Significant augmentation of regulatory T cell numbers occurs during the early neonatal period

Summary

Regulatory T cells (T_regs) control immune responses by suppressing various inflammatory cells. T_regs in newborn babies may play an important role in preventing excessive immune responses during their environmental change. We examined the number and phenotype of T_regs during the neonatal period in 49 newborn babies. T_regs were characterized by flow cytometry using cord blood (CB) and peripheral blood (PB) from the early (7–8 days after birth) and late (2–4 weeks after birth) neonatal periods. CD4⁺forkhead box protein 3 (FoxP3⁺) T cells were classified into resting T_regs (CD45RA⁺FoxP3^low), activated T_regs (CD45RA^– FoxP3^high) and newly activated T cells (CD45RA^– FoxP3^low). Compared with CB and PB during the late neonatal period, the percentage of T_regs and all T_reg subpopulations in the CD4⁺ lymphocyte population were increased significantly during the early neonatal period. Furthermore, the proportion and absolute number of activated T_regs were increased markedly compared with other T_reg subpopulations, such as resting T_regs and newly activated T cells (non‐T_regs), in the early neonatal period. Increased T_regs concomitantly expressed the suppressive molecule cytotoxic T lymphocyte antigen‐4 (CTLA‐4). The up‐regulated expression of chemokine receptor 4 (CCR4) and down‐regulated expression of CCR7 were also observed in expanded T_regs. When cord blood cells were cultured in vitro with CD3 monoclonal antibodies (mAb) for 5 days, CD4⁺CD45RA^–FoxP3^high cells were increased significantly during the culture. Thus, the presence of increased activated T_regs in early neonates may play an important role in immunological regulation by suppressing excessive T cell activation caused by the immediate exposure to ubiquitous antigens after birth.

Introduction

Regulatory T cells (T_regs) maintain immune homeostasis by suppressing the activation, proliferation and effector functions of a wide range of immune cells, including CD4⁺ and CD8⁺ T cells, natural killer (NK) cells, NK T cells, B cells and antigen‐presenting cells (APC). T_regs play an important role in diverse immune responses, with many investigations into pathophysiology identifying associations between T_regs and autoimmune disease, allergic disease and pregnancy 1.

In humans, T_regs were first characterized as CD4⁺CD25⁺ T cells, with forkhead box protein 3 (FoxP3) being identified later as the master controller of gene expression 2, 3; thus, T_regs are currently defined as CD4⁺CD25⁺FoxP3⁺ cells. However, it was reported recently that T_regs can be classified into three subpopulations based on their expression of CD4, CD45RA and FoxP3: resting T_regs (CD4⁺CD45RA⁺FoxP3^low), activated T_regs (CD4⁺CD45RA^–FoxP3^high) and non‐T_regs (CD4⁺CD45RA^–FoxP3^low) 3. Non‐T_regs are described as newly activated T cells in this report because non‐T_regs contain CD4⁺FoxP3^– T cells. Functional analysis of T_reg subpopulations have been investigated in multiple disease states 3, 4, 5, 6. Both activated and resting T_regs have suppressive functions, and once resting T_regs are stimulated they differentiate into activated T_regs and proliferate. Conversely, newly activated T cells produce inflammatory cytokines and have no suppressive function 3.

T_regs first appear during fetal development in‐utero and increase more during the fetal period than after birth; thus, T_regs play a pivotal role in feto–maternal tolerance 7, 8, 9. The proportion of T_regs among CD4⁺ T cells decreases with gestational age 10, but it is less in the cord blood (CB) of full‐term infants than in adult peripheral blood (PB). A few days after birth, the T_reg cell number increases to levels comparable to adult PB and remains stable thereafter, in the range of 5–10%. The components of the T_reg cell population also change after birth. Effector type T_regs increase depending on age and predominate by puberty; however, most of the T_regs are naive at birth 11, 12, 13. Dynamic changes in chemokine receptor expression on T_regs accompany age‐related changes in activation 11.

