Gel filtration analysis of BAFFR-Fc, TACI-Fc, Fn14-Fc and atacicept.

<p>Panel A. Superdex-200 elution profiles of the indicated receptors-Fc monitored at 280 nm. The elution position of molecular mass standards is indicated at the top of the profiles. Panel B. BMDMs were treated with TACI-Fc obtained before (total preparation) or after size exclusion chromatography (fraction 8 with high molecular mass aggregates and fraction 14 with dimeric TACI-Fc). Cells were also treated with atacicept (before size fractionation), a form of TACI-Fc unable to bind to Fc receptors. Cell activation was monitored by western blotting with an anti-phospho ERK1/2 antibody. Panel C. BAFF-sensitive BAFFR:Fas expressing cells were stimulated with the indicated concentration of BAFF 60-mer, in the presence or absence of atacicept at a fixed concentration of 300 ng/ml. Cell viability was monitored with a colorimetric assay.</p

Signalling by BAFFR-Fc and TACI-Fc also takes place in APRIL^−/− and BAFF^−/− BMDMs, and is not increased in BMDMs with uncleavable BAFF.

<p>Bone marrow-derived macrophages from various mice in the C57BL/6 background were stimulated for the indicated times with BAFFR-Fc, TACI-Fc or Fn14-Fc at 5 µg/ml, or with LPS at 50 ng/ml. Cells were lysed and analyzed by Western blotting with the indicated antibodies. Panel A: wt BMDMs. Panel B: APRIL^−/− BMDMs. Panel C: BAFF^−/− BMDMs. Panel D: BMDMs from BAFF^−/− mice overexpressing non-cleavable BAFF from a BAC transgene <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061350#pone.0061350-Bossen2" target="_blank">[23]</a>. *: as judged by the anti-tubulin blot, less sample was accidentally loaded.</p

Impaired TACI-Fc signalling in FcRγ^−/− BMDMs.

<p>Bone marrow-derived macrophages from WT and FcRγ^−/− C57BL/6 mice were stimulated for the indicated times with TACI-Fc at 10 µg/ml, atacicept at 10 µg/ml, or with LPS at 50 ng/ml. Cell extracts were analyzed by western blotting with the indicated antibodies.</p

Structure of the Fab17.2 – R13 complex.

<p>A. Superposition of the apo Fab 17.2 and R13 complex structures (grey and green respectively). VH and VL contact residues are indicated. B. Main water molecules present on the antigen binding site of Fab 17.2 apo. C. Superposition of water molecules present in the apo Fab 17.2 that are replaced by the peptide in the Fab 17.2 R13 complex. D. Superposition of R13 peptides from molecules 1 (green) and 2 (magenta). E. Structure of the Fab 17.2-R13 complex (molecule 1). All hydrogen bonds between mAb 17.2 and peptide R13 are illustrated as dotted yellow lines. The π-stacking interaction between VL Tyr101 and the epitope Phe9 is also indicated. Heavy chain CDRs are coloured in red and light chain CDRs in blue the peptide is coloured in light green.</p

Polar contacts in the model between 17.2 and β1-AR.

<p>MC: Main Chain; SC: Side Chain.</p

BAFFR-Fc and TACI-Fc, but not Fn14-Fc, induce signals in THP1 and U937 cells, but not in BAFF-expressing 293 cells.

<p>Panel A: THP1 cells were stimulated with BAFFR-Fc, TACI-Fc, Fn14-Fc (all at 5 µg/ml) or LPS (50 ng/ml) for the indicated times. In the P-IκBα blot, bands marked “*” are remnants of the P-ERK blot. The P-IκBα band is just underneath, indicated with an arrow. Bands in the P-ERK blot were quantified, normalized to the intensity of P-ERK at time zero, and plotted as a function of time. Panel B: same as panel A, except that the experiment was performed with U937 cells. Panel C: HEK-293 cells were stimulated with 5 µg/ml of BAFFR-Fc or with 100 nM PMA for the indicated time. Panel D: same as panel C, except that HEK-293 cells stably expressing full-length BAFF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061350#pone.0061350-Schneider1" target="_blank">[22]</a> were used. In all panels, all extracts were analyzed by western blotting using the indicated antibodies.</p

BAFFR-Fc and TACI-Fc, but not Fn14-Fc, induce signals in bone marrow-derived macrophages.

<p>Bone marrow-derived cells from C57BL/6 mice were stimulated for the indicated time with BAFFR-Fc, TACI-Fc or Fn14-Fc at 10 µg/ml, or with LPS at 50 ng/ml. Cells were lysed in sample buffer and analyzed by Western blotting with the indicated antibodies.</p

Functional activity of the mAb 17.2.

<p>A. Representative histogram showing the HEK and HEK-β1 cells labelled with mAb 17.2 followed by a Cy3-conjugated goat anti-mouse IgG. B. Results are expressed as means ± SD (n = 3). Inset: Binding of mAb 17.2 to HEK-β1 cells in the presence of R13 peptide. ** <i>p</i><0.01; *** <i>p</i><0.001. C. Passive transfer of mAb 17.2 to naïve mice. Repolarisation abnormalities (a) and first degree AV conduction block (b) are indicated by arrows. bpm: beats per minute.</p

Structure of the Fab 17.2.

<p>A. Apo Fab 17.2 structure (molecule 1). Heavy chain CDRs are coloured in red and light chain CDRs in blue. B. Superposition of molecules 1, VH-VL region of the two crystals asymmetric units. C. Superposition of molecules 2, VH-VL region of the two crystals asymmetric units. Apo Fab 17.2 (grey), Fab 17.2-R13 molecule 1 (green) and molecule 2 (magenta).</p

Buried surface upon complex formation, with shape complementarities (Sc) [50] and number of contacts observed between the mAb 17.2 and the R13 peptide in the crystal structure¹ and the model².

<p>Buried surface upon complex formation, with shape complementarities (Sc) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001375#pntd.0001375-Lawrence1" target="_blank">[50]</a> and number of contacts observed between the mAb 17.2 and the R13 peptide in the crystal structure¹ and the model².</p