptor membrane-lipid interactions and blocks immune synapse formation. J Immunol 180:793-9