Changes in the T_reg cell population during adulthood have been reported; however, there are few reports showing the details of the T_reg cell population during the neonatal period, when newborn babies are exposed to ubiquitous antigens after transfer from the intrauterine to the extrauterine environment. Fetuses develop in an almost sterile environment; however, newborn babies are exposed to ubiquitous antigen after birth. Excessive immune responses to environmental antigens can cause the onset of allergic diseases or inflammatory bowel disease. Indeed, affected individuals develop autoimmune disease and inflammatory bowel disease a few weeks after birth in the immunodysregulation polyendocrinopathy enteropathy X‐linked (IPEX) syndrome, which is due to a mutation in Foxp3 14, 15. T_regs in neonates probably play an important role in preventing excessive immune responses during the environmental changes faced by newborn babies. In this study, we examined the fluctuation of number and components of the T_reg cell population in the neonatal period using flow cytometry and the in‐vitro induction of T_regs from CB cells.

Materials and methods

Subjects

Forty‐nine newborn babies were admitted to the Neonatal Intensive Care Unit (NICU) of Hiroshima University Hospital from November 2013 to December 2014. Any cases administered steroids after birth or suffering congenital malformation, sepsis, gastrointestinal complications or severe intraventricular hemorrhage were not included in the study.

Blood sample collection

CB was taken in heparinized or ethylenediamine tetraacetic acid (EDTA)‐coated tubes by umbilical venipuncture. PB of neonates was taken in EDTA‐coated microtainer tubes by heel stick during the early period (7–8 days after birth) and the late period (2–4 weeks after birth). The classification of late period was based on our initial experiments showing no significant difference in T_regs in peripheral blood at 2, 3 and 4 weeks of age (data not shown). Both CB and PB samples, during the early and late periods, were collected from each newborn baby enrolled into this study. Adult PB was taken in heparinized tubes by venipuncture. Samples in EDTA‐coated tubes were used for flow cytometric analysis and samples in heparinized tubes were used for culture experiments. Samples were analysed after obtaining informed consent from the babies’ guardians. This study was approved by the Ethics/International Review Board of Hiroshima University.

White blood cells (WBC) and regulatory T cells counts

Complete blood cell counts and differential white blood counts were measured on a XT‐4000i automated haematology analyser (Sysmex Corporation, Kobe, Japan). Absolute counts for T_regs were calculated by multiplying the percentages of T_regs in the lymphocyte gate by the number of circulating lymphocytes per μl blood.

Cell staining and flow cytometry

In total, 100 μl of whole blood was used per sample. Samples were analysed within 12 hours of collection. To remove red blood cells (RBCs), samples were treated with lysing solution (Easy‐Lyse™; Dako, Carpinteria, CA, USA) and the remaining cells were washed twice with phosphate‐buffered saline (PBS) and incubated at 4°C for 10 min with anti‐human monoclonal antibodies. Cells were surface stained with anti‐CD4 monoclonal antibodies (mAb) and anti‐CD25 mAb or both anti‐CD4 mAb and anti‐CD45RA mAb and stained intracellularly with FoxP3 or CTLA‐4. Intracellular staining was performed according to the manufacturer's instructions (Human FoxP3 Buffer Set; BD, Franklin Lakes, NJ, USA). The samples were analysed on a fluorescence activated cell sorter (FACS)Calibur or FACSVerse (BD Biosciences, San Jose, CA, USA) and data were analysed using BD Cell Quest Pro software and BD FACSuite™ software. The gates were set using isotype controls and single antibody controls were used to calculate the compensation.

Anti‐human mAbs used for this study

For cell surface staining; anti‐CD4‐fluorescein isothiocyanate (FITC) [mouse (Ms)] immunoglobulin (Ig)G_1k, clone rat pancreatic amylase (RPA)‐T4; BD), anti‐CD‐25‐phycoerythrin (PE) (Ms IgG_1k, M‐A251; BD), anti‐CD45RA‐PE (Ms IgG_2b, T6D‐11; Miltenyi Biotec, San Diego, CA, USA), anti‐CD194 [CC chemokine receptor (CCR4)]‐Alexa Fluor 647 (Ms IgG₁, 1G1; BD) and anti‐CD197 (CCR7)‐Alexa Fluor 647 (Ms IgG2a, 3D12; BD). For intracellular staining; anti‐FOXP3‐Alexa Fluor 647 (Ms IgG₁, 259D/C7; BD), and anti‐CTLA‐4‐APC (Ms IgG2a, BNI3; BD).

