mAb-iB^TLR2-induced IL-6 production in Src signaling nhibited 4E-BP knockout BMMØ.

<p>mAb-iB^TLR2-induced IL-6 production in Src signaling nhibited 4E-BP knockout BMMØ.</p

TLR2 and FcγR synergize to drive the Macrophage IL-6 cytokine response to mAb-iB^TLR2.

<p><u>Panel A:</u> Flow cytometric analysis of mAb-iB^TLR2. The effect of mAb opsonization on particle size/aggregation was determined by forward light scatter (FCS, sub-panel i) and GFP levels (sub-panel ii). The level of opsonizing IgG mAb binding determined by staining with anti-mouse IgG-PE and analysis by flow cytometry (sub-panel iii, which is gated on GFP-expressing iB^TLR2). <u>Panel B:</u> BMMØ were pulsed with mAb-iB^TLR2 for 60 min. at 37°C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200764#sec002" target="_blank">methods</a>), fixed and stained for extracellular mAb- iB^TLR2 (green/yellow, arrows), intracellular mAb- iB^TLR2 (red) or DNA (DAPI, blue). Shown is a representative field of view from 1 of 3 independent experiments. <u>Panels C-E:</u> BMMØ were stimulated with mAb-iB^TLR2 or 1 μg/ml Pam3CSK4 as indicated (TLR2 blocking antibody was used at 10 μg/ml) and the resultant level of culture supernatant IL-6 cytokine determined by ELISA. Shown are IL-6 levels compared to untreated control or control wild type (WT) BMMØ stimulated with mAb-iB^TLR2, averaged across three or more independent experiments (± 1 S.D). Mean level of IL-6 detected in cultures supernatants from control mAb-iB^TLR2 stimulated BMMØ was 554 pg/ml (panel C), 3,334 pg/ml (panel D) and 1,147 pg/ml (panel E). Culture supernatants from both unstimulated BMMØ and BMMØ exposed to non-opsonized iB^TLR2 did not contain detectable levels of IL-6. <u>Panel F:</u> mAb-iB^TLR2 binding to the indicated BMMØ was determined by western blot analysis of the indicated samples for the opsonizing murine IgG mAb. The signal for the opsonizing mAb heavy chain (IgH) was determined across three independent experiments and is reported relative to WT BMMØ (± 1 S.D.). * = p<0.05, ** = p<0.01.</p

NF-κB activation by mAb-iB^TLR2 requires TLR2 but not FcγR ITAM signaling.

<p><u>Panel A:</u> WT and TLR2Δ BMMØ were stimulated with mAb-iB^TLR2 for the indicated time and total cellular IκB levels determined by western blot. <u>Panel B:</u> WT and TLR2Δ BMMØ were stimulated with mAb-iB^TLR2 for 30 minutes (arrow in Panel A). The cells were then fixed and stained for NF-κB and DNA (DAPI) and then imaged by confocal microscopy. <u>Panels C and D:</u> The indicated BMMØ were stimulated with mAb-iB^TLR2 and IκB degradation monitored as in panel A. For all panels, shown are representative results from 1 of 3 independent experiments. <u>Panel E:</u> Fraction of initial IκB levels observed 30 min. post stimulation for each sample (error bars = ± 1 S.D.). * = p<0.05. NS = not statistically significant.</p

FcγR ITAM signaling drives IL-6 mRNA incorporation into polysomes.

<p>BMMØ were stimulated for 2 hours with mAb-iB^TLR2 in the absence or presence of 10 µM PP2. Cells were then lysed under conditions that stabilize polysomes and the resulting lysates fractionated by sucrose density gradient centrifugation. <u>Panel A:</u> Plot of refractive index to demonstrates the density profile of the sucrose gradient. <u>Panel B:</u> The level of RP L26 (a ribosomal protein) and GAPDH (a soluble cytosolic protein) in each gradient fraction was determined by western blot. <u>Panels C and D:</u> The level of IL-6 and GAPDH mRNA in each gradient fraction was determined by RT-qPCR as detailed in the methods section. <u>Panel E:</u> The average level of mAb-iB^TLR2-induced IL-6 and GAPDH mRNA (with and without inhibition of FcγR signaling by PP2) in the indicated polysome fractions (panels C and D) across three independent experiments was determined. Error bars = ± 1 S.D. ** = p<0.01.</p

mAb-iB^TLR2 induction of IL-6 mRNA requires TLR2 but not FcγR ITAM signaling.

