Non-stimulatory 20-2b2 induced the down-modulation of TCR expression.

<p>(A) Jurkat cells were cultured in the presence (green) or absence (red) of 20-2b2 (50% SN) at 37°C. Cells were washed and stained with the anti-TCRVβ8 Ab two hours later. Unstained Jurkat cells are shown as a blue line. (B) Jurkat cells were cultured in the presence (green) or absence (red) of 20-2b2 (50% SN) at 4°C for 20 min, and then stained with anti-TCRVβ8 Ab conjugated with FITC. (C) The PBMCs of healthy donors were cultured with the titrated amount of 20-2b2. Cell proliferation was measured two days later. Data are expressed as the mean ± SD of triplicate cultures. The proliferation of PBMCs without a stimulus was measured as 718±346 cpm. Results are representative of three independent experiments (A–C).</p

20-2b2 inhibited the activation of T cells <i>in vitro</i>.

<p>(A–C) The PBMCs of healthy donors were cultured with an anti-CD3 Ab (OKT3) (A), PHA (B), or PWM (C) in the presence (red symbols) or absence (black) of 20-2b2 (50% SN). (D) PBMCs were pre-treated with medium (black) or 20-2b2 (red) at 37°C. Cells were washed three times and cultured with PHA two hours later. Cell proliferation was measured two days later. Values are shown as the mean ± SD of triplicate cultures. Data are representative of more than three (A–C) and three (D) independent experiments. *<i>p</i><0.01, **<i>p</i><0.001 by the Student's <i>t</i>-test.</p

20-2b2 recognized CD3ε.

<p>(A, B) Jurkat (A) or J.RT3-T3.5 cells (B) were stained with anti-CD3ε Ab (OKT3) or 20-2b2 (green lines). Unstained cells are shown as red lines. (C) Jurkat cells were pre-treated with 20-2b2 (top panel) or OKT3 (bottom panel), and were then stained with the indicated Ab (yellow lines). Jurkat cells stained without the pre-treatment are shown as green lines. Negative control staining is shown as red lines. (D) Yac-1 or Yac-1 cells transfected with human CD3ε (hCD3ε-Yac-1) were stained with the anti-CD3ε Ab (OKT3) (green) or 20-2b2 (blue). Cells stained with rat IgG as a negative control are shown as red lines. (E) Cell lysates prepared from the indicated cells were separated in SDS-PAGE and blotted with 20-2b2. Arrow: the target molecule recognized by 20-2b2. (F) The lysate from Jurkat cells was immunoprecipitated (IP) with 20-2b2 or control rat IgG. Immunoprecipitates were separated by SDS-PAGE. The blot was probed (WB) with the anti-CD3ε Ab (M-20). Results are representative of three independent experiments (A-F).</p

Comparison of the taxa showing different abundance values in BD patients and normal individuals.

<p>We analyzed the metagenomic data of bacterial taxa using LEfSe to detect major differences between BD patients (BD) and normal individuals (NI). The LEfSe provides us with cladograms of six-level (from kingdom to genus). Significantly enriched bacterial taxa in samples obtained from BD patients were demonstrated using red circles and shadings. Significantly enriched bacterial taxa in samples obtained from normal individuals were demonstrated using green circles and shadings. In patients with BD, the phylum <i>Actinobacteria</i>, namely the classes <i>Actinobacteria</i> and <i>Coriobacteria</i>, the orders <i>Bifidobacteriales</i> and <i>Coriobacteriales</i>, and the genera <i>Bifidobacterium</i>, <i>Eggerthella</i> and <i>Atopobium</i> had large effect sizes. The phylum <i>Firmicutes</i>, the class <i>Clostridia</i>, the order <i>Clostridiales</i>, the family <i>Veillonellaceae</i>, and the genera <i>Megamonas</i> and <i>Phascolarctobacterium</i> had large effect sizes in normal individuals.</p

Comparison of fecal secretory IgA concentrations and bacterial diversity between BD patients and normal individuals.

<p>(A) We evaluated the secretory IgA (sIgA) concentrations of fecal supernatants using ELISA. We observed a significant increase in sIgA concentrations of patients with BD (BD) compared with those of normal individuals (NI). (B) We counted OTU numbers (annotated species numbers) and estimated alpha diversity score (Chao 1 and Shannon indexes) of each sample. We compared the titers between BD patients and normal individuals. We did not find significant differences in the parameters between BD patients and normal individuals. These biological parameters of BD patients and normal individuals were displayed with dot plots. A box-plot and a mean level (green line) of each group of BD patients and normal individuals were indicated. (C) We estimated beta diversity between BD patients and normal individuals using PCoA of QIIME software with linear conversion formulas. We visualized the PCoA plots in a three dimensional structure where three axes and each contribution ratio (principal coordinate, PC1–3, %) were depicted. We calculated the distance between the distribution of BD patients and that of normal individuals using a two-sided Student's two-sample t-test as an exploratory analysis. We obtained a significant P value of the test of beta diversity between BD patients and normal individuals.</p

Direct comparison of bacterial taxonomic abundance between BD patients and normal individuals.

<p>We compared directly the relative abundance (expressed as parts per unit) between BD patients (BD) and normal individuals (NI). We conducted Wilcoxon rank sum test for the differences in every taxon, followed by Tukey’s honestly significant difference (HSD) test. We considered that bacterial taxa increased or decreased significantly in patients with BD as compared with those in normal individuals by fulfilling the following criteria simultaneously; #1: bacterial taxa showing significant differences by the Wilcoxon rank sum test, and #2: bacterial taxa showing positive Tukey’s HSD values. We found that there were significant differences in 11 bacterial taxa (also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153746#pone.0153746.g002" target="_blank">Fig 2</a> with asterisks). “p__”, “c__”, “o__”, “f__” and “g__” indicate phylum, class, order, family and genus, respectively. Relative abundance of bacterial taxa in BD patients and normal individuals were displayed with dot plots. A box-plot and a mean level (green line) of each group of BD patients and normal individuals were indicated.</p

Demographical and clinical characteristics of patients with Behcet’s disease and normal individuals.

<p>Demographical and clinical characteristics of patients with Behcet’s disease and normal individuals.</p