Developmentally determined reduction in CD31 during gestation is associated with CD8+ T cell effector differentiation in preterm infants

Abstract

Homeostatic T cell proliferation is more robust during human fetal development. In order to understand the relative effect of normal fetal homeostasis and perinatal exposures on CD8+ T cell behavior in PT infants, we characterized umbilical cord blood CD8+ T cells from infants born between 23-42weeks gestation. Subjects were recruited as part of the NHLBI-sponsored Prematurity and Respiratory Outcomes Program. Cord blood from PT infants had fewer naïve CD8+ T cells and lower regulatory CD31 expression on both naïve and effector, independent of prenatal exposures. CD8+ T cell in vitro effector function was greater at younger gestational ages, an effect that was exaggerated in infants with prior inflammatory exposures. These results suggest that CD8+ T cells earlier in gestation have loss of regulatory co-receptor CD31 and greater effector differentiation, which may place PT neonates at unique risk for CD8+ T cell-mediated inflammation and impaired T cell memory formation.

Keywords: Bronchopulmonary dysplasia; CD8+ T cell; Fetus; Immune dysregulation; Inflammation; Neonatal immunity; Prematurity.

Copyright © 2015 Elsevier Inc. All rights reserved.

Figures

Fig 1

Fig 1a–1f CD8+ T cells were sequentially identified by flow cytometry as intact lymphocyte/singlets/live/CD14-/CD3+/CD4-/CD8+ (a). CD8+ events were gated on CD45RO (b), CD27 (c), CD57 (d) and CD31 (e). Representative preterm (black solid line), full term (dotted line) and healthy adult donor (gray solid) subjects are shown in histogram overlays.

Fig 2

Fig 2a–f CD8+ and CD45RO+ CD8+ T cells across gestational ages. Graph shows regression analysis (with 95% CI) for CD8+ count/mL blood (a), Log10 CD45RO+ CD8+ events/mL UCB (b) and CD45RO expression as a proportion of CD8+ T cells (c), based on gestational age at birth. Points represent individuals without (circles) and with (asterisks) antenatal steroid exposure. Bar graphs compare median percent, +/- IQR and Log10 event frequency/mL UCB of CD45RO+ of CD8+ T cells from PT subjects exposed or not exposed to either antenatal steroids (d, e) or chorioamnionitis (f).

Fig 3

Fig. 3a–d Regression analysis of Log10 CD27+ (a) and CD27- (b) CD8+ T cell events (with 95% CI) as a function of gestational age in weeks. Further analysis was performed on CD127+ CD27+ CD8+ events based on gestational age, controlling for perinatal factors.

Fig 4

Fig. 4a–h Cytokine+CD8+ T cells were sequentially identified by flow cytometry as intact lymphocyte/singlets/live/CD14-/CD235-/CD3+/CD4-/CD8+/CD69+/cytokine+, as shown using representative plots for umbilical cord mononuclear cells (top), and healthy adult donor PBMC (bottom) (a). Percent CD69+/IFN-γ+ (b), MIP-1β+ (d) TNF-α+ (f), and IL-2+ (h) CD8+ T cell were used for regression analysis (with 95% CI) as a function of gestational age in weeks, with p-values prior to controlling for clinical factors. Interacting clinical variables are displayed for each cytokine as an open circle on scatter plots. Bar graphs show comparisons between PT subjects with or without exposure to membrane rupture > 18 hours (ROM >18), mode of delivery (cesarean section or vaginal) and chorioamnionitis (chorio +/-) for IFN-γ (c), MIP-1β (e) and TNF-α (g) (Mann-Whitney test).

Fig 5

Fig. 5a–f CD57+CD8+ T cells were gated on CD57 +/- expression. Plots show regression analysis (with 95% CI) for percent (b) and log-transformed number/mL UCB (c) of CD57+CD8+ T cells as a function of gestational age at birth in weeks. Bar graphs show comparison of percent and Log10 CD57+CD8+ event count/mL UCB for PT subjects by mode of delivery (d and e) and by exposed/not exposed to chorioamnionitis (f and g). (Mann-Whitney test).

Fig 6

Fig. 6a–f Regression analysis (with 95% CI) of CD31+CD8+ T cell percent (a) and Log10 CD31+ CD8+ event count/mL UCB (b) by gestational age at birth.

Fig 7

Fig. 7a–e CD27+ and CD27- CD8+ T cell populations were gated on CD31 expression. Representative preterm (black solid line), full term (dotted line) and healthy adult donor (gray solid) subjects are shown in histogram overlays (a). CD31+ percent and number/mL UCB on CD27+ (b, c) and CD27- (d, e) CD8+ T cells were analyzed as a function of gestational age at birth.

Fig. 8

CD31+ T cells were gated on CD8+ T cell events as shown in Figure 1d. CD31+ events were further subsetted on CD127 expression. Representative preterm (black solid line), full term (dotted black line), healthy adult donor (gray solid) subjects and fluorescence minus one negative control (FMO, dotted gray line) are shown in histogram overlays. Frequencies of CD127+ events within the CD31+CD8+ T cell subset are shown as a function of gestational age at birth.