Biparametric representations of B-cell maturation pathways in the lymph node.

<p>Identification of B-cell subsets in lymph node (A) and analysis of the expression of various cell surface markers versus CD27 expression (B). The specific tube of antibody combination used to analyze the expression of each marker is indicated in each graph, at the upper right. B-cell maturation is shown with arrows, drawn from the most immature. Data from one patient out of three with similar results are here represented.</p

Relative expression patterns of 17 B-cell markers based on median fluorescence intensities (MFI).

<p>Means of MFI of three stage 1 or 2 hematogone samples, nine transitional B cell samples, twelve naive B cell samples, three germinal center B cell samples, nine memory B cell samples, six plasma cell samples are represented. Expression levels between different markers cannot be compared, since the measured intensity of fluorescence depends also on the specific fluorochrome bound to the antibody. HG 1: hematogones stage 1, HG 2: hematogones stage 2, TR B: transitional B cells, N B: naive B cells, GC B: germinal center B cells, M B: memory B cells, PPC: pre-plasma cells, PC: plasma cells.</p

Gating strategy for B-cell subsets’ identification.

<p>Dot plots on blue background identify subpopulations from the antibody backbone. HG 1: hematogones stage 1, HG 2: hematogones stage 2, IMM B: immature B cells, TR B: transitional B cells, N B: naive B cells, GC B: germinal center B cells, M B: memory B cells, NM B: natural B memory cells, MM B: non-switched memory B cells, SM B: switched memory B cells, PC: plasma cells.</p

Phenotypic chatacterization of B-cell subsets.

<p>Phenotypic chatacterization of B-cell subsets.</p

Panel of seven 8-colour antibody combinations.

<p>Panel of seven 8-colour antibody combinations.</p

CD24 and CD44 expression during B cell differentiation in lymph nodes.

<p>CD24 and CD44 markers define two separate subsets within germinal center B cells. Data from one patient out of three with similar results are here represented.</p

Graphical representation of the frequency (mean ± SD) of each stage of B-cell differentiation identified in the different types of samples.

<p>Samples of different anatomical sites (bone marrow, peripheral blood, lymph node and cord blood) were stained with the antibody combination of tube 1 of the panel and analyzed with the gating strategy shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162209#pone.0162209.g002" target="_blank">Fig 2</a> in order to identify ten B-cell subsets. Cell frequency has been quantified by calculating the mean of % of cells of each subset within CD19⁺ cells (± standard deviation). HG 1: hematogones stage 1, HG 2: hematogones stage 2, IMM B: immature B cells, TR B: transitional B cells, N B: naive B cells, GC B: germinal center B cells, NM B: natural B memory cells, MM B: non-switched memory B cells, SM B: switched memory B cells, PC: plasma cells. Data from three patients for each type of sample are represented.</p

Biparametric representations of B-cell maturation pathways in the bone marrow.

<p>The modulation of the expression of various cell surface markers versus CD10 expression is represented in dot plots. The specific tube of antibody combination used to analyze the expression of each marker is indicated in each graph, at the upper right. B-cell maturation is shown with arrows, drawn from the most immature B-cell subset to the most mature one. Data from one patient out of three with similar results are here represented.</p

CHIKV infection causes severe myocarditis in mouse neonates.

<p>Histological appearance of horizontal sections of the heart through left and right ventricles of 12-day FVB (<b>A</b>, <b>C</b> and <b>E</b>) and GADD34^ΔC/ΔC mice at D5 pi (<b>B</b>, <b>D</b> and <b>F</b>). Normal appearance of heart of FVB infected mice, at low magnification (<b>A</b>, ×10) with normal cardiomyocytes (<b>C</b>, ×100) and exceptional small foci of lymphocytes (<b>E</b>, ×400). Numerous foci of necrosis in the heart of GADD34^ΔC/ΔC infected mice, at low magnification (<b>B</b>, ×10) and extensive through the ventricular wall (<b>D</b>, ×100). Higher magnification shows few residual cardiomyocytes (arrow head) and inflammation mainly composed of monocytes as well as extensive deposition of calcium (<b>F</b>, ×400). The mice were inoculated with 10⁶ PFU of CHIKV via the intradermal route.</p

Translation inhibition and IFN-β production are induced by poly I:C in MEFs.

<p><b>A</b>) Protein synthesis was monitored in poly I:C(pI:C)-stimulated MEFs using puromycin labelling followed by immunoblot with the anti-puromycin mAb 12D10. Controls are cells not treated with puromycin (No puro) and cells treated with cycloheximide (chx) 5 min prior puromycin incorporation. β-actin immunoblot is shown for equal loading control. Quantification of puromycin signal was quantified with ImageJ software and is represented above the immunoblot. Phosphorylation of eIF2α (P-eIF2α) was assessed in the same MEFs extracts. <b>B</b>) Immunofluorescence staining for puromycin, P-eIF2α and dsRNA of MEFs treated with poly I:C for 4 h and labeled with puromycin for 1 h. Scale bar, 10 µm. <b>C</b>) WT and PKR^−/− MEFs were stimulated for 8 h with poly I:C (pI:C), thapsigargin (th) or arsenite (as). PKR and P-eIF2α were detected by immunoblot. <b>D</b>) WT and PKR^−/− MEFs were stimulated for 8 h with poly I:C and protein synthesis was monitored like in (A). β-actin immunoblot is shown for equal loading control. <b>E</b>) IFN-β levels were measured, by ELISA, in cell culture supernatants of WT, PKR^−/−, eIF2αA/A and control eIF2αS/S MEFs after 4 and 8 h of poly I:C stimulation. Data are mean ± standard deviation of 3 independent experiments. <b>F</b>) Protein synthesis was measured in NIH3T3 cells by puromycin incorporation after 7 h of poly I:C treatment. Where indicated, a chase of 1 h with fresh media was performed prior to puromycin labeling and immunoblotting. Samples with cycloheximide (chx) and arsenite (as) added respectively 5 min and 30 min before the puromycin pulse are shown as controls. <b>G</b>) IFN-β was quantified by ELISA in culture supernatants in the conditions described above after 7 h of poly I:C stimulation or 7 h of poly I:C stimulation followed by 1 h with fresh media (chase). Data are mean ± standard deviation of 4 independent experiments.</p