<p>BMMØ were stimulated with mAb-iB^TLR2 under the indicated conditions. The level of cellular mRNA was determined by RT-PCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200764#sec002" target="_blank">materials and methods</a>) and the level of secreted IL-6 or TNF protein determined by ELISA (ND = not detected). The mean level of cytokine detected in cultures supernatants from control mAb-iB^TLR2 stimulated BMMØ was 554 pg/m of IL-6 (panel A), 776 pg/ml of IL-6 (panel B) and 670 pg/ml of TNF (panel C). Show are the average levels of cytokine mRNA and protein (normalized to control non-PP2 WT BMMØ) across three or more independent experiments (error bars = ± 1 S.D.). ** = p<0.01.</p

FcγR ITAM signaling is necessary for the Macrophage IL-6 cytokine response to mAb-iB^TLR2.

<p><u>Panel A:</u> BMMØ were stimulated with mAb-iB^TLR2 under the indicated conditions and the resultant level of ROS production determined by luminol assay. Shown are results averaged across 6 independent experiments. Error bars indicate ± 1 S.E.M. <u>Panel B:</u> The indicated BMMØ were stimulated with mAb-iB^TLR2 under the indicated conditions and the resultant level of IL-6 cytokine in the culture supernatant determined by ELISA. The mean level of IL-6 detected in cultures supernatants from control mAb-iB^TLR2 stimulated BMMØ was 776 pg/ml. Shown are IL-6 levels compared to untreated BMMØ across three independent experiments (± 1 S.D). ** = p<0.01.</p

Receptor Endocytosis of Isolated CD79a but not CD79b.

<p>Endocytosis of the indicated MHC class II-CD79 fusion proteins was analyzed (<u>Panel A</u>) and quantitated (<u>Panel B</u>) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g001" target="_blank">Figure 1</a>. Statistical comparisons were made between the reporter proteins with both CD79 cytoplasmic domains and other reporter proteins.</p

C-terminal Deletions Localize the Motif that Drives AP2 Binding to the Membrane Proximal YxxØ Motif of CD79a.

<p>Binding of AP2µ-btn to bead-captured GST bearing the indicated C-terminal deletions of CD79a was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Data is the mean of 3 independent experiments ± S.E.M. and data were normalized to the binding of AP2 to full length CD79a.</p

AP2µ Binds to Isolated CD79a but not CD79b.

<p><u>Panel A</u>, Amino acid sequences of CD79a and CD79b cytoplasmic domains. YxxØ putative AP2 binding motifs underlined. <u>Panel B</u>, AP2µ expressed as a Gal4 activation domain fusion protein was assayed for specific interaction with the cytoplasmic domain of either CD79a or CD79b fused to the Gal4 DNA binding domain. Growth on histidine deficient (His-) plates indicates an AP2–CD79 interaction. The cytoplasmic domain of TGN38 contains a known AP2 binding YxxØ motif and served as a positive control, while the cytoplasmic domain of OCA2 contains a dileucine motif (which does not bind AP2µ) and served as a negative control. Data are representative of 2 experiments. <u>Panel C</u>, Diagram of the GST-CD79 cytoplasmic domain–AP2µ direct binding assay. <u>Panel D</u>, The cytoplasmic domains of CD79a and CD79b were expressed as GST fusion proteins in BL21 <i>E. coli</i> cells. GST-fusion proteins were captured from cell lysates on glutathione beads and the resulting matrix was tested for binding to <i>in vitro</i> translated, biotin-labeled AP2µ. The AP2 binding motif from TGN38, (SDYQRL)₃, and a non-AP2-binding derivation containing a tyrosine to glycine substitution, (SDGQRL)₃, fused to GST served as positive and negative controls, respectively. Binding is expressed as a percentage of (SDYQRL)₃–AP2 interactions. Data is the mean of 3 independent experiments ± S.E.M. Statistical comparisons were measured between SDYQRL and other samples.</p

AP2-mediated CD79-driven Endocytosis is Regulated in Cis via ITAM-embedded DCSM and QTAT motifs.

<p><u>Panel A</u>, Binding of AP2-btn to bead-captured GST bearing the indicated CD79a or CD79b cytoplasmic domains with the indicated mutations was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g003" target="_blank">Figure 3</a>. Binding is expressed as a percentage of wild type CD79a binding and represents the mean of 3 independent experiments ± SEM. Statistical comparisons were measured between the non-mutated CD79a or CD79b_DCSM cytoplasmic domains and other samples. <u>Panels B and C</u>, Endocytosis of the indicated MHC class II-CD79 fusion proteins was analyzed and quantitated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054938#pone-0054938-g001" target="_blank">Figure 1</a>. Statistical comparisons were made between the reporter protein expressing the wild type CD79a or CD79b cytoplasmic domains and other reporter proteins. <u>Panel D</u>, Diagrammatic representation of <i>cis</i> and <i>trans</i> regulation of BCR AP2 binding and endocytosis. The open arrows indicate <i>cis</i> regulation by the DCSM and QTAT regulatory motifs. The closed arrows represent <i>trans</i> regulation of the endocytic activity of each cytoplasmic domain by the presence of the partner cytoplasmic domain.</p