The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens

Abstract

B cells are activated in vivo after the B cell receptors (BCRs) bind to antigens captured on the surfaces of antigen-presenting cells. Antigen binding results in BCR microclustering and signaling; however, the molecular nature of the signaling-active BCR clusters is not well understood. Using single-molecule imaging techniques, we provide evidence that within microclusters, the binding of monovalent membrane antigens results in the assembly of immobile signaling-active BCR oligomers. The oligomerization depends on interactions between the membrane-proximal Cmicro4 domains of the membrane immunoglobulin that are both necessary and sufficient for assembly. Antigen-bound BCRs that lacked the Cmicro4 domain failed to cluster and signal, and conversely, Cmicro4 domains alone clustered spontaneously and activated B cells. These results support a unique mechanism for the initiation of BCR signaling in which antigen binding induces a conformational change in the Fc portion of the BCR, revealing an interface that promotes BCR clustering.

Figure 1. B-cell activation and BCR clustering in response to multi- and monovalent membrane antigens

(A) Intracellular calcium in primary B cells interacting with bilayers containing ICAM-1 only or together with the indicated concentrations of antigens. Calcium on bilayers with ICAM-1 only did not differ from baseline oscillations detected in empty chambers. Data represent means of at least 20 cells from three independent experiments. (B) Upregulation of CD69 activation marker in B cells stimulated with bilayers containing the indicated antigens. (C) Distribution of the BCR labeled with IgM-specific Fab (green) as compared to plasma membrane staining with DiIC16 (red) during B-cell spreading on bilayers containing indicated antigens. Accumulation of the membrane dye in the center of the synapse was unstable and independent of the distribution of the BCR. See also movies S1-4. (D) BCR microcluster formation is coincident with the initiation of BCR signaling. Simultaneous imaging of NIP₁-H12-HyLight647 (at 15/μm²) and intracellular calcium levels. Scale bars in 5 μm.

Figure 2. Monomeric nature of the NIP₁-H12 peptide antigen

(A). Distribution of fluorescence intensities of single NIP₁-H12-HyLight647 molecules on bilayers with a gaussian fit to the data (red). (B) An example of single step bleaching of fluorescent spots of NIP₁-H12-HyLight647. (C) FRET measured between NIP₁-H12-DyLight547 and NIP₁-H12-HyLight647 (1:1 mixture) at the indicated concentrations. The concentration range used for cell assays is grayed. (D) Dispersion of NIP₁-H12 clusters in a B-cell synapse by competition of BCR binding with soluble NIP hapten. Scale bar 5 μm.

Figure 3. Microclusters arrest diffusion of BCR and Syk

(A) Cumulative probability plots of the diffusion coefficients of single BCR molecules in synapses formed with the indicated antigen. Data were acquired 3–10 min after contact of the cells with bilayers. Plots represent the cumulative frequency of diffusion coefficients of 650–1002 molecules from three experiments. (B) Microclusters are sites of BCR stopping. Cumulative probability plot of the diffusion coefficients of single BCR molecules localized inside or outside of antigen clusters from cells within 1 min of contact with the indicated antigens. Data represent 540–1323 molecules from three experiments. (C) A trajectory of single BCR molecule (green) stopping (arrow) in an NIP₁-H12 cluster (red). Plot shows instant diffusion and the corresponding localization of the BCR outside (black) or inside (red) of the antigen cluster. Scale bar 1 μm. (D) Probability of BCR stopping inside or outside of the antigen clusters, based on a search through trajectories from (B). Error bars represent SEM for cells analyzed (n in parentheses). (E) Isolated monovalent antigen binding is not sufficient to immobilize the BCR. Single molecule diffusion of NIP₁-H12-HyLight647 either free on bilayers, or bound to J558L cells expressing B1-8 IgM BCR at the indicated concentrations. At 0.05/μm² less then 15 antigens bound to each cell. Immobilization was induced by supplementing NIP₁-H12-HyLight647 with 150/μm² of unlabeled NIP₁-H12. Data are from 583–1196 molecules from three experiments. (F) Lifetime of GFP-Syk single molecules at the plasma membrane in cells stimulated with the indicated antigens. Data represent means and SEM from 15–25 cells from three experiments. Grey line represents lifetime of CD81-GFP single molecules, a transmembrane protein controlling for the rate of photobleaching. (G) Diffusion of single GFP-Syk molecules. Data represent 1311–1946 molecules from two experiments. * in (A), (B) and (E–G) indicates p<0.0001 in Kolmogorov-Smirnov tests. * in (D) p<0.01 in t-test.

