Expression of IL-4 regulated genes in HRS malignant cells.

*<p>Numbers in parentheses indicate the number of genes within the gene-set analyzed.</p><p>IL-4-regulated genes were identified experimentally by Elo <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Elo1" target="_blank">[35]</a>; of the 21014 annotated genes in the U133 plus2 Affymetrix array platform, 640 were identified as being up-regulated and 484 down-regulated by IL4 in T cells. Genes that were up- or down-regulated in microdissected HRS malignant cells from HL biopsies relative to control GC-centrocytes were identified from the data of Brune <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Brune1" target="_blank">[38]</a>. Complete gene-lists are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s002" target="_blank">Data-File S2</a>. For the determination of OR and statistical significance, equal sized gene-sets were compared by ranking probe-sets according to the magnitude of FC of the HRS cells, and selecting the top 640 or 484 unique genes.</p

Similarities in gene-expression changes induced by EBV and CD40L/IL4.

*<p>Numbers in parentheses indicate the number of genes within the gene-set. Note that for one gene (ZNF487P), one probe-set was up- regulated and one was down-regulated by EBV compared to resting B cells.</p><p>Genes altered by EBV or by CD40L/IL4 on day 7 relative to resting B cells at day 0 were determined by a paired LIMMA analysis with a significance threshold of P<0.01; FC>1.5. The Odds Ratio (OR) and significance of the overlaps were determined using the denominator, 17311, representing the total number of unique annotated genes on the HuExon 1.0 ST array. Complete gene-lists are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s002" target="_blank">Data-File S2</a>.</p

Effect of IL4 on EBV-induced B cell transformation.

<p>Purified B cells were infected with EBV at a series of multiplicities of infection (m.o.i.) in the absence or presence of added IL-4. For each infection dose, 10³ B cells were seeded into each of the 96-wells of a microtest plate. The cultures were maintained in the presence or absence of IL-4 for 6 weeks, at which point the number of wells containing viable transformed colonies was counted. The data points and error bars represent the mean ± s.d for replicate experiments performed on B cells from three separate donors. Transformation efficiency, defined as the M.O.I. at which 50% of the cultures were transformed, was 23.8±3.2 for EBV alone and 56.5±10.2 for EBV plus IL-4.</p

Expression of Interferon-regulated genes by EBV and CD40L/IL4 blasts.

*<p>Numbers in parentheses indicate the number of genes within the gene-set analyzed.</p><p>Comparisons were restricted to the 16420 genes present on both the HuExon 1.0 ST and the U133 plus2 Affymetrix array platforms. The size of each comparator gene-set was selected to ensure as far as was practical that similar-sized sets were being analyzed. For example, genes up-regulated by EBV were ranked in order of FC, and the top 370 were selected for comparison with the 370 ISGs. Complete gene-lists are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s002" target="_blank">Data-File S2</a>. (<b>A</b>) Of the 389 ISGs described by Schoggins <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Schoggins1" target="_blank">[31]</a> through meta-analysis of published arrays, 370 were present in the 16420 annotated genes common to both array platforms. (<b>B</b>) Of the 124 unique genes identified experimentally by Schoggins <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Schoggins1" target="_blank">[31]</a> as being up-regulated by IRF-1, 113 were present in the 16420 annotated genes common to both array platforms.</p

Regulation by LMP1 and LMP2A of EBV/HRS/IL-4 signature genes.

<p>The EBV/HRS/IL-4 signature was defined as the 92 genes that were identified as common to the 2084 genes up-regulated in EBV-transformed LCLs established from GC-B cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Leonard1" target="_blank">[37]</a>, the 1747 genes up-regulated in HRS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Brune1" target="_blank">[38]</a>, and the 484 genes down-regulated by IL4 in T cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Elo1" target="_blank">[35]</a>. These 92 genes were then compared to those identified as up-regulated following expression of LMP1 or LMP2A in GC-B cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Vockerodt1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Vockerodt2" target="_blank">[42]</a>. All arrays were performed on the U133 plus2 Affymetrix array platform, where the probe-sets corresponded to 21014 annotated genes. The Gene Ontology function descriptions were obtained primarily from NCBI and AmiGO databases, assisted by manual literature search. Complete gene-lists are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s002" target="_blank">Data-File S2</a>.</p

Expression of IL4-regulated genes by EBV-infected B blasts.