In‐vitro differentiation of CD4⁺CD25⁺FoxP3⁺ cells and CD4⁺CD45RA^–FoxP3^high cells

Mononuclear cells (MNCs) from CB (n = 5) and healthy adult PB (n = 5) were separated by density gradient centrifugation (Lymphoprep™; Axis‐Shield, Dundee, UK). Subsequently, MNCs were cultured in RPMI‐1640 medium (Sigma‐Aldrich, St Louis, MO, USA) supplemented with 10% heat‐inactivated fetal bovine serum (FBS) and 100 μg/ml penicillin and streptomycin. For activation and proliferation experiments, cells were plated in 24‐well flat‐bottomed plates at a density of 5 × 10⁵ cells/ml and stimulated with soluble anti‐CD3 mAb (100 ng/ml) [muromonab‐CD3 (OKT3); Miltenyi Biotec] + recombinant interleukin (rIL)‐2 (20 U/ml) (R&D Systems, Minneapolis, MN, USA) for 2 days. The cells were then washed and incubated with culture medium containing rIL‐2 (20 U/ml) for 3 more days. Cells were stained with anti‐CD4‐FITC, anti‐FoxP3‐Alexa Fluor^®647 and anti‐CD45RA‐PE or anti‐CD25‐PE on the second and fifth days after starting cultivation. The percentage of CD4⁺CD25⁺FoxP3⁺ cells and CD4⁺CD45RA^– FoxP3^high cells of CD4⁺ cells were analysed by flow cytometry. Dead cells were identified using BD Horizon™ Fixable Viability Stain780 (BD).

Statistical analysis

Statistical analysis was performed using spss version 23 software (IBM SPSS Corporation, Armonk, NY, USA). To compare the three different age groups, CB and PB in the early and late neonatal period, repeated‐measure analysis of variance (anova) followed by Bonferroni correction or Friedman's test followed by Wilcoxon t‐test with Bonferroni correction were used. Spearman's correlation was used to analyse the correlation between gestational age or birth weight and T_regs or T_reg subpopulations. For comparisons between groups, the Mann–Whitney U‐test was used. A value of P < 0·05 was considered statistically significant.

Results

Clinical characteristics of neonate participants

Forty‐nine neonates, including four sets of dichorionic diamniotic twins and two sets of monochorionic diamniotic twins, were enrolled into this study. Their clinical characteristics are shown in Table 1. All neonates were admitted to the NICU of Hiroshima University Hospital, accommodated in an infant incubator and received infusion of glucose and electrolyte solutions. None of the neonates received glucocorticoid steroid or immunoglobulin preparations, which may have affected their immune system after birth. Eighteen neonates received preventive antibiotics, none developed infectious disease and the administration of antibiotics was discontinued within 3 days. Enteral nutrition started soon after birth and establishment of nutrition was not delayed in any cases.

Table 1.

Characteristics of participating neonates

n = 49
Gestational age (weeks, mean ± s.d.)	30·9–37·0 (34·0 ± 1·6)
Birth weight (g, mean ± s.d.)	1306–2947 (1983 ± 361)
APGAR score 5 min (mean ± s.d.)	4–10 (9·08 ± 1·2)
Gender (%)
Male	24 (49)
Female	25 (51)
Mode of delivery (%)
Caesarean section	29 (59)
Vaginal	20 (41)
Antibiotics administration (%)	18 (37)
Initiation time of feeding (day‐old, mean ± s.d.)	0–1 (0·6 ± 0·5)
Feeding mode (%)
Breastfeeding mainly	20 (41)
Formula mainly	29 (59)
Antenatal administration of corticosteroids (%)	17 (35)
Antenatal administration of antibiotics (%)	19 (39)
Antenatal administration of magnesium sulphate (%)	15 (31)
Maternal allergic disease (%)	13 (27)
Maternal smoking in pregnancy (%)	6 (12)
Histological chorioamnionitis	5 (10)

APGAR = Appearance, Pulse, Grimace, Activity, Respiration; s.d. = standard deviation.

