Phenotypic Differences in Leucocyte Populations among Healthy Preterm and Full-Term Newborns

Authors

  • C. Quinello,

    Corresponding author
    1. Department of Pediatrics, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
    2. Laboratory of Medical Investigation (LIM-36), Instituto da Criança, Hospital das Clínicas, São Paulo, Brazil
    • Correspondence to: C. Quinello, LIM-36, Department of Pediatrics, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, Av. Dr. Enéas Carvalho de Aguiar, 647 - Instituto da Criança – 5° andar, CEP: 05403-900, São Paulo,  SP,  Brazil.  E-mail: caquinello@hotmail.com

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  • A. L. Silveira-Lessa,

    1. Laboratory of Medical Investigation (LIM-36), Instituto da Criança, Hospital das Clínicas, São Paulo, Brazil
    2. Department of Parasitology, Instituto de Ciências Biomédicas da Universidade de São Paulo, São Paulo, Brazil
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  • M. E. J. R. Ceccon,

    1. Department of Pediatrics, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
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  • M. A. Cianciarullo,

    1. Department of Pediatrics, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
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  • M. Carneiro-Sampaio,

    1. Department of Pediatrics, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
    2. Laboratory of Medical Investigation (LIM-36), Instituto da Criança, Hospital das Clínicas, São Paulo, Brazil
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  • P. Palmeira

    1. Department of Pediatrics, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
    2. Laboratory of Medical Investigation (LIM-36), Instituto da Criança, Hospital das Clínicas, São Paulo, Brazil
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Abstract

The immune system of neonates has been considered functionally immature, and due to their high susceptibility to infections, the aim of this study was to analyse the phenotypic differences in leucocyte populations in healthy preterm and full-term newborns. We evaluated the absolute numbers and frequencies of dendritic cells (DCs) and DC subsets, monocytes and T and B lymphocytes and subsets in the cord blood of healthy moderate and very preterm (Group 1), late preterm (Group 2) and full-term (Group 3) newborns and in healthy adults, as controls, by flow cytometry. The analyses revealed statistically higher absolute cell numbers in neonates compared with adults due to the characteristic leucocytosis of neonates. We observed a lower frequency of CD80+ myeloid and plasmacytoid DCs in Group 1 and reduced expression of TLR-4 on myeloid DCs in all neonates compared with adults. TLR-2+ monocytes were reduced in Group 1 compared with Groups 2 and 3, and TLR-4+ monocytes were reduced in Groups 1 and 2 compared with Group 3. The frequencies and numbers of naïve CD4+ T and CD19+ B cells were higher in the three groups of neonates compared with adults, while CD4+ effector and effector memory T cells and CD19+ memory B cells were elevated in adults compared with neonates, as expected. Our study provides reference values for leucocytes in cord blood from term and preterm newborns, which may facilitate the identification of immunological deficiencies in protection against extracellular pathogens.

Introduction

At birth, neonates have an increased susceptibility to infectious agents due to the functional immaturity of their immune systems. Some immune functions in neonates are particularly immature, while other aspects of neonates' immune systems are functional at birth, even in extremely preterm infants. Innate immune impairment in the neonate is marked by quantitative and qualitative neutrophil defects, including a small storage pool of neutrophils at birth and reduced complement levels. Monocytes/macrophages are functionally adequate in neonates but are limited in their chemotactic responses. The lack of adaptive immunity in neonates is reflected in the decreased number of memory T cells expressing the CD45RO isoform and the increased number of naïve T cells expressing the CD45RA isoform compared with adults as well as low production of immunoglobulin (Ig) G, IgA and IgE by newborn B cells [1-7].

The immune system of preterm neonates is thought to be less developed at birth than term ones, but very little is known about the actual size of lymphocyte subpopulations in preterm neonates or the maturation of these subpopulations during the first months after a premature birth [8]. Studies of leucocytes from preterm newborns have been limited, and there are little data available concerning the development and function of the innate immune system during gestational ageing [9]. Preterm infants are at a heightened risk of acquisition of recurrent bacterial infections during their first weeks of life due to frequent exposure to micro-organisms, frequent invasive procedures such as catheterization and intravascular or assisted ventilation, in addition to the immaturity of the immune system [10].

Immunophenotyping of blood cell populations to identify these cells and their expression of pattern recognition and activation molecules is an important tool in the diagnosis and follow-up of children with immunodeficiencies and other immune disorders. Correct interpretation of the results obtained from patients requires knowledge of the normal development of the immune system during the first years of life. Most studies on the human immune system have been conducted with adults, and many questions about lymphocyte development in children have not been answered. This deficiency is due to the difficulty in obtaining blood from babies in large quantities, thus representing a practical problem. Therefore, most studies of newborns have been conducted using neonatal umbilical cord blood, which can be easily obtained in larger quantities [11].

Preterm infants born before 28 weeks' gestation are at greater risk of invasive bacterial infections [12], but current studies indicate that late preterm infants also have a significantly higher rate of complications of prematurity and experience more difficulties with feeding, hypoglycaemia, jaundice, temperature instability, apnoea and respiratory distress [13]. This suggests that the last month of gestation is a critical culminating period for the maturation of the immune system [13-15].

