B cells are activated to proliferate and differentiate into antibody secreting cells by antigen binding to the B cell receptor (BCR). We have applied high resolution live cell imaging technologies to the investigation of the earliest events in the antigen-driven initiation of BCR signaling. Our results provided evidence for an ordered series of discrete events following antigen binding that are initially BCR intrinsic and then become dependent on the BCR signaling apparatus. We imaged B cells as they first encountered antigen incorporated into fluid lipid bilayers, mimicking the surface of an antigen presenting cell, the apparent physiologically relevant mode of antigen recognition for B cells in vivo. Our results fit a model in which in the absence of antigen the BCRs ectodomain, particularly the membrane proximal domain, Cmu4 or Cgamma3, is not receptive to oligomerization. Binding antigen on the opposing lipid bilayer exerts a force or torque on the BCR such that the membrane proximal domain becomes oligomerization competent. The random bumping of antigen-bound BCRs results in their oligomerization and immobilization as shown by single molecule tracking analyses. The microclusters then grow in both size and area resulting in an increased recruitment of the first kinases in the pathway to the BCR clusters. Oligomerization and the initial growth of the BCR microclusters are BCR intrinsic events that are sensitive to both the affinity and the isotype of the BCRs mIg. Later cluster growth depends on kinases in the BCR signaling cascades and the actin cytoskeleton and microtubule network. The BCR microclusters perturb the local lipid environment causing the transient coalescing of lipid rafts around the microclusters. An important repercussion of the association of the BCR clusters with raft lipids is the recruitment of the first kinase in the BCR signaling cascade, the lipid-raft tethered kinase, Lyn. Simultaneously, the BCR cytoplasmic domains undergo a transition from a closed to an open conformation and are phosphorylated on tyrosines by Lyn. These events stimulated the assembly of a signalosome, composed of the kinases Syk and Btk, and the adaptor BLNK that together recruited PLC-gamma2. This early signalosome subsequently recruited a number of downstream kinases, including cRaf and the MAP kinases. We hypothesize that mutations in the BCR or changes in the B cell that affect any step in this process could result in a lowered threshold for activation and hyperactivation such as in systemic autoimmune disease or in chronic activation leading to B cell tumors. In collaboration with Dr. Louis Staudt in the NCI we provided evidence that in the ABC subtype of DLBCL B cell tumors that are dependent on the BCR for survival the BCRs are constitutively in immobile, signaling active clusters. Because other B cell tumors that did not depend on their BCRs for survival did not spontaneously form signaling active BCR microclusters, these data strongly suggest that spontaneous BCR clustering plays a role in B cell tumorigenesis. We are continuing this collaboration to identify the changes in these tumors that result in spontaneous BCR clustering and signaling. These results are important as they may provide new strategies for treatment of these tumors. With the Staudt group we are also imaging ABC, Burkitts Lymphomas (BLs) and germinal center B cell (GBCs) tumors that are dependent on the BCR for survival and in addition are either dependent or not on the B cell coreceptors, CD19, for their survival. We will determine if the BCRs are clustered, whether the BCR and CD19 colocalize, if CD19 is active and phosphorylated and if these cells exhibit enhanced BCR and CD19 signaling by determining the colocalization of phosphorylated PI3K and Syk with the BCR and CD19. These studies have the potential to identify new targets for B tumor therapy. Over the last year we also initiated studies to address a longstanding puzzle in B cell tumor biology namely that endemic Burkitt lymphomas (BLs) arise exclusively in malaria endemic areas of the world and unlike sporadic BLs that occur outside of malaria endemic regions, uniformly carry Epstein Barr Virus (EBV). This suggests that EBV-infected B cells are at greater risk of transformation in individuals experiencing clinical cases of malaria. We are using a FISH-based assay coupled with flow cytometry to characterize EBV-infected B cells in children living in a high malaria transmission region in Mali before and immediately after cases of acute malaria. We are also establishing a protocol to collect BLs from children in the same area for molecular characterization. Together results of these studies should provide new insight into the dynamics of B cell infection with EBV and the molecular nature of BLs. Our efforts in understanding B cell signaling mechanisms that contribute to autoimmunity have focused on a collaboration with Dr. Joshua Milner in Laboratory of Allergic Disease, to analyze B cells from patients with an immunodeficiency and autoimmunity syndrome. Dr. Milner discovered that individuals with one copy of a mutant PLCG2 gene, lacking the autoinhibitory domain, cSH2, show PLCgamma2-associated antibody deficiencies and immune dysregulation (PLAID). Paradoxically, even though the mutant PLCgamma2 is constitutively active in vitro, PLAID B cells show deficient intracellular calcium responses upon BCR crosslinking. We provided a molecular explanation for this paradox showing that the cSH2 domain of PLCgamma2 plays a critical role in stabilizing the early signaling complex induced by BCR crosslinking. The binding of antigen to the B cell receptor (BCR) triggers the assembly of a signaling complex composed initially of the kinases LYN, SYK, and BTK and the adaptor, BLNK, that together recruit and activate PLCgamma2, a critical effector that triggers increases in intracellular Ca2+ and activates a variety of vital, downstream signaling pathways. In the presence of the mutant PLCgamma2, SYK, BTK and BLNK are only weakly phosphorylated and fail to stably associate with the BCR resulting in the failure of the BCR to stably cluster and dysregulation of downstream signaling and trafficking of the BCR. Thus, the cSH2 domain functions not only to inhibit the active site of PLCgamma2 but to directly or indirectly stabilize the early BCR signaling complex.
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