Extremely Preterm Infants Have Significant Alterations in Their Conventional T Cell Compartment during the First Weeks of Life

Abstract

Extremely preterm neonates are particularly susceptible to infections, likely because of severely impaired immune function. However, little is known on the composition of the T cell compartment in early life in this vulnerable population. We conducted a comprehensive phenotypic flow cytometry-based longitudinal analysis of the peripheral conventional T cell compartment of human extremely low gestational age neonates (ELGAN) with extremely low birth weight (ELBW; <1000 g) participating in a randomized placebo-controlled study of probiotic supplementation. PBMCs from ELGAN/ELBW neonates were collected at day 14, day 28, and postmenstrual week 36. Comparisons were made with full-term 14-d-old neonates. Total CD4⁺ and CD8⁺ T cell frequencies were markedly lower in the preterm neonates. The reduction was more pronounced among the CD8⁺ population, resulting in an increased CD4/CD8 ratio. The preterm infants were also more Th2 skewed than the full-term infants. Although the frequency of regulatory T cells seemed normal in the ELGAN/ELBW preterm neonates, their expression of the homing receptors α4β7, CCR4, and CCR9 was altered. Notably, ELGAN/ELBW infants developing necrotizing enterocolitis before day 14 had higher expression of CCR9 in CD4⁺T cells at day 14. Chorioamnionitis clearly associated with reduced T regulatory cell frequencies and functional characteristics within the preterm group. Finally, probiotic supplementation with Lactobacillus reuteri did not impose any phenotypic changes of the conventional T cell compartment. In conclusion, notable immaturities of the T cell compartment in ELGAN/ELBW neonates may at least partially explain their increased susceptibility to severe immune-mediated morbidities.

FIGURE 1.

Flow chart showing the number of subjects recruited, time points for blood sampling, number of PBMC samples analyzed by flow cytometry, and the Ab panels used for the experiments. A total of 134 ELBW preterm infants and 29 FT control subjects were recruited for the study. PBMCs were isolated from 76 ELBW infants from D14, 50 ELBW infants from D28, and 59 ELBW infants from PMW 36 + 0 wk. PBMCs from 28 infants were available from all three time points, and PBMCs from 48 neonates were available from at least two time points. FACS panels were designed to characterize conventional T cells, their corresponding subsets, and the homing markers. The number of PBMC samples analyzed by FACS varied in different panels depending on the adequacy of the cells.

FIGURE 2.

The frequencies of total and viable lymphocyte populations are decreased in the PBMCs of ELBW preterm infants compared with FT controls. Blood samples from 29 FT infants (D14 after birth) and 134 ELBW preterm infants (D14, D28, and PMW 36 + 0 after birth) were collected, and phenotypic characterization of the T lymphocyte compartment was performed by multicolor flow cytometry. The figure shows the compiled frequencies of (A) total, (B) viable, (C) CD4⁺, and (D) CD8⁺ lymphocytes among the PBMCs. The CD4/CD8 ratio is shown in (E), and the frequency of CD25^hi cells within the CD4⁺ population is shown in (F). Bars show medians with interquartile range. Kruskal-Wallis test with Dunn multiple comparison tests was used for group comparisons. PCA comparing the T cell phenotype at D14 of FT neonates (black) with the ELBW neonates born week 23–27 (in color) is shown in (G). The correlation between the percentage of lymphocytes at D14 and birth weight is shown in (H) for the ELBW neonates.

FIGURE 3.

Proportions of NA, effector (EC), and effector memory (EM) subsets of CD4⁺ and CD8⁺ T cells are similar in ELBW preterm and FT infants. The frequencies of CM, NA, EC, and EM cells within (A) CD4⁺ T cells and (B) CD8⁺ T cells. Bars show medians with interquartile ranges.

FIGURE 4.

ELBW preterm infants have a reduced T-bet/GATA3 ratio in CD4⁺ T cells at 14 and 28 d of age. The expression levels of (A) GATA3, (B) T-bet, and (C) RORγt in the CD4⁺ T cells of control FT neonates at D14 and the ELBW preterm infants at D14, D28, and at PMW 36 + 0. The presented data of geometric mean fluorescence intensity (geo MFI) for transcription factors analyses was done following gating on CD4 cells that are positive for these factors. (D) Ratio between the percentages of T-bet– and GATA3-expressing CD4⁺ T cells. Bars show medians with interquartile range. Kruskal-Wallis test with Dunn multiple comparison tests was used to make group comparisons.

FIGURE 5.

Tregs express lower levels of the transcription factor Helios, and their frequency is inversely correlated with gestational age in ELBW preterm infants. Relative proportions of (A) total Tregs (CD25⁺FOXP3⁺CD127⁻) and (B) the frequencies of CD45RA⁺ and CD45RA⁻ Tregs are shown. (C and D) Expression level of (C) CTLA4 among all Tregs and frequencies of (D) CD45RA⁺ and CD45RA⁻ cells within CTLA4⁺ Tregs. (E) The level of Helios expression among Tregs and (F) the frequencies of CD45RA⁺ and CD45RA⁻ cells within Helios⁺ Tregs. Bars show medians with interquartile ranges. Kruskal-Wallis test with Dunn multiple comparison tests was used to make group comparisons. (G) Correlation between Treg percentages from ELBW neonates at D14 and the gestational age at birth.

FIGURE 6.

CD4⁺ T cells and Tregs from 14-d-old ELGAN/ELBW preterm infants have a markedly altered homing capacity. The frequencies of (A and D) CCR4⁺, (B and E) α4β7⁺, and (C and F) CCR9⁺ within total (A–C) CD4⁺ T cells and (D–F) Tregs, respectively, are shown. Bars show medians with interquartile ranges. Kruskal-Wallis test with Dunn multiple comparison tests was used for group comparisons. (G) PCA of the influence of gestational age on the frequencies of CCR4-, CCR9- and α4β7-expressing CD4⁺ T cells and Tregs at D14 in the ELBW (color) neonates. FT infants (black) are shown as a reference.

FIGURE 7.

Chorioamnionitis and NEC affect the Tregs and CD4 T cells, respectively, whereas no association of cause and effect of sepsis is observed on T cell compartment. PCA plot of the proportions of (A) total, CD4⁺, and CD8⁺ lymphocytes and their ratio and (B and C) frequencies of CCR4⁺, CCR9⁺, and α4β7⁺ CD4⁺ T cells and Tregs at D14 from ELBW neonates in sepsis and NEC cases, respectively. The infants were grouped according to if the sepsis/NEC onset was before or after the sample was collected at 14 d of life. (D) Proportions of CCR9⁺ CD4 T cells in ELBW neonates at D14 of life in NEC cases. Frequencies of (E) total CD4⁺ T cells, (F) Tregs, (G) CTLA4⁺, and (H) Helios⁺ Tregs at D14 for ELGAN/ELBW infants from chorioamnionitis cases. Scatter plots show medians with interquartile ranges. Mann-Whitney U test was used for group comparisons.

FIGURE 8.

L. reuteri supplementation has no effect on CD4 and Tregs and on their homing properties. PCA plot of the proportions of the frequencies of CCR4⁺, CCR9⁺, and α4β7⁺ CD4⁺ T cells and Tregs from ELGAN/ELBW neonates supplemented with L. reuteri (green) or placebo (blue) at (A) D14, (B) D28, and (C) PMW 36 + 0.