Figure 1: TLR priming increases the sensitivity of B cells to membrane-bound antigens. | Nature Communications

Figure 1: TLR priming increases the sensitivity of B cells to membrane-bound antigens.

From: Toll-like receptor ligands sensitize B-cell receptor signalling by reducing actin-dependent spatial confinement of the receptor

Figure 1

(a) Pseudocolored scanning EM image of B cells (purple) adhering to an APC (green) expressing a transmembrane rat anti-mouse Igκ antibody (surrogate antigen (Ag)). Scale bar, 50 μm. (b) Confocal images of ex vivo B cells that were added to APCs for 3 min before staining with Alexa488-anti-rat IgG to detect the surrogate antigen. Scale bar, 20 μm. Cells were visualized by F-actin staining (inset). To categorize APCs as having low or high antigen density, the gain was increased and the intensity of Alexa488-anti-rat IgG staining in regions of the APC that were not engaged by B cells was quantified (mean±s.e.m; n=30 cells from three experiments). (c,d) Ex vivo B cells, or B cells that had been cultured for 16 h with 5 ng ml−1 BAFF, BAFF+5 μg ml−1 LPS or BAFF+0.5 μg ml−1 CpG DNA, were added to APCs for 3 min and stained for surrogate antigen (Ag) and either pTyr (c) or pERK (d). At the settings used, only clustered antigen is detected. Confocal slices of pTyr staining at the contact site between B cells and APCs expressing low amounts of surrogate antigen (c). Staining for pERK, antigen and nuclei (4′,6-diamidino-2-phenylindole (DAPI) staining; outlined on pERK images) is shown as three-dimensional projections reconstructed from confocal slices (d). Scale bars, 5 μm. pTyr intensity at the B cell:APC contact site, as well as total pERK intensity, were quantified from images and normalized to those in BAFF-cultured B cells that had bound to APCs with a low density of surrogate antigen (mean±s.e.m. for three experiments; n>30 B cells per point). (e,f) B cells that had been cultured overnight with BAFF or BAFF+LPS were incubated for 10 min in wells coated with the indicated densities of anti-Igκ or 2 μg cm−1 anti-MHCII (−). pERK and total ERK (e) or pAkt and total Akt (f) blots are shown. pERK/ERK and pAkt/Akt ratios, normalized to those for LPS-cultured cells plated on 2 μg cm−2 anti-Igκ, are graphed (means for greater than three experiments). **P<0.01 using Student’s two-tailed unpaired t-test.

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