Alterations in regulatory T cell subpopulations seen in preterm infants

Abstract

Regulatory T cells are a population of CD4+ T cells that play a critical role in peripheral tolerance and control of immune responses to pathogens. The purpose of this study was to measure the percentages of two different regulatory T cells subpopulations, identified by the presence or absence of CD31 (Recent thymic emigrants and peripherally induced naïve regulatory T cells), in term and preterm infant cord blood. We report the association of prenatal factors, intrauterine exposure to lipopolysaccharide and inflammation and the percentages of these regulatory T cell subpopulations in term and preterm infants. Cord blood samples were collected from both term and preterm infants and mononuclear cells isolated over a Ficoll-Hypaque cushion. Cells were then stained with fluorochrome-labeled antibodies to characterize regulatory T cell populations and analyzed with multi-color flow cytometry. Cord blood plasma C-reactive protein, and lipopolysaccharide were also measured. Placental pathology was also examined. We report a gestational age-dependent difference in the percentage of total regulatory T cells, in which preterm infants of lower gestational ages have an increased percentage of regulatory T cells. We report the presence of two populations of regulatory T cells (CD31+ and CD31-) in cord blood of term and preterm infants and their association with different maternal and fetal characteristics. Factors associated with differences in the percentage of CD31- Tregs included the use of prenatal antibiotics, steroids and magnesium sulfate. In addition, the percentage of CD31- Tregs was significantly higher in cord blood of preterm pregnancies associated with inflammation and prenatal lipopolysaccharide exposure. The peripheral Treg pool of preterm infants could be altered by prenatal exposure to inflammation and chorioamnionitis; however, the clinical implications of this finding are not yet understood.

Figure 1. Cord blood flow cytometry analysis of regulatory T cells.

Cord blood mononuclear cells were surface stained with fluorochrome labeled monoclonal antibodies followed by intranuclear staining for FOXP3, fixation and flow cytometric analysis. (A) Box-plot with lymphocytes gated based on size (FSC) and granularity (SSC). (B) Neonatal CD4⁺ T cells are selected based on CD3 and CD4 co-expression (box-plot). (C) Neonatal regulatory T cells (CD3⁺CD4⁺) are selected based on CD25 and FOXP3 co-expression (box-plot). (D) Regulatory T cells (CD3⁺CD4⁺CD25⁺FOXP3⁺) subpopulations selected by the expression of CD45RA and CD31 (CD45RA⁺CD31⁺ recent thymic emigrant, CD45RA⁺ CD31⁻ peripherally induced naïve regulatory T cell) (box-plot).

Figure 2. Increased percentage of Tregs in CB of preterm infants and correlates with gestational age.

(A) Vertical scatter plot comparing mean percentage of Tregs (CD3⁺CD4⁺CD25⁺FOXP3⁺) in CB of term (n = 31) and preterm (n = 40) infants. Statistical significance was determined by independent sample T-test (*p = 0.015) (B) Scatter plot graph showing a negative correlation between gestational age (X-axis, in weeks) and CB Tregs percentage (Y-axis). Pearson's correlation test used (r = −0.414, p = 0.0001)

Figure 3. Tregs subpopulations percentages are different between term and preterm infants.

(A) Term CB (first box plot) and preterm CB (second box plot). Tregs were examined for expression of the surface markers CD45RA and CD31. (B) Vertical scatter plot comparing mean percentage of CD3⁺CD4⁺CD25⁺FOXP3⁺CD45RA⁺CD31⁺ in CB of term and preterm infants. Statistical significance was determined by independent sample T-test (*p = 0.0001). (C) Vertical scatter plot comparing median percentage of CD3⁺CD4⁺CD25⁺FOXP3⁺CD45RA⁺CD31⁻ in CB of term and preterm infants. Statistical significance was determined by independent sample T-test (*p = 0.0001). (D) Scatter plot graph showing a positive correlation between gestational age (X-axis, in weeks) and CB CD31⁺ Tregs percentage (Y-axis). Pearson's correlation test used (r =  0.54, p = 0.0001). (E) Scatter plot graph showing a positive correlation between gestational age (X-axis, in weeks) and CB CD31⁻ Treg percentage (Y-axis). Pearson's correlation test used (r = −0.62, p = 0.0001).

Figure 4. Maternal factors affect the percentages of Treg phenotypes in CB.

(A) 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) 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). (C) 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).

Figure 5. CB LPS correlates with the percentage of CD31⁻ Tregs of preterm infants.

Scatter plot graph showing correlation between CB LPS levels (X-axis) and CD31⁻ Tregs (Y-axis, percentage). Spearman rank correlation test used (r = 0.373, p = 0.046).

Figure 6. Histological Chorioamnionitis and Tregs Homeostasis.

(A) 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) 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.