The B cell response to antigen is regulated by a variety of co-receptors that convey information to the B cell about the quality of the antigen and the status of the ongoing immune response. Over the last year we have made progress in defining the mechanisms by which the inhibitory receptor, FcgammaRIIB, function to regulate the initiation of BCR signaling. In addition we initiated studies to determine the function of the Fc like receptor 4 (FcRL4) present on a distinct subset of memory B cells (MBCs) observed to be greatly expanded in HIV infected viremic individuals and in children and adults in malaria-endemic areas. Our progress using high resolution live cell imaging to delineate the very early antigen driven events in B cell activation has provided a new context in which the impact of coreceptors can be evaluated. Using high resolution fluorescence resonance energy transfer (FRET) coupled with total internal reflection microscopy (TIRFM) and single molecule tracking we provided evidence for an ordered process that occurs within seconds to minutes of the BCR binding antigen. Antigen bound BCRs form immobile clusters that then grow in size by molecular trapping. The clusters perturb the local lipid environment causing lipid rafts to coalesce around the BCR clusters. As a consequence of the membrane perturbation the first kinase in the pathway, Lyn, that is tethered to the membrane by raft lipids is brought into close molecular proximity to the BCR clusters. Simultaneously, Lyn phosphorylates the Ig alpha beta cytoplasmic domain of the BCR and the Ig alpha beta chains undergo a conformational change from a closed to an open form. Syk is recruited to the phosphorylated BCR and the signaling cascades are triggered. Over the last year we investigated the effect of coligating the FcgammaRIIB and BCR by the binding of immune complexes (ICs) on these early events. We discovered that the binding of ICs induced the colocalization of the BCR and FcgammaRIIB in microscopic clusters but that within these clusters the BCR and Fcgamma RIIBs were not in close molecular proximity as measured by FRET. Nonetheless, binding of ICs resulted in a block in the earliest events in BCR signaling beginning with the oligomerization of the BCR into immobile signaling active clusters. Following IC binding the BCRs failed to associate with a lipid raft probe in contrast to the FcgammaRIIB that stably associated with the raft lipid probe. The ability of the FcgammaRIIB to block the initiation of BCR signaling appeared to be dependent on its ability to associate with raft lipids as loss of function mutant FcgammaRIIBs that are unable to associate with lipid rafts did not block BCR signaling. Taken together these studies indicate that the FcgammaRIIB affects BCR signaling at a much earlier point than previously appreciated. Over the last year we initiated studies to define the function of FcRL4, a member of an ancient family of transmembrane proteins, the FcRLs, that share ancestors with the classical FcRs, like FcgammaRIIB, and are preferentially expressed in the B cell lineage. FcRL4 is expressed on a distinctive subset of MBCs located in mucosal lymphoid tissues in healthy individuals. In addition, FcRL4+ MBCs are greatly expanded in numbers in the blood of HIV-infected viremic individuals and in individuals chronically re-infected with malaria. We provided evidence that the expression of FcRL4 in human B cell lines inhibits antigen-induced BCR signaling at the level of Syk phosphorylation. Inhibition did not require coligation of the FcRL4 with the BCR but depended on the two immunoreceptor inhibitory motifs (ITIMs) in FcRL4s cytoplasmic domain. Remarkably, FcRL4 expression simultaneously enhanced signaling through the innate immune toll-like receptor 9 (TLR9). These findings suggest that FcRL4 may act as molecular switch in B cells to dampen adaptive immune signaling and enhance signaling in response to chronic antigenic stimulation.

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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
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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