Due to the high incidence of extracellular bacterial infections in neonates, it is important to investigate the neonatal ability to combat these pathogens. In this study, we used flow cytometry to evaluate monocyte and dendritic cell populations and their pattern recognition and activation markers as well as helper T and B lymphocytes and their subpopulations in umbilical cord blood from healthy preterm and full-term newborns and, as controls, in peripheral blood from healthy adults.

Materials and methods

Study population and blood samples

We performed a cross-sectional descriptive study using umbilical cord blood from healthy adequate-for-gestational-age preterm and full-term newborns. Newborns (n = 56) were classified into three groups according to their post-menstrual age at birth: newborns born at 30–336/7 gestation weeks were classified as moderate and very preterm newborns (Group 1, n = 13); newborns born at 34–366/7 gestation weeks were classified as late preterm newborns (Group 2, n = 21); and newborns born at 37–41 gestation weeks were classified as full-term newborns (Group 3, n = 22) [16]. Gestational age (GA) was calculated based on last menstrual period or ultrasound measurement of foetal length in the first trimester. Exclusion criteria were genetic syndromes, Apgar score of <5 at 5 min, clinical or pathological evidence of chorioamnionitis or clinical and laboratory signs of infection (elevated infection markers such as C-reactive protein, IL-8 and IL-6; rupture of membranes for more than 18 h; and confirmed early-onset sepsis).

There were two sets of twins within both preterm groups. The subset data were examined for concordance and discordance in the twins. The range of subset percentages in the twins was similar to that of the singleton population.

Neonatal cord blood was collected from the foetal side of the placenta immediately after delivery by umbilical cord venipuncture after clamping in EDTA-coated tubes and in special clot activator tubes for serum separation and cytokine measurement. Blood in EDTA tubes was maintained at room temperature and processed within 24 h of collection.

The cytokines IL-8 and IL-6 were measured with a cytometric bead array (Becton Dickinson BD Biosciences, San Jose, CA, USA) using sera from cord blood from the three groups of neonates.

Peripheral blood was collected from healthy adult volunteers as controls (n = 39). The age range of the adults is 20 to 40 years. They were selected from the laboratory's group and were chosen by the absence of clinical signs of infection.

The neonates were recruited from the Obstetric Center, University Hospital, University of São Paulo, and the Obstetric Center of the Central Institute of the Hospital das Clínicas. The study was approved by the Research and Ethics Committee of the Department of Pediatrics of São Paulo University School of Medicine and by the Ethics Committee for the Analysis of Projects and Research of the Hospital das Clínicas (0101/10). Informed consent was obtained from each participant.

Immunophenotyping by flow cytometry

The number of total blood leucocytes from the umbilical cord blood and peripheral blood samples was determined with an automatic counter (Abbott Cell-Dyn CD-1700 Hematology Analyzer, Abbott Diagnostics, Abbott Park, IL, USA). To analyse cell populations, 1 × 106 leucocytes were placed in a tube and subjected to two 20-min erythrocyte lysis steps using BD Pharm Lyse (BD Biosciences). After centrifugation and washing with staining buffer (phosphate-buffered saline with 1% foetal calf serum and 0.1% sodium azide), the cells were stained with fluorochrome-conjugated monoclonal antibodies against each cell population under study for 30 min. After washing, the cells were resuspended in staining buffer and immediately analysed to prevent the loss of fluorescence. Data were acquired with a BD LSRII Flow Cytometer (BD Biosciences) with FACSDiva software (Becton Dickinson), and the analysis was conducted using FlowJo software (Tree Star, Ashland, OR, USA). Data are represented as the means of absolute numbers or frequencies.

The same fluorochromes were used for all experiments: fluorescein isothiocyanate (FITC) or Alexa Fluor 488; phycoerythrin (PE); phycoerythrin–cyanine 5 (PE-Cy5) or peridinin–chlorophyll–protein–cyanine 5.5 (PerCP-Cy5.5); phycoerythrin–cyanine 7 (PE-Cy7); allophycocyanin (APC) or Alexa 647; allophycocyanin–cyanine 7 (APC-Cy7); Pacific blue or V450 or Brilliant Violet 421 (BioLegend, San Diego, CA, USA); AmCyan or V500, all from BD Biosciences unless otherwise stated.

Dendritic cells

Dendritic cells were defined as lineage cocktail (Lin) negative and (HLA-DR+) positive according to Koumbi et al. [17] and Shin et al. [18], and myeloid DC populations (mDC: LinHLA-DR+/CD11c+) and plasmacytoid DC populations (pDC: LinHLA-DR+/CD123c+) were characterized as described by Sorg et al. [19] and Schreibelt et al. [20]. Within each subpopulation, the dendritic cell pattern recognition, maturation and activation markers were analysed using CD1a, CD80, CD86, TLR-2 and TLR-4. The results were expressed as the percentage of total DCs of all nucleated cells. Absolute numbers of DCs were calculated from complete blood leucocyte counts. Subpopulations of mDCs and pDCs were expressed as percentages and absolute numbers of total DCs (Fig. 1A, B).

