B cell activation is regulated by B cell antigen receptor (BCR) signaling and antigen internalization in immune synapses. Using large-scale imaging across B cell subsets, we found that, in contrast with naive and memory B cells, which gathered antigen toward the synapse center before internalization, germinal center (GC) B cells extracted antigen by a distinct pathway using small peripheral clusters. Both naive and GC B cell synapses required proximal BCR signaling, but GC cells signaled less through the protein kinase C-β–NF-κB pathway and produced stronger tugging forces on the BCR, thereby more stringently regulating antigen binding. Consequently, GC B cells extracted antigen with better affinity discrimination than naive B cells, suggesting that specialized biomechanical patterns in B cell synapses regulate T cell–dependent selection of high-affinity B cells in GCs.
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Supported by the European Research Council (Consolidator Grant 648228 to K.M.S. and P.T.), the EMBO Young Investigator Programme (P.T.), and the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council and the Wellcome Trust (C.R.N., K.M.S. and P.T.).
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Integrated supplementary information
Supplementary Figure 1 Schematic workflow and identification of cells in large-scale imaging using CellScore.
(a) Schematic of large-scale imaging workflow, including substrate preparation, loading with B cells, fixing, staining and imaging. (b) Mean intensity projection of a cell stained with anti-B220. Plot below shows intensities in the B220 and brightfield channels as mean-normalized radial profiles. Correlation coefficients of the profiles to predetermined ideal profiles of the B220 and brightfield channels are calculated for each object; the average of the two correlations is termed CellScore. (c) Plot of CellScore values versus B220 intensity in a typical large-scale experiment. The three gates show correctly identified cells (B220 positive, CellScore high), incorrectly identified cells or cell-associated debris (B220-positive, CellScore low), and non-cellular debris and artifacts (B220-negative, CellScore low). Images show B220 staining (green), brightfield (grey) and outlines of the identified objects (red). Scale bars, 5 µm.
Left, galleries of synaptic planes of naive and GC B cells interacting with antigen (anti-Igκ) on PLBs. Intensity of the tubulin signal was equalized between the two subsets to allow visibility of the individual microtubules in the face of the brighter tubulin staining in GC cells. Right, quantification of overall levels of tubulin staining and of tubulin polarization calculated as the ratio of mean tubulin intensity in the synapse to the mean tubulin intensity in the whole cell. Bar graphs show means and s.e.m. from n = 4429 naive and 3138 GC cells from one out of two experiments. Scale bar, 5 µm. ****, p < 10-4.
(a) Intracellular staining in unstimulated and anti-Igκ stimulated naive and GC B cells analyzed by flow cytometry. (b) Specificity of phospho-specific intracellular staining in large-scale imaging controlled by inhibitor treatment. Naive and enriched GC B cells were treated with DMSO or the indicated inhibitors, incubated with PMSs containing anti-Igκ antigen, fixed and stained with the phospho-specific antibodies. Staining was analyzed in synapses (p-Syk, p-BLNK, p-JNK, p-SHIP1) or whole cells (p-Erk, p-Akt, p-MLC) gated on naive (N) or GC B cells outside and on the PMSs. FU fluorescence units. Data are means and s.e.m. from one out of two independent experiments.
Synapse planes (top panels) and side views (bottom panels) of naive and GC B cells on PMSs showing intracellular staining with the indicated reagents (green). Red, antigen (anti-Igκ), blue, B220 staining. Scale bars, 5 µm.
Supplementary Figure 5 Reduced PKCβ activity in GC B cells and consequences of PKCβ inhibition on synaptic architecture and antigen extraction.
(a, b) Quantification of synaptic staining and synaptic recruitment using antibodies recognizing phospho-PKCβ (a) and total PKCβ (b). Data are from cells recognizing antigen (anti-Igκ) on PMSs and were analyzed as in Fig. 5. (c) Representative images of antigen (anti-Igκ) in synapses of naive and GC cells interacting with PLBs and quantification of antigen centralization with or without inhibition of PKCβ with Go6976. (d) Quantification of antigen extraction from PMSs with or without inhibition of PKCβ. All bar plots show means and s.e.m. from n = 130-809 naive and 88-184 GC B cells in (a, b) and 103-115 naive and 17-27 GC cells in (d) representing one out of three experiments. *, p < 10-4 in non-parametric tests between the GC and corresponding naive cells or between the bracketed naive B cell groups. Scale bar, 5 µm.
Supplementary Figure 6 Internalization and degradation of soluble antigen by naive and GC B cells, as determined by flow cytometry.
(a) Internalization of soluble biotinylated anti-Igκ determined by surface accessibility to streptavidin binding. (b) Percentage of degradation of a DNA-based degradation sensor conjugated to anti-Igκ. Data are means and s.e.m. from three experiments.
Supplementary Figure 7 Force sensor design and synapses of naive and GC B cells with or without inhibition of myosin II by blebbistatin.
(a) Schematic explaining the design of sensors used to measure force of BCR pulling. Black ‘ladders’ depict DNA, with F1/2 corresponding to the force required to open the DNA hairpin. The antigen conjugated to the sensor was anti-Igκ. (b) Images of antigen (anti-Igκ) in synapses of naïve and GC B cells with or without inhibition of myosin II by blebbistatin. Data are from one out of three experiments analyzed in Fig. 7d. Scale bar, 5 µm.
(a) Staining with NP15 or NIP15 antigens analyzed by flow cytometry in splenic cells of immunized mice that received transferred B1-8 B cells. Staining is analyzed after gating on the transferred B1-8 B cells that differentiated into GC B cells (CD45.2+, Fas+, CD38-) compared to host-derived GC B cells (CD45.1+, Fas+, CD38-). (b) Staining of B1-8 naive and GC B cells with monovalent NP and NIP antigens using the indicated concentrations. MFI, mean fluorescence intensity. Data are from one out of two experiments.
Supplementary Figures 1–8 and Supplementary Table 1 (PDF 1488 kb)
Antigen (anti-Igκ, grey) in naive (left) and GC (right) B cell synapses with PLBs observed using timelapse TIRF microscopy. Time is shown in minutes and seconds. (AVI 2895 kb)
Synapses with anti-Igκ coupled to a 9 pN force sensor on PLBs in naive (upper panel) and GC (lower panel) B cell synapses. Signals from the force-insensitive label Atto550 (green) and the force-sensitive Atto647N (red) are overlaid on the left panels, and the red-to-green ratio defining opening of the sensor are shown on the right panels. Time is shown in minutes and seconds. (AVI 2296 kb)
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Nowosad, C., Spillane, K. & Tolar, P. Germinal center B cells recognize antigen through a specialized immune synapse architecture. Nat Immunol 17, 870–877 (2016). https://doi.org/10.1038/ni.3458
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