Pre-Clustering of the B Cell Antigen Receptor Demonstrated by Mathematically Extended Electron Microscopy

Abstract

The B cell antigen receptor (BCR) plays a crucial role in adaptive immunity, since antigen-induced signaling by the BCR leads to the activation of the B cell and production of antibodies during an immune response. However, the spatial nano-scale organization of the BCR on the cell surface prior to antigen encounter is still controversial. Here, we fixed murine B cells, stained the BCRs on the cell surface with immuno-gold and visualized the distribution of the gold particles by transmission electron microscopy. Approximately 30% of the gold particles were clustered. However the low staining efficiency of 15% precluded a quantitative conclusion concerning the oligomerization state of the BCRs. To overcome this limitation, we used Monte-Carlo simulations to include or to exclude possible distributions of the BCRs. Our combined experimental-modeling approach assuming the lowest number of different BCR sizes to explain the observed gold distribution suggests that 40% of the surface IgD-BCR was present in dimers and 60% formed large laminar clusters of about 18 receptors. In contrast, a transmembrane mutant of the mIgD molecule only formed IgD-BCR dimers. Our approach complements high resolution fluorescence imaging and clearly demonstrates the existence of pre-formed BCR clusters on resting B cells, questioning the classical cross-linking model of BCR activation.

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“…It is nevertheless possible to tell if dimers are present or not. From the literature is it known, that even a labeling efficiency around 15% is enough to detect dimers with sufficient statistical significance 32 .…”

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“…Therefore, activation was assumed to rely on crosslinking of BCRs in order to create clusters [7]. However, recent evidence indicates that in the absence of stimuli, BCRs are clustered in an autoinhibitory conformation and antigen binding disperses these clusters [20,21]. Consequently, Src kinases Lyn, Fyn, and Blk phosphorylate the now accessible immunoreceptor tyrosine-based activation motifs (ITAMs) on Igα (CD79A) and Igβ (CD79B), thereby create a docking site for the tyrosine kinase SYK [22–24].…”

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“…However, these papers offer new viewpoints, which emerged thanks to the immunological “data revolution”, in particular next-generation sequencing of lymphocyte repertoires. Others address new methods of extracting ( 13 – 15 ) and analyzing ( 16 – 18 ) comprehensive T and B cell phenotype and repertoire data, and delineate some of the first insights gleaned from sequencing studies regarding how these repertoires emerge, evolve, and function ( 19 – 25 ). Natural killer cells ( 26 ), myeloid cells ( 27 ), and structural immunology ( 28 – 31 ) are also represented.…”