Monocytes

To analyse monocyte populations, a P1 gate on CD14+/HLA-DR+ cells was established. The expression levels of the costimulatory molecules CD80, CD86 and CD40, and the pattern recognition receptors TLR-2 and TLR-4 were analysed on cells within this P1 gate (Fig. 1C).

T lymphocytes

T cell subsets were identified based on the expression of CD45RA and CCR7. Characterization of the CD4+ T cell compartment was performed using combinations of markers gating on CD3+/CD4+ cells within the total lymphocyte population. Within the CD3+⁄CD4+ helper T lymphocyte gate, CD4+/CD27+/CD45RA+/CCR7+/CD62L+ cells were naïve T lymphocytes; CD4+/CD62Llow/CD69+ cells were effector T lymphocytes; CD4+/CD27+/CD45RA/CCR7+ cells were central memory T lymphocytes; and CD4+/CD27+/CD45RA/CCR7 cells were effector memory T lymphocytes (Fig. 1D, E, F, G).

B lymphocytes

B cells were identified as CD19+ cells within the total lymphocyte population. Definitions of B cell subsets were performed using the following markers: CD19+/CD27 cells as naïve B lymphocytes; CD19+/CD27+ cells as memory B lymphocytes; and CD19+/CD69+/CD40+ cells as activated B lymphocytes. Within the CD19+ B cell population, TLR-2 and TLR-4 expressions were also analysed (Fig. 1H, I).

Statistical analysis

Statistical analyses were conducted using MedCalc 12.7.0 (MedCalc Software, Ostend, Belgium). Kruskal–Wallis nonparametric statistical test followed by Dunn's post hoc test for multiple comparisons was performed to compare groups that were not normally distributed. A significance threshold of 0.05 was used for all tests unless otherwise stated.

Results

Characteristics of preterm and term newborns

A total of 56 neonates, 13 moderate and very preterm neonates born before 34 gestation weeks, 21 late preterm neonates born between 34–366/7 gestation weeks and 22 full-term neonates born after 37 gestation weeks were enrolled in the study as shown in Table 1. These groups were divided according to the classification proposed by Engle [21]. Leucocyte subset data from these neonates were compared with the leucocyte subset data from 39 adult controls. All 56 newborns included in this study were born without signs of infection; they were excluded if any indication of a pathologic background in the mother's clinical history was detected. The C-reactive protein, IL-8 and IL-6 levels in the newborns' sera also indicated that they were healthy.

Table 1. Demographic characteristics and total leucocyte counts of study subjects
CharacteristicsGroup 1 (G1)Group 2 (G2)Group 3 (G3)P value
(N = 13)(N = 21)(N = 22)G1 × G2G1 × G3G2 × G3
  1. NSVD, normal spontaneous vaginal delivery.

  2. a

    Frequency (percentage).

  3. b

    Mean ± standard deviation.

Male/femalea9 (69.2)/4 (30.8)13 (61.9) / 8 (38.1)13 (59.1) / 9 (40.9)
Birth weight (g)b1606 ± 294.32166 ± 4543317 ± 431.4P < 0.001P < 0.0001P < 0.0001
Gestational age (weeks)b32 ± 12/7351/7 ± 06/7391/7 ± 11/7P < 0.001P < 0.0001P < 0.0001
Apgar at 1 minb7 ± 28 ± 29 ± 1P < 0.001P < 0.05
Apgar at 5 minb9 ± 19 ± 110 ± 1P < 0.001P < 0.05
Deliverya NSVD /C-section3 (23.1)/10 (76.9)6 (28.6)/15 (71.4)11 (50.0)/11 (50.0)
IL-8b68.7 ± 104.7142.5 ± 402.4276.7 ± 568.4
IL-6b32.7 ± 79.925.2 ± 56.214.0 ± 40.8
Leucocyte numbers (103/μl)b9.1 ± 4.110.4 ± 3.914.8 ± 5.3P < 0.05P < 0.05

Given the increased risk of infection in preterm infants and the pathophysiological role of cytokines as mediators of infection, measurement of neonatal cytokine levels may provide an earlier biomarker for the diagnosis of infection than downstream markers such as C-reactive protein and absolute changes in white blood cell counts. The concentration of IL-8 depended on the mode of delivery and was higher in neonates delivered vaginally than those delivered by elective caesarean section, which resulted in slightly higher, although not statistically significant, IL-8 levels in Group 3 compared with the other groups. IL-6 concentrations were low in all newborn samples and were statistically equivalent between groups.

Figure 1.

