Developmental regulation of Th17-cell capacity in human neonates

Abstract

Human neonates are at significantly greater risk of serious infection than immunocompetent adults. In particular, very low birth weight infants in the neonatal intensive care nursery are at high risk of developing life-threatening bacterial and fungal infections. Recent studies have identified Th17 cells as critical mediators of immunity to bacterial and fungal infections at epithelial barriers. Little is known, however, about the ontogeny of Th17-cell responses in humans. The frequency of serious bacterial infections in preterm infants and the importance of Th17 cells in providing protection against such infections in animal studies prompted us to study Th17-cell development in human neonates. Naïve CD4(+) T cells from extremely preterm infants, term infants, and adults were assayed for their capacity to develop into Th17 effector cells. Surprisingly, Th17-cell capacity was inversely related to developmental age. Neonates expressed higher levels of IL-23R, RORγt, and STAT3 prior to activation and showed a significant Th17-cell bias after activation. In contrast, adult cells expressed more TBX21 with a corresponding Th1-cell bias. CD161 expression on Th17-cell precursors was also developmentally regulated. Our results suggest there is significant developmental regulation of CD4(+) effector lineages with a strong bias toward Th17-cell development early in life.

Figure 1

Preterm and term infant naÔve CD4 T cells secrete significantly more Th17 and Th2 cytokines. Purified naÔve CD4 T cells from preterm infants (○), term infants (□), or adults (∆), or memory CD4 T cells from adults (∇) were stimulated in vitro with CD3/CD28 beads under Th0 conditions with only IL-2 added (open symbols) or under Th17-polarizing conditions with IL-1, IL-2, IL-6, IL-23 and low-dose TGF-β added (closed symbols). On day 3, supernatants were collected and assayed by multiplex cytokine bead array analysis. Each symbol represents a separate patient. Bars indicate mean values and significant differences between groups (Mann-Whitney test) are indicated.

Figure 2

Th17 cells develop from neonatal but not from naÔve adult T cells. Purified (A) VLBW preterm, (B) term, (C) adult naÔve, and (D) adult memory CD4⁺ T cells were stimulated with CD3/CD28 beads under Th17 polarizing conditions. On day 6 of culture, the cells were re-stimulated with PMA, ionomycin, and brefeldin A for 5 hours, then fixed, permeabilized, and stained for IL-17 and IFN-γ. Shown are representative plots of 10 or more similar experiments.

Figure 3

Th17 gene expression in resting naÔve neonatal CD4⁺ cells relative to adult cells. Real time PCR for the indicated genes was performed on samples from purified naÔve preterm, term, or adult T cells prior to activation. Fold differences in gene expression were calculated using the ΔΔCt method. Bars indicate averages of 4 independent pairings of neonatal and adult samples.

Figure 4

Inhibition of IFN-γ does not increase IL-17 production from adult naÔve cells. Purified adult CD4⁺RA⁺RO⁻ cells were cultured under Th17 conditions with or without 10 µg/ml of anti-IFN-γ added. On day 3, IL-17 in the supernatant was measured by multiplex cytokine bead array.

Figure 5

Preterm infants generate significant Th17 responses but have fewer CD161⁺ CD4⁺ Th17 precursor cells than term infants. CD4 cells were purified from preterm and term cord blood samples as described then preserved in liquid nitrogen. On the day of analysis, cells were thawed, washed, and stained with antibodies for CD3, CD4, CD161, CD56, and CD25 and with vital dye then analyzed by flow cytometry (A). Shown are representative plots of term and preterm samples with the percentages of CD161⁺CD4⁺CD3⁺ cells indicated after gating off of dead cells and CD56⁺ or CD25⁺ cells. The data for all the patient samples are summarized in (B), with bars representing the mean. To determine if Th17 cells could develop from CD161⁻ precursors, samples were depleted of CD161⁺ cells using magnetic beads. Unfractionated cells and the CD161⁺ and CD161⁻ fractions were stimulated separately for 6 days under Th17 conditions then re-stimulated and analyzed for IFN-γ and IL-17 production by flow cytometry. Recovery and growth of preterm CD161⁺ cells was consistently poor and is omitted from the figure. Shown are representative data from one of four experiments.