The Mechanism of Co-Receptor Regulation of B-cell Activation

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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 Over the last year we carried out extensive analyses of the metabolic reprogramming of B cells following BCR and/or TLR9 activation. We discovered that B cell activation either through the BCR or through TLR9 lead to an instantaneous boost in both metabolic glycolysis and respiration. Dual stimulation of B cells through BCR and TLR lead to an additive affect which was independently governed by different signaling pathways. We also learned that in the quiescent state and shortly after stimulation, B cell function and survival depended heavily on oxidative phosphorylation fueled almost exclusively by fatty acid oxidation. In time, while the role of fatty acid oxidation diminished the role of glucose metabolism increased. This switch was accompanied by additive changes in gene expression as well as cellular alterations that facilitated increased glucose uptake. Although the initial boost in energy production was similar for both BCR and TLR9 stimulated B cells, we determined that in the long term, the TLR mediated boost was far more sustainable than BCR mediated boost which in turn failed to keep up with the energy needs of the cell in the absence of a secondary signal. We also found that the long term inadequacy of BCR-mediated metabolic activation goes hand in hand with pathologic changes in mitochondria including swelling, loss of cristae architecture and production of large amounts of mitochondria-produced reactive oxygen species. We demonstrate that this mitochondrial pathology was a result of continuous calcium leak into the cell following BCR stimulation which altered the permeability of the mitochondria in a gradual fashion and generated a futile cycle with ROS production. Our findings provided a unified perspective on how B cell metabolism and morphological changes evolve in relation to each other following different activation modalities and also how these alterations lead to either survival or death of the B cell depending on the fate decision implemented by check points of B cell activation and differentiation.