Representative phenotypic analysis of leucocyte subsets by flow cytometry. (A) Lin-negative but HLA-DR-positive dendritic cells were gated in Q3; (B) myeloid dendritic cells (mDCs) were identified as CD11c positive and CD123 negative, and plasmacytoid dendritic cells (pDCs) were identified as CD11c negative and CD123 positive within LinHLA-DR+ cells; (C) monocytes were identified as CD14-positive and HLA-DR-positive cells; (D) CD4 T cells were identified by gating on CD3CD4bright and within the total lymphocyte population; (E–F) CD4+ T cell subsets were identified within the CD27+ gate and then based on the expression of CD45RA and CCR7, as shown. Q1: CD4+ central memory T cells; Q4: CD4+ effector memory T cells; and Q2: CD4+ naive T cells were further analysed within the CD62L+ gate (G); (H) B cells were identified by gating on CD19-positive cells and within the total lymphocyte population; (I) naïve and memory B cell subsets were identified based on the expression of CD27, within CD19+ cells, as shown.

Statistical analysis demonstrated that newborns from Group 3 had higher leucocyte numbers than the other groups (P < 0.05) (Table 1) and that adults had significantly lower leucocyte numbers than the three groups of newborns (mean = 6.6 × 103/μl) (P < 0.05).

We determined the absolute leucocyte subpopulation counts in preterm and full-term infants by 8-colour flow cytometry. We compared these data with data obtained from healthy adults.

Dendritic cell subsets

The data in Table 2 and Fig. 2 show specific comparisons among full-term neonates and preterm neonates after determination of statistical significance across these study groups using the Kruskal–Wallis test as described elsewhere. The frequencies and absolute numbers of dendritic cells among all nucleated cells were equivalent across neonatal groups. Group 3 had lower percentages of mDCs and pDCs than both preterm groups. The numbers of mDCs and pDCs did not differ among the neonate groups. Myeloid and plasmacytoid DCs expressing the activation markers CD80 and CD86 were also present at the same frequencies among neonatal groups, with the exception of Group 1, which displayed a reduced frequency of CD80+ cells in both DC subsets compared with Group 3. No differences in frequencies were detected among the groups with regard to immature mDCs and pDCs expressing CD1a. Group 1 had significantly less absolute numbers of CD80+ mDCs and CD80+ pDCs compared with Groups 2 and 3. These results were influenced by the low frequencies of these cell subsets in neonates from Group 1.

Table 2. Frequencies (%) and absolute numbers (/μl) of total dendritic cells and dendritic cell subtypes
DC subsetsGroup 1 (N = 13) GA = 30–336/7Group 2 (N = 21) GA = 34–366/7Group 3 (N = 22) GA = 37–41Adult (N = 39)P value
Median [P25–75]Median [P25–75]Median [P25–75]Median [P25–75]G1 × G2% (/μl)G1 × G3% (/μl)G2 × G3% (/μl)
(%)(/μl)(%)(/μl)(%)(/μl)(%)(/μl)
  1. GA, gestational age; DC, dendritic cell; mDC, myeloid dendritic cell; pDC, plasmacytoid dendritic cell; P, percentile; %, frequency of cells; /μl, absolute number per microlitre blood; –, no statistically significant differences were found.

  2. Differences in frequencies (%) between groups are shown first and differences in absolute numbers (/μl) are in parenthesis.

Total DC (LIN/HLA-DR+)1.5 [0.9–3.5]149 [92–282]1.4 [1–2.1]150 [91–221]1.8 [1.2–3.3]304 [116–528]0.5 [0.4–0.7]31 [21–58]– (–)– (–)– (–)
mDC (CD11c+)28 [17.2–34.7]30 [17–51]17.4 [12–26.7]23 [13–64]13.4 [9–19.7]38 [13–81]28.5 [20.2–40.8]11 [5–17]– (–)0.0001 (–)0.05 (–)
mDC CD1a+19.7 [10.9–36.5]38 [6–84]20.3 [7.3–31.8]23 [9–58]20.6 [11.7–45.6]54 [19–170]20.9 [7–35.6]6 [2–24]– (–)– (–)– (–)
mDC CD80+25.3 [6.5–44.6]25 [8–81]33.7 [7.4–60.4]53 [13–102]49.3 [30.7–66.8]154 [37–241]41.7 [21.8–61.2]16 [9–22]– (–)0.01 (0.01)– (0.01)
mDC CD86+68.2 [46.8–84.5]76 [41–185]58.6 [47–81.7]72 [59–172]71.4 [35.5–83.8]129 [67–309]67.4 [51.9–77.4]20 [15–37]– (–)– (–)– (–)
mDC TLR-2+19.5 [11.7–39.4]32 [13–66]25.0 [11–64.2]50 [20–89]45.7 [15.8–63.2]95 [44–317]44.5 [14.5–62.4]12 [4–21]– (–)– (0.05)– (0.05)
mDC TLR-4+24 [10.8–60.2]35 [9–78]28.4 [18.5–40.8]49 [23–72]25.3 [10–37.6]52 [31–122]48.6 [25.1–81.5]17 [6–33]– (–)– (–)– (–)
pDC (CD123+)42.7 [24.2–52.2]68 [42–102]14.7 [8.8–45.5]32 [13–78]11.1 [7.3–15.8]19 [14–71]29.8 [18–48.3]10 [4–21]– (–)0.01 (–)0.05 (–)
pDC CD1a+33 [15.3–62.2]55 [17–103]44 [24.9–57.2]49 [37–102]28.6 [16.9–44.4]59 [25–173]38 [23.9–54.8]13 [6–25]– (–)– (–)– (–)
pDC CD80+6.1 [4.7–15.8]9 [4–23]13.5 [4.2–33.5]16 [9–60]18.1 [10–34.4]48 [29–109]15.9 [6.8–30.7]6 [2–12]– (–)0.05 (0.01)– (–)
pDC CD86+31.3 [26.7–67.6]43 [34–109]33 [20.4–47]47 [27–101]37 [25.4–56]103 [43–213]36.1 [24.3–42.5]11 [8–22]– (–)– (–)– (–)
pDC TLR-2+10.6 [4.6–17.7]16 [5–31]7.5 [3.4–16.4]19 [5–29]15.6 [7.7–36.8]51 [6–149]17.2 [5.1–29.6]7 [2–13]– (–)– (–)– (–)
pDC TLR-4+15.4 [8–35.9]19 [8–60]25.6 [14.9–35.8]36 [22–67]20.7 [10.4–30.4]39 [25–89]37.8 [10.7–61.7]12 [2–30]– (–)– (0.01)– (–)
Figure 2.