WBC and T_reg counts

Absolute counts of T_regs in CB and PB during the early and late neonatal periods were measured using differential WBC counts in 36 newborn babies. Absolute counts for WBC and lymphocytes were not increased in the early neonatal period, whereas absolute counts of T_regs were increased significantly in the early neonatal period compared with those in CB and the late neonatal period (Fig. 1).

Figure 1.

Absolute counts for white blood cells, lymphocytes and regulatory T cells (T_regs). Absolute counts for T_regs were calculated by multiplying the percentage of T_regs in the lymphocyte gate by the absolute lymphocyte count and dividing by 100 (n = 36). **P < 0·01 (Friedman's test with post‐hoc tests). n.s., not significant.

Analysis of circulating total T_regs and T_reg subpopulations in neonates

The percentage of total T_regs and T_reg cell subpopulations in CD4⁺ lymphocytes in CB and PB of neonates is shown in Fig. 2a. Lymphocytes were identified initially by forward‐ and side‐scatter. Total T_regs were defined as CD4⁺CD25⁺FoxP3⁺ T cells. In the analysis of T_reg cell subpopulations, CD4⁺ cells were classified by the expression of CD45RA and FoxP3 into three subpopulations, resting T_regs (CD4⁺CD45RA⁺FoxP3^low), activated T_regs (CD4⁺CD45RA^–FoxP3^high) and newly activated T cells (CD4⁺CD45RA^–FoxP3^low) based on the differential expression of CD45RA and FoxP3. The percentage of total T_regs in CD4⁺ lymphocytes was increased significantly during the early neonatal period compared with T_regs in CB and PB during the late neonatal period (Fig. 2b). Next, we investigated changes within the T_reg cell subpopulations (Fig. 2c). The proportion of all subpopulations in CD4⁺ lymphocytes was increased significantly in PB in the early neonatal period compared with CB and PB in the late neonatal period. Furthermore, activated T_regs were increased predominantly among the T_reg cell subpopulations (Fig. 2d). Additionally, resting T_regs predominated during the late neonatal period, while the number of activated T_regs was comparable with resting T_regs during the early neonatal period. Newly activated T cells were increased in number during the early neonatal period and were reduced during the late neonatal period; however, the fluctuation in the number of newly activated T cells is small compared with other subpopulations. Thus, total T_regs were increased significantly in the early neonatal period through the predominant expansion of activated T_regs. Additionally, we investigated the proportion of effector (CD45RA^–FoxP3^–) CD4⁺ T cells and naive (CD45RA⁺FoxP3^–) CD4⁺ T cells. The proportion of effector and naive CD4⁺ T cells decreased transiently in the early neonatal period, contrary to that of activated T_regs (Supporting information, Fig. S1).

Figure 2.

Fluctuation of regulatory T cells (T_regs) and T_reg subpopulations during the neonatal period. T_regs (a) and T_reg subpopulations (c) in cord blood (CB) or peripheral blood (PB) obtained during the early and late neonatal periods were measured by flow cytometry (n = 49). Data are also presented as box‐plots which display the minimum value, 25th, 50th and 75th percentiles and maximum value (b,d). *P < 0·05; **P < 0·01 [repeated‐measure analysis of variance (anova) post‐hoc test]. ##P < 0·01 (Friedman's test with post‐hoc tests). [Colour figure can be viewed at wileyonlinelibrary.com]

