No significant effect of a blocking anti-CD25 antibody on the proportion of human FoxP3^<b>+</b> CD4 T cells in vivo.

<p>The proportion of CD25⁺FoxP3⁺ (<b>1A</b>), CD25⁻FoxP3⁺ (<b>1B</b>) and total FoxP3⁺ cells (<b>1C</b>) among CD4 T lymphocytes was determined before liver grafting and basiliximab injection, and for more than 9 months thereafter; data are means (curves) and SEM at the different time points. In <b>1D</b>, the FoxP3⁺/FoxP3⁻ and FoxP3^hi/FoxP3⁻ MFI ratios were used to compare the level of intracellular FoxP3 expression (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011784#s3" target="_blank">Methods</a>). Means and SEM are shown. The Mann Whitney test was used for statistical analysis. The number of patients tested at each time point is indicated. Only statistically significant differences are indicated.</p

Human FoxP3^<b>+</b> CD4 T cells express high levels of IL-2 Rβ.

<p>IL-2Rβ (<b>Fig. 2A, 2B</b>), IL-2Rγ (<b>Fig. 2A, 2C</b>), IL-15Rα (<b>Fig. 2A, 2D</b>) and IL-7Rα (<b>Fig. 2A, 2E</b>) expression was analyzed by flow cytometry in FoxP3⁺ and FoxP3⁻ CD4 T cells before liver grafting and basiliximab injection (Figs. 2B to 2E), and at various times thereafter (2B to 2E). Fig. A shows a representative pre-graft staining profile. Figs. 2B to 2E represent the mean (SEM) MFI ratios (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011784#s3" target="_blank">Methods</a>). The Wilcoxon test was used to compare FoxP3⁺ and FoxP3⁻ CD4 T cells. The Mann Whitney test was used to analyze the time course of expression in a given cell subset. Only significant differences are indicated.</p

IFN-α increased IFN-γ gene responsiveness to IL-12. 3A, B cells were treated for various times with IFN-γ, and T-bet mRNA was quantified by RT-PCR.

<p>The results are expressed as -fold increases versus untreated cells. <b>3B</b>: T-bet mRNA -fold increases are shown for the naive and memory subsets at the 6-hour time point. <b>3C</b>: Purified naive and memory B cells were activated for 6 hours with IFN-α or IFN-γ. IL-12Rβ2 mRNA expression was analyzed by quantitative PCR. <b>3D</b>: B cells were pretreated with IFN-α for 18 h and treated with IL-12 for 24 h, then IFN-γ and GAPDH cDNAs were quantified by PCR. The results are mean ± SEM of values obtained with cells from 4 (<b>3B</b>, <b>3C</b>) or 6 (<b>3A</b>, <b>3D</b>) donors. Statistically significant differences are indicated by asterisks.</p

IFN-α induces STAT4 activation in human B cells.

<p>In <b>1A</b>, IFNAR1 and IFNAR2 expression was analyzed in the CD3⁻CD19⁺CD27⁺ and CD3⁻CD19⁺CD27⁻ lymphocyte gates. A representative staining profile is shown in the left-hand graph. The right-hand graph represents corrected mean fluorescence intensities (MFI) of IFNAR1 and IFNAR2 after substraction of MFI values obtained in isotype control in naive (CD27⁻) and memory (CD27⁺) B cell subsets. The results correspond to the mean ± SEM of the values obtained with cells from 6 healthy donors. <b>1B</b> shows the purity of B cell preparations (see methods). In <b>1C</b>, purified B cells were activated with IFN-α for 1 h then lysed. Western blotting was performed on whole-cell lysates by using anti-phospho-STAT2, and the membranes were reprobed with anti-STAT2. The data shown in <b>1C</b> are representative of 2 independent experiments. <b>1D, left panel</b>: B cells were activated for 1 hour with IFN-α. Western blotting was performed on whole-cell lysates by using anti-phospho-STAT1 or anti-phospho-STAT4. The membranes were then reprobed with anti-STAT1 or anti-STAT4. The data shown in <b>1D</b>, <b>left panel</b> are representative of 2 independent experiments. <b>1D, right panel</b>: B cells were activated with IFN-α for 1 hour or left untreated. They were then fixed, permeabilized, and stained with anti-STAT4 or anti-STAT1 (green) plus propidium iodide (nuclear staining, red). Nuclear translocation was examined by confocal microscopy. Yellow spots indicate nuclear STAT. Similar results were obtained in three other experiments. In <b>1E</b>, the kinetics of STAT1 and STAT4 phosphorylation was analyzed in B cells by flow cytometry with phospho-STAT-specific antibodies. A representative staining profile is shown in the left-hand graph. The right-hand graph represents corrected mean fluorescence intensities (MFI), after subtraction of MFI values obtained in isotype controls, of phospho-STAT1 and phospho-STAT4 in B cells. The data shown in 1E are the mean ± SEM for cells from 4 healthy donors.</p

IFN-α induces T-bet expression in B cells and primes B cells for IFN-γ production.

