Regulatory T Cells in Human Cord Blood of Preterm and Term Infants

Regulatory T (Treg) cells are a subset of CD4⁺ T cells that play key roles in the maintenance of immunologic self-tolerance and negative control of immune responses. Treg cells are defined based on expression of CD4, CD25, and transcription factor forkhead box P3 (FoxP3). FoxP3 controls the development and function of Treg cells and can be used as a reliable marker of Treg cells, however, its intracellular localization prohibits its use for the isolation of viable Treg cells. Most FoxP3⁺ Treg cells express low levels of CD127 in adult peripheral blood, however, the expression of CD127 in cord blood (CB) Treg cells of preterm and term infants at each gestational age remains unknown. Being that CD127 is a cell surface marker, it enables the isolation of viable Treg cells by flow cytometric sorting for further analysis. We analyzed CD4+CD25+FoxP3+ and CD4+CD25+CD127− T cells in preterm and term CB at various gestational ages.

CB samples were obtained at Iwate Medical University hospital from preterm and term neonates who had no hereditary disorders, hematologic abnormalities, or immunodeficiency diseases. Of 103 total samples, 36 were obtained from preterm (24–36 gestational weeks) neonates and 67 were obtained from term (37–41 gestational weeks) neonates. Out of 36 preterm infants, 9 were diagnosed with chorioamnionitis histologically. None of the mothers involved in the study had any specific underlying diseases. CB mononuclear cells were isolated and analyzed by FACSCalibur flow cytometer and CellQuestPro software (BD Biosciences).

No differences were observed in the proportions of CD25+FoxP3+ (median 4.5%) and CD25+CD127− (median 5.0%) cells in the CD4⁺ T-cell population. A strong correlation was found between the proportions of CD25+FoxP3+ and CD25+CD127− cells in the CD4⁺ T-cell population (r=0.96). The proportion of CD25⁺FoxP3⁺ cells in the CD4⁺ T-cell population in the CB of neonates at 24-26, 27-29, and 30-32 gestational weeks was significantly higher than in the CB of neonates at 37-38 and 39-41 gestational weeks. The proportion of CD25⁺CD127⁻ cells in the CD4⁺ T-cell population in the CB of neonates at 24-26, 27-29, and 30-32 gestational weeks was also higher than in the CB of neonates at 37-38 and 39-41 gestational weeks. The absolute number of both CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁺CD127⁻ T cells did not differ between the CB of preterm and term neonates. Absolute lymphocyte counts in preterm CB were significantly lower than term infants (P<0.05). The proportions of CD25⁺FoxP3⁺ and CD25⁺CD127⁻ cells in the CD4⁺ T-cell population did not differ between preterm infants with histological chorioamnionitis (n=9) and no histological chorioamnionitis (n=27).

We showed that CD127 is preferable to FoxP3 as a marker for identifying Treg cells in CB of various gestational ages and would be useful for separating Treg cells by flow cytometry for therapeutic applications. The proportions of Treg cells in the CB of preterm infants were significantly higher than in the CB of term infants. The high Treg proportion during early gestation suggests that fetal naive T cells have a high propensity to differentiate into Treg cells in response to antigenic stimulation, such as by maternal alloantigens. The functional implications of the high proportion of Treg cells in preterm CB are unknown but may be important for the suppression of undesirable alloimmune reactions to maternal derivatives in utero and the maintenance of tolerance during pregnancy.