Expression of CTLA‐4 in CD4⁺CD25⁺ T cells and CD4⁺CD25⁺FoxP3⁺ T_regs

CTLA‐4 is a member of the immunoglobulin superfamily, which is expressed on CD4⁺ lymphocytes, transmitting an inhibitory signal to T cells. Similarly, CTLA‐4 is also expressed in T_regs and is important for T_reg cell function 16. To confirm that the T_regs increased transiently in the early neonatal period potentially have suppressive function, the expression of CTLA‐4 on CD4⁺CD25⁺ lymphocytes was investigated (Fig. 3a). The numbers of CD4⁺CD25⁺CTLA‐4⁺ T cells, as well as T_regs and activated T_regs, increased significantly during the early neonatal period (Fig. 3b). The mean fluorescence intensity (MFI) of CTLA‐4 in CD4⁺CD25⁺FoxP3⁺ T_regs was measured in an additional four newborn babies (Fig. 3c). They were two male and two female babies, and their gestational ages were 33 weeks 6 days, 34 weeks 0 days, 36 weeks 1 day and 36 weeks 2 days, respectively (mean: 34 weeks 6 days). CTLA‐4 MFI of CD4⁺CD25⁺FoxP3⁺ T_regs was higher in the early neonatal period compared with that in CB and the late neonatal period (Fig. 3d). The expression of CTLA‐4 in T_regs increased similarly in all four cases.

Figure 3.

Cytotoxic T lymphocyte antigen‐4 (CTLA‐4) expression in CD4⁺CD25⁺ T cells and CD4⁺CD25⁺forkhead box protein 3 (FoxP3⁺) regulatory T cells (T_regs) during the neonatal period. CTLA‐4 expression was measured on CD4⁺CD25⁺ T cells from cord blood (CB) or peripheral blood (PB) obtained during the early or late neonatal periods (n = 49, a). Data are presented in the right‐hand box plot (b). The histograms show the CTLA‐4 expression on CD4⁺CD25⁺FoxP3⁺ T_regs in four additional newborn babies (c). Data are also presented as box‐plots (d). **P < 0·01 [repeated‐measure analysis of variance (anova) post‐hoc test]. [Colour figure can be viewed at wileyonlinelibrary.com]

Expression of CCR4 and CCR7 on CD4⁺CD25⁺ T cells

CC chemokine receptors, CCR4 and CCR7, are markers of mature or naive phenotype in T_regs 11. The expression of CCR4 increases while the expression of CCR7 decreases during the activation of T cells. To characterize the function of T_regs, CCR4 and CCR7 expression on CD4⁺CD25⁺ T cells and CD4⁺CD25^high T cells was investigated by flow cytometry (Fig. 4a and Supporting information, Fig. S3). CD4⁺CD25^high T cells were defined as the 2% of CD4⁺ T cells with the highest expression of CD25, which was reported previously as being a T_reg cell‐enriched population 17. The expression of CCR4 on CD4⁺CD25⁺ T cells and CD4⁺CD25^high T cells was increased significantly (Fig. 4b), while the expression of CCR7 was decreased during the early neonatal period compared with the late neonatal period (Fig. 4c). A significantly greater change was observed in the expression of CCR4 and CCR7 in CD4⁺CD25^high T cells than CD4⁺CD25⁺ T cells during the neonatal period (Mann–Whitney U‐test, P < 0·01).

Figure 4.

Expression of CC chemokine receptor (CCR4 or CCR7) on CD4⁺CD25^high T cells during the neonatal period. CCR4 and CCR7 expression on CD4⁺CD25^– T cells, CD4⁺CD25⁺ T cells and CD4⁺CD25^high T cells was investigated (n = 49). Data are displayed as representative histograms from the same baby (a) and box‐plots (b). **P < 0·01 (Friedman's post‐hoc tests). [Colour figure can be viewed at wileyonlinelibrary.com]

In‐vitro differentiation of CD4⁺CD25⁺FoxP3⁺ cells and CD4⁺CD45RA^–FoxP3^high cells

A marked change in the total T_reg cell numbers, including T_reg cell subpopulations, and CTLA‐4, CCR4 and CCR7 expression in CD4⁺CD25⁺ cells was observed during the neonatal period. Based on these results, we hypothesized that CD4⁺ T cells from CB might differentiate efficiently into FoxP3⁺ T_regs.

CB and adult PB MNCs were activated in the presence of anti‐CD3 mAb and rIL‐2 for 2 days, followed by culture with rIL‐2 for a further 3 days. The induction of FoxP3 expression in CD4⁺ cells was observed in MNCs derived from both CB and adult PB by stimulation with anti‐CD3 mAb (Supporting information, Fig. S4). The induction of FoxP3 was more pronounced in CB compared with adult PB (Fig. 5a,b). Analysis of the T_reg cell subpopulations revealed that CD4⁺ FoxP3⁺ cells from CB MNCs differentiated from naive to effector cells following stimulation with anti‐CD3 mAb and CD4⁺CD45RA^–FoxP3^high cells from CB MNCs increased significantly compared with adult PBMNCs after 2 and 5 days of culture (Fig. 5b).