Frequencies of dendritic cells and their subsets in cord blood. (A) Total dendritic cells; (B) myeloid dendritic cell (mDC); and (C) plasmacytoid dendritic cell (pDC); immature (CD1a+); mDCs or pDCs expressing activation markers CD80 and CD86; and TLR-2 and TLR-4 in cord blood from preterm (Groups 1 and 2) and term (Group 3) newborns and in adult blood, as controls. Box plots: black crosses are the mean, black horizontal lines are medians, the solid lines of the box represent the 75th and 25th percentiles, and the short lines outside the top and the base of the box represent the highest and the lowest values, respectively.

The percentages of TLR-2+ mDCs gradually increased across the neonatal groups, but no significant differences were detected among them. All newborns had lower frequencies of TLR-4+ mDCs than adults, while TLR-4+ pDCs were present in similar percentages in neonates and adults.

The majority of the analyses revealed statistically higher absolute numbers of DCs and DC subsets in neonates compared with adults, as was also observed for TLR-2 and TLR-4 expressions in both DC subsets, but these results reflected the higher numbers of leucocytes in the umbilical cord blood.

Monocytes

The percentages of monocytes were similar among all neonate groups and the adults with the exception of Group 2, which had a higher frequency of these cells than adults (Fig. 3). However, the absolute counts revealed a lower number of these cells in Group 1 compared with the other neonates (Table 3). The analyses of the frequency of activation markers CD80 and CD86 on the surface of monocytes revealed no significant differences with the exception of Group 2, which showed a higher frequency of CD86+ monocytes compared with Group 3 (P < 0.01). The frequencies of TLR-2- and TLR-4-expressing monocytes were similar among all groups. In addition, Group 1 had a lower number of TLR-2+ monocytes compared with other newborns, while Group 3 had a higher number of TLR-4+ monocytes compared with both preterm newborn groups.

Table 3. Frequencies (%) and absolute numbers (/μl) of total monocytes and monocytes CD80+, CD86+, TLR-2+ and TLR-4+
MonocytesGroup 1 (N = 13) GA = 30–336/7Group 2 (N = 21) GA = 34–366/7Group 3 (N = 22) GA = 37–41Adult (N = 39)P value
Median [P25–75]Median [P25–75]Median [P25–75]Median [P25–75]G1 × G2% (/μl)G1 × G3% (/μl)G2 × G3% (/μl)
(%)(/μl)(%)(/μl)(%)(/μl)(%)(/μl)
  1. GA, gestational age; P, percentile; %, frequency of cells; /μl, absolute number per microlitre blood; –, no statistically significant differences were found.

  2. Differences in frequencies (%) between groups are shown first and differences in absolute numbers (/μl) are in parenthesis.

Monocyte (CD14+/HLA-DR+)3.2 [2.5–4.0]252 [139–442]3.9 [3.0–6.8]455 [247–629]3.2 [1.6–6.5]437 [325–676]2.7 [1.7–3.9]154 [88–260]– (0.05)– (0.05)– (–)
Monocyte CD80+13.5 [5.7–17.3]22 [14–47]8.1 [4.8–16.3]35 [17–47]12.6 [9–18.4]43 [28–99]15.0 [6.3–28.8]19 [10–38]– (–)– (0.05)– (–)
Monocyte CD86+96.2 [90.6–98.1]231 [125–421]98.4 [95.7–99.3]453 [213–603]92 [85.3–97.1]414 [303–626]96.2 [90.1–98.7]139 [82–256]– (0.0001)– (0.0001)0.01 (–)
Monocyte TLR-2+79.8 [51.2–94.3]180 [116–260]95.1 [85.1–98.6]411 [200–591]91.6 [78.1–98.5]412 [176–601]93.5 [82–98.5]114 [61–255]– (0.05)– (0.05)– (–)
Monocyte TLR-4+12.0 [7.7–18.3]30 [16–43]9.1 [3.8–23]31 [13–86]16.0 [8.7–24.9]84 [34–125]10.6 [5.7–17.9]15 [9–26]– (–)– (0.01)– (0.05)
Figure 3.

