Global Foxp3⁺ T cell numbers are reduced in HD patients.

<p>(A) Representative FACS analysis of CD4⁺CD25⁺Foxp3⁺T cells and their distinct subsets on PBMC from control (top) and HD patients (bottom). (B) CD4⁺CD25⁺Foxp3⁺T cell numbers are reduced in HD patients. (C to E) Among these gated cells, cytokine-secreting CD45RA⁻Foxp3^lo nonsuppressive (C), resting suppressive CD45RA⁺Foxp3^lo (D) and activated suppressive CD45RA⁻Foxp3^hi (E) cells, all these subsets are reduced in HD patients compared to controls. Bars represent the median. *, P<0.01; ****, P<0.0001.</p

Both ILT and conventional T cells are equally deficient in HD and KD patients.

<p>(A) HD patients had no significant differences in their number of iNKT, MAIT, CD4⁺ and CD8⁺ T cells when compared to kidney transplanted (KT) patients. Bars represent the median. (B) CD4⁺CD25⁺Foxp3⁺T cell numbers are similarly reduced in HD as in KD patients. Among these gated cells, cytokine-secreting CD45RA⁻Foxp3^lo nonsuppressive, resting suppressive CD45RA⁺Foxp3^lo and activated suppressive CD45RA⁻Foxp3^hi cell levels, are also similar in HD and KD patients. (C and D) The percentage of IFN<b>γ</b>⁺, IL-4⁺ or IL-17⁺ cells among gated CD4⁺ (C) or CD8⁺ (D) T lymphocytes was assessed after 6 h stimulation with PMA and ionomycin. Box-and-whisker plots are used to represent the distributions. The bottom and the top of a box represent the 5th and 95th percentiles, and the bar in the box shows the median. (A and B) Bars represent the median. *, P<0.01.</p

Patient demographic characteristics.

<p>Two hemodialysed patients had glomerulonephritis and vascular nephropathy at the same time.</p

CD4⁺ and CD8⁺ T cell numbers are reduced in HD patients.

<p>(A) CD4⁺ and CD8⁺T cell numbers are significant decreased in the peripheral blood of HD patients compared to healthy donors. Bars represent the median. (B) The percentage of IFN<b>γ</b>⁺ cells among gated CD8⁺ or CD4⁺ T lymphocytes or (C) the percentage of IL-4⁺ and IL-17⁺ cells among gated CD4⁺ T lymphocytes was assessed after 6 h stimulation with PMA and ionomycin. Box-and-whisker plots are used to represent the distributions. The bottom and the top of a box represent the 5th and 95th percentiles, and the bar in the box shows the median. *, P<0.01; **, P<0.001; ***, P<0.0005 versus controls. Representative FACS analysis of IL-4, IL-17 and IFN<b>γ</b> production by gated CD4⁺ (E) or CD8⁺ (F) T cells from health donor controls (top) or HD patients (bottom) are represented.</p

iNKT cell deficit in HD patients.

<p>(A) iNKT cells were double stained by anti-CD3 and the PBS57-loaded CD1d-tetramer. Representative FACS profile showing the percentage of iNKT and MAIT cells in control versus HD patients. (B and C) HD patients had significant low numbers (B) and percentage (C) of peripheral blood iNKT and MAIT cells when compared to healthy donors. Bars represent the median. (D) The frequency of CD4⁺ and CD8⁻ subsets respectively among gated iNKT and MAIT cells in control versus HD patients is represented. (E) The percentage of IL-4⁺ or IFN<b>γ</b>⁺ cells among gated iNKT lymphocytes was assessed after 5 h stimulation with PMA and ionomycin. Box-and-whisker plots are used to represent the distributions. The bottom and the top of a box represent the 5th and 95th percentiles, and the bar in the box shows the median. **, P<0.001; ***, P<0.0005; ****, P<0.0001 versus controls.</p

<i>L</i>. <i>monocytogenes</i> infection modulates a number of IFN signaling pathway and IFN response genes in the blood and spleen.

<p><b>(A)</b> The IFN signaling pathway (QIAGEN Ingenuity® Pathway Analysis) was overlaid with the day 3 post infection blood genes shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g001" target="_blank">Fig 1A</a>. Genes (IFNαβ and JAK1) with opposite expression patterns between blood and spleen are highlighted with black circles. Red: upregulated, Blue: downregulated. <b>(B)</b> IFN response genes (type I, type II, and type I and II) associated with blood and spleen transcripts reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g001" target="_blank">Fig 1A and 1B</a> and the Interferome database (<a href="http://www.interferome.org/" target="_blank">www.interferome.org</a>) were quantitated.</p

Transcriptional response following <i>L</i>. <i>monocytogenes</i> infection of WT mice in blood and spleen.