Figure 5.

In‐vitro differentiation of cord blood (CB) CD4⁺ T cells into CD25⁺FoxP3⁺ T cells. CB or peripheral blood (PB) mononuclear cells were cultured in the presence of anti‐CD3 monoclonal antibodies (mAb) and recombinant interleukin (rIL‐2) for 2 days followed by rIL‐2 for a further 3 days. The expression of CD25 and forkhead box protein 3 (FoxP3) was analysed by flow cytometry after a total of 5 days in culture. Data are displayed as representative flow cytometry plots (a) and as the mean ± standard deviation (s.d.) (b). *P < 0·05 (Mann–Whitney U‐test). [Colour figure can be viewed at wileyonlinelibrary.com]

Factors associated with changes in T_regs

Various factors potentially associated with the change in T_reg cell numbers were examined in this study. We compared gestational age, birth weight, APGAR (Appearance, Pulse, Grimace, Activity, Respiration) score 5 min, gender, mode of delivery, antibiotic administration, initiation time of feeding, feeding mode, antenatal administration of corticosteroids, antenatal administration of antibiotics, antenatal administration of magnesium sulphate, maternal allergic disease, maternal smoking in pregnancy and histological chorioamnionitis to identify potential factors affecting T_reg cell numbers. T_regs in CB were correlated negatively with birth weight and gestational age, although T_regs in the early and late neonatal periods were not correlated with these factors. In T_reg cell subpopulations, activated T_regs in CB were correlated negatively with gestational age. Resting T_regs in CB were correlated negatively with birth weight (Fig. 6a and Supporting information, Fig. S5). Considering other factors, administration of antenatal corticosteroid increased T_regs significantly in CB and PB during the late neonatal period. Administration of antenatal antibiotics decreased T_regs significantly and activated T_regs in PB during the early neonatal period (Fig. 6b). Histological chorioamnionitis decreased T_regs significantly and activated T_regs PB during the early neonatal period (Supporting information, Fig. S6). In contrast, we did not see an obvious difference in the total number of T_regs and their subpopulations in CB and PB during the late neonatal period.

Figure 6.

Factors associated with the change in regulatory T cells (T_regs) and T_reg subpopulations. Association analysis of the percentages of T_regs in CD4⁺ lymphocytes and gestational age or birth weight were performed (a). The effect of corticosteroid or antibiotic administration on the percentage of total T_regs and activated T_regs was analysed. Data are shown as box‐plots (b). *P < 0·05; **P < 0·01 (Mann–Whitney U‐test). n.s., not significant.

Discussion

T_regs are a subpopulation of the CD4⁺ T cell subset, which suppress the immune responses of inflammatory cells, particularly during autoimmune and allergic diseases. T_regs emerge at approximately 13 weeks of gestation 18, 19, and the increased number of T_regs present in the second trimester plays a pivotal role in feto–maternal tolerance 7. Subsequently, the number of T_regs decreases with gestational age 10 and increases after birth with the attainment of adult proportions within a few days 11, 12. In this study, the T_reg cell population of total CD4⁺ cells during the early neonatal period was increased transiently when compared with cells from both CB and PB during the late neonatal period. This is the first report to show a transient, but striking, increase in T_regs in the early neonatal period.