<p>In <b>2A</b>, T-bet mRNA expression at rest, assessed by RT-PCR, in untreated sorted naive and memory B cell subsets is shown. T-Bet expression was also quantitatively analyzed quantitative RT-PCR in total B cells after IFN-α treatment. Results are expressed as fold increases versus untreated total B cells. In <b>2B</b>, T-bet mRNA expression was analyzed in sorted naive and memory B cell subsets after IFN-α treatment for 6 hours. <b>2C</b>, B cells were treated for 24 h with IFN-α, and cDNAs were amplified with primers for IFN-γ, IL-12 p40, IL-12 p35, IL-4, IL-5, IL-13 and GAPDH. Positive controls consisted of the EBV-transformed B cell line RPMI-8866 (which constitutively expresses IL-12p35 and IL-12p40) and anti-CD3-activated T cells. <b>2D, 2E</b>: B cells pretreated with IFN-α for 18 hours were treated with SAC for 24 hours (for mRNA quantification, 2D) or 48 hours (for ELISA, 2E). The corresponding cDNA and supernatants were assayed for IFN-γ expression by quantitative PCR and ELISA, respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019366#pone-0019366-g002" target="_blank"><b>Fig. 2F</b></a> shows IFN-γ expression in naive and memory B cell subsets after IFN-α pretreatment and SAC activation for 12 hours, as determined with flow cytometry. Dashed lines correspond to isotype control. Data are representative of 4 different donors (<b>2C, 2F</b>) or are mean± SEM of 6 to 7 different donors (<b>2A, 2B, 2D, 2E</b>). Statistically significant differences are indicated by an asterisk.</p

Additional file 1: Table S1. of Serum IL-33, a new marker predicting response to rituximab in rheumatoid arthritis

Baseline characteristics of RA patients from Clermont and from Leeds. (DOC 33 kb

Combined Linkage and Association Studies Show that HLA Class II Variants Control Levels of Antibodies against Epstein-Barr Virus Antigens

<div><p>Over 95% of the adult population worldwide is infected with Epstein-Barr virus (EBV). EBV infection is associated with the development of several cancers, including Hodgkin lymphoma (HL). Elevated levels of anti-EBV antibodies have been associated with increased risk of HL. There is growing evidence that genetic factors control the levels of antibodies against EBV antigens. Here, we conducted linkage and association studies to search for genetic factors influencing either anti-viral capsid antigen (VCA) or anti-Epstein Barr nuclear antigen-1 (EBNA-1) IgG levels in a unique cohort of 424 individuals of European origin from 119 French families recruited through a Hodgkin lymphoma (HL) patient. No major locus controlling anti-VCA antibody levels was identified. However, we found that the HLA region influenced anti-EBNA-1 IgG titers. Refined association studies in this region identified a cluster of HLA class II variants associated with anti-EBNA-1 IgG titers (e.g. p = 5×10–5 for rs9268403). The major allele of rs9268403 conferring a predisposition to high anti-EBNA-1 antibody levels was also associated with an increased risk of HL (p = 0.02). In summary, this study shows that HLA class II variants influenced anti-EBNA-1 IgG titers in a European population. It further shows the role of the same variants in the risk of HL.</p></div

Genome-wide linkage analysis and association study of anti-EBNA-1 IgG levels in the linked 6p region.

<p>Top panel: Multipoint LOD score (left y-axis) and information content (right y-axis) are plotted along the 22 autosomes (x axis). Mid panel: Results of association study for 824 SNPs located in the 90% confidence linkage interval are given as −log₁₀ (pvalue) (y-axis) and plotted against SNP positions on chromosome 6 (x-axis in megabases). Bottom panel: Association results as −log₁₀ (pvalue) (y axis) with 1) the cluster of SNPs in strong linkage disequilibrium (r²>0.8) with rs9268403 (squares), and 2) the SNPs reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102501#pone.0102501-Rubicz1" target="_blank">[3]</a>(circles); the two genotyped SNPs are shown under dominant model (plain symbol), and all the others SNPs are imputed and shown under additive model (open symbols); chromosome 6 position of SNPs and genes in the region are given on the x axis (in megabases).</p

Anti-EBNA-1 IgG distributions among offspring and parent samples as a function of their genotype.

<p>Mean titers (black circles) with 95% confidence interval for anti-EBNA-1 IgG levels are shown as a function of the genotype at rs9268403 under a dominant model for the minor allele C (TT vs CC/CT).</p

Autonomous NK commitment of PB-HP: morphology and expression of cytolytic granules.

<p>(A) Ral 555 staining on 3-weeks-old PB-HP cultured in in STEMα Α medium supplemented with 100 ng/ml of FL and SCF. PB-NKP exhibit a lymphoid morphology. (B) Intracytoplasmic expression of perforin and granzyme B in PB-NKP and in PB activated conventional NK cells was analyzed by confocal microscopy. Both cell types were fixed, permeabilized, and stained for perforin and granzyme B. As negative controls, cells were incubated with mouse IgG, and the second reagent. (C) Analysis of the cytolytic activity of freshly purified NK cells from healthy donors (open boxes) and from PB-NKP (grey boxes) against ⁵¹Cr-labelled K562 cells. NK cell effectors, treated or not for 18 hours with IL-12 (5 ng/ml), were assayed in a 4-h ⁵¹Cr-release assay against K562 cells in a dilution of E/T cell ratios (10∶1) in duplicate wells. Data are representative of three experiments performed.</p