Frequencies of monocytes in cord blood. Total monocytes and monocytes expressing activation markers CD80 and CD86 and TLR-2 and TLR-4 in cord blood from preterm (Groups 1 and 2) and term (Group 3) newborns and in adult blood, as controls. Box plots: black crosses are the mean, black horizontal lines are medians, the solid lines of the box represent the 75th and 25th percentiles, and the short lines outside the top and the base of the box represent the highest and the lowest values, respectively.

T Lymphocyte subsets

The analysis of total lymphocytes revealed a lower frequency of this population in term newborns from Group 3 compared with both preterm groups. Based on the expression of CD45RA, CCR7 and CD62L, the CD4+ T cell compartment was divided into naïve, central memory and effector memory T cells. The data in Table 4 and Fig. 4 show that the percentages of CD3+CD4+ helper T cells varied across the neonatal groups but were not significantly different, and the same was found for absolute numbers.

Table 4. Frequencies (%) and absolute numbers (/μl) of total lymphocytes, CD4+ T cells and CD4+ T cell subsets
CD3+CD4+ T lymphocyte subsetsGroup 1 (N = 13) GA = 30–336/7Group 2 (N = 21) GA = 34–366/7Group 3 (N = 22) GA = 37–41Adult (N = 39)P value
Median [P25–75]Median [P25–75]Median [P25–75]Median [P25–75]G1 × G2% (/μl)G1 × G3% (/μl)G2 × G3% (/μl)
(%)(/μl)(%)(/μl)(%)(/μl)(%)(/μl)
  1. GA, gestational age; naïve T lymphocyte (CD4+CD27+CD45RA+CCR7+CD62L+); effector T lymphocyte (CD4+CD62LlowCD69+); central memory T lymphocyte (CD4+CD27+CD45RACCR7+); effector memory T lymphocyte (CD4+CD27+CD45RACCR7); P, percentile;%, frequency of cells; /μl, absolute number per microlitre blood; –, no statistically significant differences were found.

  2. Differences in frequencies (%) between groups are shown first and differences in absolute numbers (/μl) are in parenthesis.

Lymphocyte (total)52.7 [41.1–57.9]4002 [3053–5106]49.3 [34.7–55.1]4404 [3029–5700]31.3 [25.5–39.4]4100 [2579–5132]33.3 [29.7–44]2162 [1709–2679]– (–)0.01 (–)0.01 (–)
CD3+CD4+ T lymphocyte43.3 [30.3–45.1]1606 [1191–2114]42.1 [36.5–48.8]1823 [1204–2565]36.8 [31.1–42.6]1502 [803–1881]41.9 [35–49.9]904 [709–1197]– (–)– (–)– (–)
Naïve T lymphocyte41.1 [20.1–58]421 [290–951]45.5 [32–63.5]803 [456–1670]41.5 [19.4–63.6]498 [237–816]24.3 [8.9–32]195 [57–358]– (–)– (–)– (0.05)
Effector T lymphocyte0.8 [0.2–1.3]11 [3–23]0.6 [0.2–1.0]10 [3–18]0.7 [0.3–2.3]10 [5–31]1.6 [1.1–2.4]12 [8–30]– (–)– (–)– (–)
Central memory T lymphocyte23.5 [20–30.2]339 [204–598]21.7 [13.3–31.3]380 [235–670]15.4 [9.7–23.6]167 [113–266]18.1 [13.4–24.2]154 [104–263]– (–)– (–)– (–)
Effector memory T lymphocyte9.5 [5.7–12.9]135 [90–182]6.7 [4.8–10.7]131 [92–225]5.3 [2.9–10]53 [30–182]33.6 [26.9–44.4]301 [259–384]– (–)– (–)– (–)
Figure 4.

Frequencies of total lymphocytes, TCD4+ lymphocytes and their subsets in cord blood. Total lymphocytes; TCD4+ lymphocytes and their subsets: naïve, effector, central memory and effector memory TCD4+ lymphocytes in cord blood from preterm (Groups 1 and 2) and term (Group 3) newborns and in adult blood, as controls. Box plots: black crosses are the mean, black horizontal lines are medians, the solid lines of the box represent the 75th and 25th percentiles, and the short lines outside the top and the base of the box represent the highest and the lowest values, respectively.

Study of the central memory, effector memory and naïve subsets of CD4+ T cells revealed no differences among neonates, with the exception of the higher number of naïve T cells found in Group 2 compared with Group 3. As expected, higher percentages and numbers of naïve T cells and strikingly lower frequencies and numbers of effector memory T cells and effector T cells were observed in all newborns compared with adults.

B Lymphocyte subsets

As shown in Table 5 and Fig. 5, the percentages and numbers of blood CD19+ B lymphocytes were equivalent among all neonatal groups. Naïve B cell percentages and numbers were higher in neonates than in adults, indicating that these cells constituted the largest peripheral B cell subset in preterm and full-term neonates, and by contrast, full-term and preterm groups had a strikingly lower percentage of memory B cells compared with adults, as expected. The activated B cell subset was present in similarly low percentages in newborns and adults. TLR-2 and TLR-4 expressions on B lymphocytes were insignificant (data not shown).