To study the suppressive function of the T_regs in early neonates, T_regs were separated further based on phenotypical differences. It has been reported that CD4⁺ cells are classified phenotypically and/or functionally into three subpopulations, resting T_regs, activated T_regs and newly activated T cells. Both resting and activated T_regs are capable of suppressing an immune response 3. The resting T_regs, once stimulated, proliferate, becoming activated T_regs possessing strong suppressive ability. Conversely, newly activated T cells typically produce inflammatory cytokines without immunosuppressive activity 3. Our results demonstrated that T_reg cell numbers augmented in early neonates comprised predominantly activated T_regs, as shown in Fig. 1. Therefore, the increased T_regs in early neonates may be responsible for suppression of the immune response during the neonatal period. Mayer et al. 20 demonstrated that CB‐derived T_regs possess the ability to become highly suppressive upon antigen exposure and that early exposure to innocuous antigen is vital to develop T cell tolerance. The proportion of effector and naive CD4⁺ T cells was decreased transiently during the early neonatal period, contrary to that of activated T_regs. Therefore, we hypothesize that increased activated T_regs during the early neonatal period cannot be explained by global T cell activation. Thus, the increase of T_regs with suppressive function in early neonates may play a pivotal role in regulating immune response for adapting to environmental antigens as early as a week after birth.

CTLA‐4 is a member of the immunoglobulin superfamily that is expressed on CD4⁺ cells to transmit an inhibitory signal to T cells. In addition, CTLA‐4 is expressed highly on T_regs and comprises one of the mechanisms of T_reg cell‐mediated immune suppression 16. Recently, germline heterozygous mutations in the CTLA4 gene were identified in patients with primary immunodeficiency presenting as the dysregulation of FoxP3⁺ T_regs. Thus, CTLA‐4 is considered to play an important role in T_reg cell‐mediated host immune regulation in humans 21, 22. The increase of CD4⁺CD25⁺CTLA‐4⁺ T cells, as well as T_regs and activated T_regs, observed in this study suggests the requirement of immunosuppression through these T cells during the early neonatal period.

T_regs undergo programmed switches in migratory behaviour during their development and activation 23. T_regs generated in the thymus highly express homing receptors for emigration to secondary lymphoid tissues. Simulating antigen presentation was shown to induce a decrease in the expression of second lymphoid tissue homing receptors and an increase in the expression of memory/effector chemokine receptors 23. To confirm the functional change in T_regs in the neonatal period, the expression of chemokine receptors CCR4 and CCR7 on CD4⁺CD25^high T cells was examined. The expression of homing receptors CCR4 and CCR7 on T_regs is associated with the differentiation of T_regs from naive to effector cells 11, 23. Our results showed an increase in the expression of CCR4 and a concomitant decrease in the expression of CCR7 on CD4⁺CD25^high T cells during the early neonatal period. This evidence confirms further that augmented CD4⁺CD25^high T cell numbers in early neonates possess an immune phenotype consistent with activated T_regs expressing CTLA‐4.

Recently, Lee et al. proposed a layered immune system hypothesis for the change of T cells from tolerogenic to immunogenic during the development of a fetus to an adult. Gene expression profiles of T_regs and T cells in a fetus differ from expression in adults, suggesting that fetal T cells play a role in immunotolerance 7, 24. CD4⁺CD25^–CD45RO^– naive T cells isolated from peripheral blood in neonates were shown to be more functionally active T_regs than those present during adulthood, when naive T cells were cultured with APCs in vitro 25, 26. Our study showed that the expression of FoxP3 in CD4⁺ lymphocytes induced by the stimulation of CB MNCs with anti‐CD3 mAb was much higher than FoxP3 expression induced in adult PB MNCs. Collectively, T cells in CB consisting primarily of naive T cells are probably predisposed to respond to stimulation by environmental antigens, resulting in the immunotolerance seen in neonates through the functionally activated T_regs possessing strong immunosuppressive tendencies.

Various factors have the potential to modulate the ontogenic development of T_regs. Such factors in neonates include gestational age, intestinal microbiota, Toll‐like receptor (TLR)‐2 polymorphisms and IL‐10 polymorphisms, while the maternal factors include smoking in pregnancy, allergic disease and chorioamnionitis 10, 27, 28, 29, 30, 31, 32, 33, 34, 35. A negative correlation between gestational age and T_reg cell development shown in this study using CB was consistent with previous reports. This study showed the involvement of antenatal maternal administration of antibiotics and histological chorioamnionitis in the decreased T_reg cell number in early neonates. Thus, further studies are necessary to elucidate the factors, which may affect the development of T_regs during the fetal and neonatal period.

