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. 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. Staudt and colleagues observed that the BCR-dependent B cell tumors were also dependent on MYD88 the TLR signaling adaptor. Remarkably nearly 30% of the BCR-dependent B cell tumors had a gain of function mutation in the MYD88 TLR/TIR domain at an evolutionary invariant residue. The mutant MYD88 appeared to promote tumor survival by spontaneously activating NF-B. We will investigate the intracellular interaction of TLRs and BCRs in these tumors using live cell imaging. We have also determined that a subset of B cell myeloma tumors express BCRs and are dependent on these for their survival. Using live cell total internal reflection microscopy we determined that these BCR were constitutively signaling. This novel observation may provide for new strategies of the development of therapies for myelomas.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2012
Total Cost
$194,284
Indirect Cost
City
State
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Yooseph, Shibu; Kirkness, Ewen F; Tran, Tuan M et al. (2015) Stool microbiota composition is associated with the prospective risk of Plasmodium falciparum infection. BMC Genomics 16:631
Davis, R Eric; Ngo, Vu N; Lenz, Georg et al. (2010) Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature 463:88-92