Table 5. Frequencies (%) and absolute numbers (/μl) of total CD19 B cells and B cell subsets
CD19+ B lymphocyte subsetsGroup 1 (N = 13) GA = 30–336/7Group 2 (N = 21) GA = 34–366/7Group 3 (N = 22) GA = 37–41Adult (N = 39)P value
Median [P25–75]Median [P25–75]Median [P25–75]Median [P25–75]G1 × G2% (/μl)G1 × G3% (/μl)G2 × G3% (/μl)
(%)(/μl)(%)(/μl)(%)(/μl)(%)(/μl)
  1. GA, gestational age; naïve B lymphocyte (CD19+CD27); memory B lymphocyte (CD19+CD27+); activated B lymphocyte (CD19+CD40+CD69+); P, percentile;%, frequency of cells; /μl, absolute number per microlitre blood; –, no statistically significant differences were found.

  2. Differences in frequencies (%) between groups are shown first and differences in absolute numbers (/μl) are in parenthesis.

CD19+ B lymphocyte16.1 [12–21.2]594 [493–844]11.5 [8.6–15.4]507 [311–701]12.6 [9.1–18.2]537 [362–634]11.1 [8.4–13.8]244 [152–324]– (–)– (–)– (–)
Naïve B lymphocyte83.7 [73.9–90.9]537 [334–758]84.6 [74–89.5]374 [201–628]91 [81.7–94.2]422 [271–526]63.5 [54.1–72.5]138 [92–216]– (–)– (–)– (–)
Memory B lymphocyte16.3 [9.1–26.1]98 [54–219]15.4 [10.5–26.1]94 [41–132]9 [4.8–18.2]40 [22–90]36.5 [27.5–45.9]85 [49–119]– (–)– (–)– (–)
Activated B lymphocyte1.6 [0.3–2.1]10 [2–15]1.3 [0.6–3.4]7 [3–14]2.5 [1.2–4.2]11 [5–29]1.4 [0.8–2.8]3 [1–7]– (–)– (–)– (–)
Figure 5.

Frequencies of B lymphocytes and their subsets in cord blood. B lymphocytes and their subsets: naïve, memory and activated B lymphocytes in cord blood from preterm (Groups 1 and 2) and term (Group 3) newborns and in adult blood, as controls. Box plots: black crosses are the mean, black horizontal lines are medians, the solid lines of the box represent the 75th and 25th percentiles, and the short lines outside the top and the base of the box represent the highest and the lowest values, respectively.

Discussion

Previous studies have compared leucocyte subsets from full-term neonates with adults, but these studies rarely include samples from preterm newborns. Generally, the composition of circulating white blood cells numerically differs among neonates of different gestational ages, likely reflecting dynamic developmental phases [15]. The proportions of both lymphoid and myeloid cells are higher in the peripheral blood of a healthy-term neonate than a preterm [22-25]. Furthermore, it is difficult to compare previous data because of differences in methodology. Both relative and absolute counts have been used to illustrate the changes in leucocyte [26-28]. However, it is important to compare absolute counts to assess the actual size of a leucocyte subpopulation [29]. In the present work, we consistently observed differences in absolute numbers of different leucocyte populations among neonates and adults, but these results reflected the higher number of circulating leucocytes found in the three groups of neonates.

The induction of different types of innate and adaptive immune responses based on the nature of the antigens and the environmental context is crucial to cope with a variety of pathogens and to concurrently avoid pathological reactions to self-antigens. Several studies have shown that the diverse immune responses are critically controlled by dendritic cells (DCs). DCs are widely distributed, potent antigen-presenting cells (APCs) that are unique based on their prominent role in the activation, polarization and regulation of adaptive immune responses [19, 30-32].

Depending on their developmental origin, cytokine activation, surface antigens and functional capacity, DCs can be subdivided into two major distinct populations: myeloid and lymphoid/plasmacytoid DCs [33]. Myeloid DCs may be further split into CD1c+ and CD141+ fractions. CD1c+ mDCs are the major population of human mDCs in blood, tissues and lymphoid organs and are good stimulators of naïve CD4 T cells. mDCs secrete tumour necrosis factor-α (TNF-α), IL-8, IL-10 and high levels of IL-12, and a small amount of IL-23 can be detected after a range of stimuli. This implies a dual role in T helper type 1 (Th1) and Th17 sensitization and highlights the plasticity of DCs in different contexts. Plasmacytoid DCs are distinguished by expression of CD123, CD303 and CD304, and their propensity to release abundant amounts of type I interferons (IFNs) in response to viruses was one of the first specialized DC functions to be described. It has been demonstrated that freshly isolated blood pDCs do not prime naïve T cells efficiently and appear less mature than mDCs until activated, in agreement with our findings that showed higher frequencies of immature CD1a+ pDC than mDC, but no differences across the study groups. Nevertheless, the ability of pDCs to polarize CD4 responses towards Th1 or Th2 is variable and may be context dependent [33, 34]. In the present work, although we observed significantly higher relative and absolute numbers of total DCs in preterm and full-term newborns compared with adults, the latter had an increased frequency of mDCs compared with newborns, which could contribute to a more efficient activation of T cell response.