Figure 4. Cμ4 domain and the transmembrane region of the mIgM are required for BCR clustering in response to monovalent membrane antigens

Schematic structures of the BCR mutants that were expressed in J558L cells and the cumulative distribution of their single molecule diffusions in synapses with the indicated antigens. YS/VV mutation is indicated in red, TM mutation in orange. Data are from 789–2387 molecules from three experiments. *, p<0.0001 in Kolmogorov-Smirnov tests.

Figure 5. Accumulation in synapses, antigen binding, and type of movement of BCR constructs in synapses

(A) Quantification of the amount of CFP-tagged BCR constructs in synapses expressed as average CFP fluorescence. (B) Binding of antigens expressed as a ratio of fluorescence of bound antigen per BCR-CFP in the synapses. Data in (A) and (B) represent means and SEM from 65–79 cells from four experiments. (C) A trajectory of a YS/VV Cμ4ΔTM-CFP molecule (green) entering and confined in a cluster defined by YS/VV Cμ4ΔTM-CFP fluorescence (red). Plot shows instant diffusion and the corresponding localization of the molecule outside (black) or inside (red) of the BCR cluster. Scale bar 1 μm. (D) Confined diffusion of the YS/VV Cμ4ΔTM construct in synapses with NIP₁-H12 antigen. Plots show MSDs over time steps up to 0.7 s in BCR trajectories of the indicated constructs with or without binding to the NIP₁-H12 antigen. Data were acquired at 1–3 min of cell spreading when clusters were well resolved. Linearity of the plots indicates random walk, plateau indicates confined diffusion. Data represent means and SEM from 1619–1784 trajectories from three experiments.

Figure 6. Fc region of mIgM is important for the initiation of BCR signaling in response to membrane antigens

(A) Schematic representation of the fusion constructs of the mIgM with Igα/β. (B) Timecourse of tyrosine phosphorylation in synapses of J558L cells expressing the indicated fusion constructs. Phosphorylation was determined as a ratio of anti-phosphotyrosine staining and CFP fluorescence of the BCR constructs. Data points represent median fluorescence ratios and 95% confidence intervals from 14–75 cells measured in three independent experiments. **, p < 0.001, *, p < 0.05 in Mann-Whitney tests. (C) CD69 upregulation in primary B-cell blasts expressing the indicated constructs. Data show percentage of CD69 positive cells after gating on NIP-binding cells. The data represent means and SEM of eight experiments. *, p < 0.001 in paired t-tests.

Figure 7. Spontaneous clustering and B-cell activation by the Cμ4 domain

(A) Schematic structures of constructs of the Fc region and TIRF images of their distribution on the cell surface after spreading onto empty bilayers. Constructs were visualized via their intracellular CFP tag. (B) Single molecule diffusion of the Fc region constructs. Data are from 789–2662 molecules from at least three experiments. *, p<0.0001 in Kolmogorov-Smirnov tests. (C) Colocalization of the CFP-tagged Fc region constructs coexpressed with Igα-YFP in J558L cells that express endogenous Igβ. (D) Localization and lifetime of GFP-Syk molecules at the plasma membrane in J558L cells expressing the indicated constructs. Grey line represents bleaching of CD81-GFP single molecules as a control. *, p<0.0001 in Kolmogorov-Smirnov tests. Inset shows co-localization of Cμ4-CFP with GFP-Syk. See also Fig S8D. (E) CD69 levels in primary B cell blasts two days after transfection with the indicated constructs tagged with YFP. Data are gated on YFP positive cells. The mean and SEM of CD69 positive cells from three experiments were 20.3 ± 1.9% for Cμ4 versus 10.4 ± 0.9%, 9.4 ± 1% and 10.9 ± 1.1%, for the control, Cμ2-Cμ4 and Cμ3-Cμ4, respectively.