PP-DC express a distinct molecular footprint.

<p>(A) Dot plots show MACS sorted CD11c⁺ DC isolated from the PP or PLN and stained with antibodies against CD11c and CD11b. (B) Histrogram plots show cells from (A) gated for CD11c expression and stained with antibodies against MHC class II, CD40 or CD80 as indicated. All plots show expression levels of the indicated activation marker (solid line) as compared to unstained CD11c⁺ control populations (shaded area). All data shown are from one experiment and are representative of at least three independent experiments. (C) Relative mRNA levels to GAPDH for the indicated genes as determined for CD11c⁺ DC isolated from the PP or PLN by quantitative real time PCR. Results shown are from representative measurements. (D) Same as (C) shown as fold change for PP-DC (black bars) relative to PLN-DC (open bars) for which mRNA expression was normalized to a value of 1. Data represent the mean±S.E.M. of combined values from at least three independent experiments.</p

PP-DC promoted IgA production requires BCMA and TACI signaling in the absence of CD40- or LPS-mediated signals.

<p>Naïve splenic IgD⁺ B cells from C57BL/6 (plain bars) or TACIxBCMA^o/o mice (striped bars) were cultured together with CD11c⁺ DC from the PP (black and white bars) or PLN (grey bars). (A–C) Anti-CD40 mAb (2 µg/ml) was added to all cultures in the absence or presence of additional LPS (1 µg/ml) as indicated. (D–F) Cultures were performed in the absence of anti-CD40 mAb but contained LPS (1 µg/ml) where indicated. At the end of 7 days of culture, supernatants were collected and (A&D) IgA, (B&E) IgG1 and (C&F) IgM antibody concentrations determined by standard ELISA assay. Cultures were performed in triplicate and the mean±S.E.M. are shown. B.D. depicts those samples where the antibody concentration was below the detection limit of the ELISA assay. No IgA was detected in cultures containing DC alone. The data shown are from one experiment and are representative of two (D–F) and three (A–C) independent experiments.</p

Intestinal commensal bacteria are partially responsible for the ability of PP-DC to promote IgA production.

<p>Naïve splenic IgD⁺ B cells from C57BL/6 mice were cultured alone (open bars) or together with CD11c⁺ DC from the PP (black bars) or PLN (grey bars). CD11c⁺ DC were isolated from mice maintained under SPF or GF conditions as indicated. Anti-CD40 mAb (5 µg/ml) was added to all cultures in the absence or presence of additional LPS (1 µg/ml) as indicated. At the end of 7 days of culture, supernatants were collected and (A) IgA, (B) IgG1 and (C) IgM antibody concentrations determined by standard ELISA assay. Cultures were performed in triplicate and the mean±S.E.M. are shown. B.D. depicts those samples where the antibody concentration was below the detection limit of the ELISA assay. No IgA was detected in control cultures containing DC alone. The data shown are from one experiment and are representative of three independent experiments.</p

PP-DC promote IgA production by B cells.

<p>(A) The dot plot (left panel) shows IgM and IgD expression by MACS sorted splenic IgD⁺ B cells. Gates depict putative B2 and B1 cell populations. The histogram plot (right panel) shows CD5 expression by splenic IgD⁺ B cells (shaded area) or peritoneal B cells (solid line) gated for CD19 expression. (B–D) Splenic IgD⁺ B cells from naïve C57BL6 mice were cultured alone (open bars) or together with CD11c⁺ DC isolated from the PP (black bars) or PLN (grey bars). Anti-CD40 mAb (2 µg/ml) was added to all cultures in the absence or presence of additional LPS (1 µg/ml) as indicated. At the end of 7 days of culture, supernatants were collected and (B) IgA, (C) IgG1 and (D) IgM antibody concentrations determined by standard ELISA assay. Cultures were performed in triplicate and the mean±S.E.M. are shown. B.D. depicts those samples where the antibody concentration was below the detection limit of the ELISA assay. No IgA was detected in control cultures containing DC alone. The data shown are from one experiment and are representative of at least three independent experiments.</p

RA and TGF-β are involved in PP-DC mediated IgA production.

<p>Naïve splenic IgD⁺ B cells from C57BL/6 mice were cultured together with CD11c⁺ DC from the PP. Anti-CD40 mAb (5 µg/ml) was added to all cultures in the absence (A) or presence (B) of additional LPS (1 µg/ml). LE135, anti-TGF-β or the combination of both were added where indicated. At the end of 7 days of culture, supernatants were collected and IgA concentration determined by standard ELISA assay. The inhibitory effect of each reagent and their combination was calculated by comparing their IgA concentrations to control cultures containing medium alone, and are depicted as percent inhibition, medium being equivalent to 0% inhibition. Cultures were performed in triplicate and the mean±S.E.M. are shown. The data shown are from one experiment and are representative of two independent experiments.</p

DataSheet_1_Kinetics of free and ligand-bound atacicept in human serum.pdf

BAFF (B cell activation factor of the TNF family/B lymphocyte stimulator, BLyS) and APRIL (a proliferation-inducing ligand) are targeted by atacicept, a decoy receptor consisting of the extracellular domain of TACI (transmembrane activator and calcium-modulator and cyclophilin (CAML) interactor) fused to the Fc portion of human IgG1. The purpose of the study was to characterize free and ligand-bound atacicept in humans. Total and active atacicept in serum of healthy volunteers receiving a single dose of subcutaneous atacicept or in patients treated weekly for one year were measured by ELISA, Western blot, or cell-based assays. Pharmacokinetics of free and bound atacicept were predicted based on total atacicept ELISA results. Persistence of complexes of purified atacicept bound to recombinant ligands was also monitored in mice. Results show that unbound or active atacicept in human serum exceeded 0.1 µg/ml for one week post administration, or throughout a 1-year treatment with weekly administrations. After a single administration of atacicept, endogenous BAFF bound to atacicept was detected after 8 h then increased about 100-fold within 2 to 4 weeks. Endogenous heteromers of BAFF and APRIL bound to atacicept also accumulated, but atacicept-APRIL complexes were not detected. In mice receiving intravenous injections of purified complexes pre-formed in vitro, atacicept-BAFF persisted longer (more than a week) than atacicept-APRIL (less than a day). Thus, only biologically inactive BAFF and BAFF-APRIL heteromers accumulate on atacicept in vivo. The measure of active atacicept provides further support for the once-weekly dosing regimen implemented in the clinical development of atacicept.</p

Additional file 1: of CART cells are prone to Fas- and DR5-mediated cell death

Supplementary Figures 1-3. (PDF 18433 kb

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

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

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