Maternal and fetal characteristics.

<p>Maternal and fetal characteristics.</p

Preterm infants demonstrate enhanced memory T cell induction.

<p>Memory T cell frequency was compared between the term group (n = 23) and the preterm group (n = 15). Preterm neonates had an increased median percentage of memory CD4+ T cells (9.40%, IQR: 6.40, 28.10), CD45R0+/CD45RA-, compared to the median percentage of memory CD4+ T cells in term infants (6.10%, IQR: 2.37, 9.50) in term infants (<i>P</i> = 0.0032).</p

Cell surface activation marker expression on neonatal CD4⁺ T cells.

<p>Cord blood samples obtained simultaneously from an uncomplicated term delivery (control) and a delivery complicated by preterm labor were stained with fluorochrome labeled antibodies for cell surface activation markers (CD25, HLA-DR, and CD69, y axis of each dot plot) and subjected to three-color flow cytometric analysis. Cells were gated based on size and CD3 expression, quadrants were set based on isotype controls, and samples were compensated electronically for overlap in fluorescent emission. The percentage of cells in each quadrant is indicated. These data are representative of 34 term infants and 12 preterm infants.</p

Toll-like receptors 3 and 5 are expressed by a subset of T cells, but expression is not always sufficient for activation by TLR ligands.

<p>(A) Whole PBMCs were isolated from healthy donors and expression of TLRs 2, 3, and 5 were evaluated by flow cytometry on gated CD3+ T cells. (B) Whole PBMCs, purified T cells (>95% CD3⁺), or purified T cells separated from whole PBMCs by a transwell, were cultured in medium alone, or in medium supplemented with plate bound anti-CD3, poly I:C or flagellin A. Expression of CD38 on memory (CD45RO⁺CD45RA⁻) CD4⁺ T cells was assessed by flow cytometry following overnight culture. Dotplots shown are representative of 8 separate experiments.</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

Activation as a result of TLR ligand exposure preferentially induces T cell death especially among CD4+ T cells.

<p>PBMCs were labeled with CFSE and incubated for 6 days in the presence of anti-CD3 antibody, medium alone or TLR ligands alone (PGN, poly-I:C, LPS (10 or 20 ng/ml), Flagellin A, Flagellin B or imiquimod). After 6 days of incubation, the CD4⁺ and CD8⁺ T cells were examined for dilution of CFSE dye and for binding of Annexin V. Numbers in right upper corners represent percentage of Annexin V-binding cells. Numbers in the left lower corner represent percentage of cells that diluted dye without Annexin-V binding. This experiment is representative of three separate experiments.</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

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

Histological Chorioamnionitis and Tregs Homeostasis.

<p>(<b>A</b>) Column bar graph representing means and standard deviations of CD31⁺ Treg percentage in CB with different staging of histological chorioamnionitis and no chorioamnionitis. Statistical significance between all groups (p = 0.018) was determined by one way ANOVA with Bonferroni post hoc test. (<b>B</b>) Column bar graph representing means and standard deviations of CD31⁻ Treg percentage in CB with different staging of histological chorioamnionitis and no chorioamnionitis. Statistical significance between groups (p = 0.013) was determined by one way ANOVA with Bonferroni post hoc test.</p

Maternal factors affect the percentages of Treg phenotypes in CB.

<p>(<b>A</b>) Column bar graph comparing median and confidence intervals of the percentage of CD31⁻ Tregs in infants (both term and preterm) whose mothers had received prenatal antibiotics. “Yes” indicates mother received prenatal antibiotics and “No” indicates no antibiotics were given. Statistical significance was determined by Mann-Whitney U test (*p<0.0001). (<b>B</b>) Column bar graph comparing median and confidence intervals of the percentage of CD31⁻ Tregs in infants (both term and preterm) whose mothers had received prenatal steroids. “Yes” indicates mother received prenatal steroids and “No” indicates no steroids were given. Statistical significance was determined by Mann-Whitney U test (*p<0.0001). (<b>C</b>) Column bar graph comparing median and confidence intervals of the percentage of CD31⁻ Tregs in infants (both term and preterm) whose mothers had received prenatal magnesium sulfate. “Yes” indicates mother received prenatal magnesium sulfate and “No” indicates no magnesium sulfate was given. Statistical significance was determined by Mann-Whitney U test (* p = 0.005).</p