This study was designed with the care of premature infants as the end objective. The limited results from mature neonates who were hospitalized in NICU have demonstrated a similar fluctuation of T_regs to that presented in this study (data not shown). These findings suggest that the characteristic change of proportion of T_regs may be observed during the first 14 days in neonates. Further studies are required to confirm the conclusion of a transient increase of activated T_reg in mature and full‐term infants.

This study clearly showed changes in the proportion and absolute number of T_regs in the early neonatal period. However, we have not investigated the suppressive function of these increased T_regs during the early neonatal period because of the limited number of cells available. Previous studies revealed that T_regs in CB from preterm infants have lower activity than those in CB from term infants and adult PB 36, 37, 38. The difference in the function of T_reg due to prematurity may affect the frequency of T_regs and its subpopulations during the neonatal period. Additionally, a previous study suggested that the function of T_regs in CB was impaired in the case with severe chorioamnionitis 35. In the current study, cases with histological chorioamnionitis showed a significantly lower frequency of T_regs and activated T_regs during the early neonatal period. The decrease of T_regs and activated T_regs may reflect the impaired T_reg function of CB in chorioamnionitis cases. The evaluation of qualitative and quantitative change of T_regs during the early neonatal period may be necessary to clarify the role of T_regs in neonates because phenotypical differences and differences in function are not always the same.

In conclusion, the numbers of total T_regs were increased significantly during the early neonatal period, comprising predominantly activated T_regs. CD4⁺CD25⁺CTLA‐4⁺ T cells were also increased significantly during the early neonatal period, consistent with elevated T_regs and activated T_reg cell numbers, suggesting suppressive immune regulation by T cells in early neonates. The increased expression of CCR4 and decreased expression of CCR7 on CD4⁺CD25^high T cells implied the activation of T_regs in the early neonatal period. CD4⁺CD25⁺ FoxP3⁺ T cells were induced transiently when MNCs from CB and from adult PB were cultured in vitro with anti‐CD3 mAb and rIL‐2. The percentage of CD4⁺CD25⁺FoxP3⁺ T cells induced from CB was higher than from adult PB. The naive nature of CB suggests that lymphocytes in CB may be more attuned to becoming T_regs when encountering the appropriate stimulation. These results suggest that the increase in T_regs and their function in early neonates may play an important role in early immune suppression required for adapting to environmental change encountered after birth.

Disclosure

The authors have declared no disclosures.

Supporting information

Additional Supporting information may be found in the online version of this article at the publisher's web‐site:

Fig. S1. Fluctuation of naive CD4⁺ T cells and effector CD4⁺ T cells during the neonatal period. Naive T cells are defined as CD4⁺CD45RA⁺forkhead box protein 3 (FoxP3^–) cells and effector T cells are defined as CD4⁺CD45RA^–FoxP3^– cells in this figure. Data are presented as box plots. #P < 0·05; ##P < 0·01 (Friedman's test with post‐hoc tests).

Fig. S2. Association analysis between the percentages of CD4⁺CD25⁺cytotoxic T lymphocyte antigen‐4 (CTLA‐4⁺) cells and gestational age were performed. Rs = correlation coefficients (Spearman's rank correlation coefficient).

Fig. S3. Expression of CC chemokine receptor (CCR)4 or CCR7 on CD4⁺ T cells during the neonatal period. Data are displayed as representative dot‐plots.

Fig. S4. In‐vitro differentiation of the mononuclear cells (MNCs) stimulated by anti‐CD3 monoclonal antibodies (mAb) and unstimulated CD4⁺ cells in cord blood (CB) or adult peripheral blood (PB) into CD25⁺forkhead box protein 3 (FoxP3⁺) T cells. Data are displayed as the mean ± standard deviation (s.d.).

Fig. S5. Association analysis between the percentages of resting or activated regulatory T cells (T_regs) and gestational age or birth weight were performed. Rs = correlation coefficients (Spearman's rank correlation coefficient).

Fig. S6. Effect of histological chorioamnionitis on the percentage of total regulatory T cells (T_regs) and activated T_regs was analysed. Data are shown as box‐plots. *P < 0·05; **P < 0·01 (Mann–Whitney U‐test).