<p><b>(A, B)</b> Heatmaps of the significantly regulated blood and spleen transcripts at day 3 following intravenous injection of C57BL/6 WT mice with 5 × 10³ of <i>L</i>. <i>monocytogenes</i> (<i>p</i> < 0.01 after unpaired <i>t</i>-test with Benjamini–Hochberg multiple testing correction on transcripts passing quality control filtering, <i>n</i> = 10 mice/group). The top five QIAGEN Ingenuity® Pathway Analysis (IPA®) canonical pathways by significance (Fisher’s exact test) for upregulated (red bars) or downregulated (blue bars) genes are listed below the heatmaps. <b>(C)</b> qRT-PCR data on selected transcripts normalized relative to <i>Hprt1</i> expression levels (mean with SD, <i>n</i> = 5 mice/group). Samples are from the same experiment as the microarray data. <b>(D)</b> CD3⁺Thy1⁺ cells in blood and spleen as a percentage of total live cells. Pooled results are from triplicates of 3 independent experiments (mean with SEM, <i>n</i> = 3 mice/group/experiment).</p

Tissue-specific transcriptional responses of WT and <i>Ifnar1</i>^-/- mice in blood, spleen and liver following <i>L</i>. <i>monocytogenes</i> infection at different time points and bacterial loads.

<p><b>(A)</b> Colony forming unit (CFU) values were assessed from the spleen and liver of C57BL/6 WT and <i>Ifnar1</i>^<b>-/-</b> mice at day 1, day 2 and day 3 following intravenous injection with 5 × 10³ of <i>L</i>. <i>monocytogenes</i> (mean with SEM, <i>n</i> = 4 or 5 mice/group). <b>(B)</b> Heatmap representations of differentially expressed transcripts at different times (days 1, 2 and 3) in blood, spleen, liver from WT and <i>Ifnar1</i>^<b>-/-</b> infected mice relative to uninfected controls (<i>p</i> < 0.05 after 2-way ANOVA with Benjamini–Hochberg multiple testing correction on transcripts passing quality control filtering, <i>n</i> = 3 or 4 mice/group). <b>(C)</b> Percentage of transcripts that were significant for infection alone, strain alone or involving a combination of strain and infection across the different tissues and the infection time course. <b>(D)</b> Venn diagrams showing temporal differences and similarities in gene expression in blood, spleen and liver after infection.</p

<i>Ifnar1</i>^-/- uninfected mice show a lowered expression of several IFN regulated genes as compared with WT uninfected mice.

<p><b>(A–C)</b> The strain-associated subsets of transcripts identified from the 2-way ANOVA as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g003" target="_blank">Fig 3B</a> for an independent experiment were filtered to identify baseline differences (fold-ratio of 1.5 between day 3 uninfected <i>Ifnar1</i>^<b>-/-</b> to day 3 uninfected WT mice) for blood, spleen and liver. Heatmaps and a list of top three IPA® canonical pathways are shown. <b>(D)</b> Venn diagram of above detailed transcripts identifies 50 transcripts that are commonly shared between blood, spleen and liver. These transcripts map to 35 genes in IPA® and include a number of Interferome-based type I IFN responsive genes that are marked in red. <b>(E)</b> Heatmap of mean-normalised expression values for selected (<i>Irf1</i>, <i>Irf3</i>, <i>Irf7</i>, <i>Irf9</i>, <i>Stat1</i> and <i>Stat2</i>) IFN transcriptional regulator transcripts. <b>(F)</b> qRT-PCR validation of <i>Irf7</i> gene normalized relative to <i>Hprt1</i> gene in blood (left panel), spleen (middle panel) and liver (right panel) at indicated times post infection (mean with SD). Data from one experiment with four mice per group.</p

Canonical pathways associated with blood transcripts that are differentially expressed in <i>L</i>. <i>monocytogenes</i> infected WT versus <i>Ifnar1</i>^-/- mice against control uninfected WT mice.

<p>Top IPA® canonical pathways that are associated with differentially expressed blood day 1, day 2 and day 3 transcripts shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150251#pone.0150251.g003" target="_blank">Fig 3B</a> and that pass a further 1.5-fold change filter ratio between <b>(A)</b> WT infected to WT uninfected; <b>(B)</b> KO infected to WT uninfected; and <b>(C)</b> (WT infected to WT uninfected) as compared to (KO infected to WT uninfected). Percent pathway modulation relative to each dataset and pathway size is indicated in red for up-regulated and blue for down-regulated genes. Pathway rank for pathways passing <i>p<0</i>.<i>05</i> after Fisher’s Exact test at each time-point is marked. <b>(D and E)</b> Detailed heat map of differentially expressed genes found in the <b>(D)</b> Interferon Signaling Pathway; and <b>(E)</b> Antigen Presentation Pathway.</p