*<p>Numbers in parentheses indicate the number of genes within the gene-set analyzed. Comparisons were restricted to the 16420 genes present on both the HuExon 1.0 ST and the U133 plus2 Affymetrix array platforms. The IL-4 regulated genes were identified experimentally by Elo <i>et al</i> who arrayed IL-2 stimulated T cells treated with or without IL-4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Elo1" target="_blank">[35]</a>. Complete gene-lists are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s002" target="_blank">Data-File S2</a>.</p

Relationships between genes regulated by Interferon, IRF-1, and IL-4.

<p>Venn diagrams showing the overlaps of 370 ISGs and 113 IRF-1 up-regulated genes defined by Schoggins et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Schoggins1" target="_blank">[31]</a> with IL-4-regulated genes (567 up-regulated and 452 down-regulated) identified by Elo <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Elo1" target="_blank">[35]</a>. Only genes that were annotated and common to both U133 plus 2 and Human Exon 1.0 ST Affymetrix array platforms (i.e. 16420 genes) were analysed here. Complete gene-lists are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s002" target="_blank">Data-File S2</a>.</p

Ontology analysis of genes differentially expressed between EBV blasts and CD40L/IL-4 blasts.

<p>Ontology analysis of genes differentially expressed between EBV and CD40L/IL-4 blasts by paired LIMMA (p<0.01, FC>2) was performed using the DAVID v6.7 bioinformatics resource <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Huang1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Huang2" target="_blank">[29]</a> to identify Panther Biological Processes terms for which there was a significant enrichment of genes. The lists of probe-sets entered into DAVID v6.7 and the complete analyses readouts are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s001" target="_blank">Data-File S1</a>. The probe-sets analyzed were those which were: (A) differentially expressed between EBV and CD40L/IL-4 blasts at day 7, (B) the subset of differentially expressed genes that was expressed higher in EBV blasts than in CD40L/IL-4 blasts, and (C) the subset of differentially expressed genes that was expressed lower in EBV blasts than in CD40L/IL-4 blasts.</p

Similarities in the transcriptomes of blasts induced by EBV infection or by CD40L/IL4 treatment of resting peripheral blood B cells.

<p>Significant changes in transcript expression were identified by paired LIMMA (p<0.01; FC>1.5) and complete gene-lists are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s002" target="_blank">Data-File S2</a>. (<b>A</b>) Venn diagram indicating the overlap of the 4197 genes changed by EBV and the 1666 genes changed by CD40L/IL4, irrespective of the direction of the change. (<b>B</b>) Venn diagram showing the 3519 genes that were up-regulated by EBV, and the overlap with the 1394 genes up-regulated by CD40L/IL4 and the 272 down-regulated by CD40L/IL4. (<b>C</b>) Venn diagram showing the 679 genes that were down-regulated by EBV, and the overlap with the 1394 genes up-regulated by CD40L/IL4 and the 272 down-regulated by CD40L/IL4.</p

Expression of IL-4 regulated genes in GC-B cell LCL.

*<p>Numbers in parentheses indicate the number of genes within the gene-set analyzed.</p><p>Of the 21014 annotated genes in the U133 plus2 Affymetrix array platform, 484 were identified experimentally by Elo <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Elo1" target="_blank">[35]</a> as being down-regulated and 640 up-regulated by IL-4 in T cells. Gene-sets up- or down-regulated in EBV-transformed LCL from GC-B cells relative to uninfected GC-B cells were identified by Leonard <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868-Leonard1" target="_blank">[37]</a>. For the determination of OR and statistical significance, equal sized probe-sets were compared by ranking probe-sets according to the magnitude of FC of the GCB-LCL relative to uninfected GC-B cells, and selecting the top 484 or 640 unique genes. Complete gene-lists are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064868#pone.0064868.s002" target="_blank">Data-File S2</a>.</p