Hydrogen peroxide impairs IL-7 induction of P-STAT5 in T cell subsets.

<p>PBMC from healthy control donors were pre-incubated with H₂O₂ at the indicated concentrations for 30 minutes prior to stimulation with IL-7 (1 ng/ml). The sequential gating strategy included gating on live lymphocytes by forward and side scatter properties, removal of doublets (FSC-A vs FSC-H), gating on CD3+CD4+ cells or CD3+CD8+ cells and then gating on cells based on CD45RA and CD27 co-expression patterns to define subsets. Medians and inter-quartile ranges of P-STAT5+ cells within indicated subsets are represented by box-and-whisker plots for CD4+ T cells (n = 10) and CD8+ T cells (n = 6). Differences between H₂O₂-treated cells and cells incubated in medium alone were statistically significant at each concentration and for each cell type (p values <0.05). (A) P-STAT5 expression is shown in representative histograms of CD4+ (left column) and CD8+ (right column) T cells from either PBMC cell cultures (top) or purified T cells (>98% purity; bottom). Cells were incubated with H₂O₂ at the indicated molar concentrations for 30 min. or incubated in medium alone. Cells were then stimulated with IL-7 and assessed for intracellular P-STAT5 expression (1 ng/ml). Dashed histograms represent P-STAT5 expression in unstimulated cells. Data are representative of experiments from 3 different donors (B). PBMC from 5 different donors were used to assess the effects of H₂O₂ on T cell apoptosis and P-STAT5 induction. The percentages of total apoptotic CD4+ and CD8+ T cells were determined by annexin V staining during culture of PBMC with 98 µM H₂O_2. These percentages are compared to the percent inhibition of P-STAT5 signaling in PBMC that was observed after stimulation with IL-7 in the presence of 98 µM H₂O₂ (C). Note that the proportions of cells that show evidence of apoptosis are far lower than the proportions of cells with impaired P-STAT5 induction.</p

P-STAT5 induction is related to CD127 expression in CD8+ T cells.

<p>The relationship between IL-7-induced P-STAT+ cells and the percentage of CD127+ T cells in whole blood are shown for CD4 cells (left column) and CD8 cells (right column) within the indicated subsets. Open symbols represent aviremic subjects and closed symbols represent viremic subjects. Correlation coefficients and P values were determined by Spearman's correlations.</p

Franke, Marian Alois (Marjan Alojzy)

(1877 - 1944), Patholog

Diminished IL-7 induction of P-STAT5 in T cell subsets from HIV+ donors.

<p>PBMC were incubated for 15 min with rIL-7 (1 ng/ml) and cells were assessed by flow cyomtery for expression of P-STAT5. Responses of CD4 lymphocytes (A and B) and CD8 lymphocytes (C and D) are shown as box-and-whiskers plots indicating the median, the inter-quartile values (box), the range (whiskers) and the outliers (symbols). Data are represented for all HIV+ donors (A and C) or for viremic and aviremic donors (B and D). The percentage of P-STAT5 expression represents the percentages of P-STAT5+ cells that increased P-STAT5 expression above levels observed in unstimulated cells.</p

Spearman correlations for relationships between MDA serum concentrations and measures of CD127 expression in T cell subsets.

<p>Spearman correlations for relationships between MDA serum concentrations and measures of CD127 expression in T cell subsets.</p

Serum concentrations of MDA adducts are inversely related to IL-7-induction of P-STAT5.

<p>Percent induction of P-STAT5 in the indicated T cell subsets was plotted against serum MDA adducts for each subject. Open symbols represent aviremic subjects and closed symbols represent viremic subjects. Correlation coefficients and P values were determined by Spearman's correlations.</p

Increased sCD14 and MDA adducts in serum of HIV+ donors.

<p>Serum samples from HIV+ and HIV- donors were stored at −80°C until analyzed in batch with commercial ELISA kits. Data are shown comparing cytokine levels in all HIV+ donors to controls (A) or comparing viremic, aviremic and controls (B). Statistical significance was determined by Mann Whitney tests (A) or by Kruskal-Wallis Test for multi-group analysis and Mann Whitney (B).</p

Stimulation of peripheral blood cells by Toll-like Receptor ligands increases expression of CD38 on CD4+ and CD8+ T lymphocytes.

<p>PBMC were cultured overnight in medium alone, or stimulated with individual TLR ligands (Poly-I:C, LPS, Flagellin, or CpG DNA) or with plate-bound anti-CD3 antibody. Expression of CD38 and HLA-DR was monitored by flow cytometry among: (A) CD4+ T cells and (B) CD8+ T cells. Percentages of cells expressing each marker are shown. This experiment is representative of 5.</p

TLR ligands preferentially activate CD4+ central memory and effector memory T cells and CD8+ effector memory T cells.

<p>Intracellular Ki-67 expression (3A, 3C) and cell surface CD69 (3B, 3D) were analyzed after 7 days' incubation of PBMC in medium alone, or in medium supplemented with plate bound anti-CD3 antibodies, or the indicated TLR ligand. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001915#pone-0001915-g003" target="_blank">Figures 3A and B</a> are representative results among phenotypically defined naïve (CD45RA⁺CD45RO⁻CCR7⁺), central memory (CD45RA⁻CD45RO⁺CCR7⁺), and effector memory (CD45RA⁻CD45RO⁺CCR7⁻) CD4⁺ and CD8⁺ T cells. Values represent percentages of cells staining for Ki-67 or CD69. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001915#pone-0001915-g003" target="_blank">Figures 3C and 3D</a> reflect the mean data from 15 separate experiments. Values nominally significantly different from medium alone values (Wilcoxon Sign Rank Test) are shown with an asterisk.</p

TLR ligands induce high level CD69 expression on CD8+ T cells and Ki- 67 expression in CD4+ T cells.

<p>Intracellular expression of Ki-67 (A) and surface expression of CD69 (B) were monitored after 7 days of cell culture in medium or with the stimuli as indicated. Bars represent means and the lines standard errors of the mean (SEM) of 15 separate experiments using PBMC of healthy controls. Black boxes = CD4+ T cells and gray boxes = CD8+ T cells. * = nominally significantly different (p<0.05 by Wilcoxon Sign Rank test when compared to results obtained in medium alone.</p