TLRs play a crucial role in the induction of adaptive immunity as well as innate immunity. DC subsets recognize different microbial pathogens by expressing distinct TLRs on their surface and induce different types of innate and adaptive immune responses, depending on environmental factors [35-37]. Dendritic cells from cord blood normally express TLR-2 and TLR-4 [32, 38]. Expression levels of TLR-2 and TLR-4 were previously shown to be normal on neonatal mDCs, in contrast to the lower levels of expression of these molecules on neonatal monocytes [39]. These results are in agreement with our data, at least with regard to TLR-4. We observed the same expression pattern on pDCs as we did not observe significant differences in TLR expression on pDCs among neonates and adults, although we observed increasing TLR-2 expression on mDCs and pDCs according to gestational age. The lower expression of TLR-4 on neonatal mDCs found in our work and the regulation of critical molecules involved in TLR signalling [32], which has not yet been evaluated in neonatal DCs, could account for the relative unresponsiveness of neonatal DCs to TLR stimulation by Gram-negative rods.

Although neonatal monocytes express similar mRNA levels of different TLRs compared with adult monocytes, these data do not exclude possible differences in the protein expression of these receptors [32, 40, 41]. In the present work, we observed similar expression of TLR-2 and TLR-4 in monocytes from all study groups, but the numbers of TLR-2+ and TLR-4+ monocytes were slightly higher in Groups 2 and 3. These observations agree with previous data that describe constitutive TLR-4 expression and cytokine secretion upon LPS stimulation that increases with gestational age [9].

Previous studies have shown that neonatal monocytes exhibit low baseline expression of the costimulatory molecules CD40 and CD86, potentially contributing to deficient APC activity, in contrast to our data [32]. We observed similar frequencies of monocytes expressing CD80 and CD86 across the groups, but the percentages of CD80+ monocytes were slightly lower in neonates than adults, although significantly different only for Group 2. We also observed reduced CD80 expression on mDCs and pDCs in neonates from Group 1 compared with Group 3 and adults, supporting the notion of deficient activity of APCs from very preterm newborns.

Upon contact with cognate MHC–peptide complexes on APCs, naïve T cells become effector cells that are highly functional. These effector cells can differentiate into effector memory T cells and, later, into central memory T cells based on the expression of different cell surface molecules underlying homing markers, chemokines and cytokine receptors [42-47].

We observed higher frequencies of total lymphocytes in both groups of preterm neonates compared with full-term neonates and adults. However, the percentages of T helper cells were equivalent among all groups, with the exception of the comparison between full-term neonates and adults. These results have been shown by others [6, 15], and it has been discussed that this probably indicates that newborns with higher gestational age, which are ready to leave the protective environment of the uterus, need to mobilize a greater number of cells to the periphery to respond to possible infectious agents. This fact could also explain the lower frequencies of mDCs and pDCs in term neonates compared with preterm ones.

Naïve T cells are characterized by the expression of CD45RA and CCR7, the costimulatory receptors CD28 and CD27, and the lack of expression of cytolytic molecules. Long-lived central memory T cells home preferentially to lymph nodes because they express the lymph node-homing markers CD62L and CCR7 and share several phenotypic properties with naïve T cells but do not express CD45RA. Central memory T cells, as opposed to naïve T cells, can rapidly differentiate into cells endowed with effector function upon exposure to antigen [43], can upregulate CD40L to a greater extent than naïve cells and are characterized by their ability to proliferate and secrete high levels of IL-2.

In the present work, no significant differences in naïve T cells were observed among the newborns, although newborns from Group 1 showed a slightly lower relative number compared with the other neonates. An overall increase in naïve T cells in neonates has been demonstrated previously, and it has also been shown that preterm neonates have reduced naïve T cell populations compared with full-term neonates [6, 8]. It has been speculated that this difference is due to decreased thymic output or to decreased peripheral expansion of naïve T cells because of more limited functional T cell capacities in extreme preterm infants [8]. The thymic output of naïve T cells in term neonates has already been shown to be normal [48]. In disagreement with others [8, 49], the term group's naïve T helper cells were significantly lower than those of late preterm newborns, and the number and frequency of CD3+CD4+ T lymphocytes were also somewhat reduced compared with the preterm groups. Pérez et al. [15] showed that the frequencies of neonatal CD3+ and CD4+ T cells followed a trend similar to ours, with the lowest values at 38 and 41 gestation weeks. These authors discuss that this fact might be due to the recruitment of the major number of cells at ganglia, the place where the mature cells wait until a stimulus causes them to leave to the periphery again, just at the ‘normal’ period of birth.

Surprisingly, central memory T cell frequencies were equivalent among preterm neonates and adults. These finding are in contrast to previous reports of a reduced frequency of memory T cells in neonates compared with adults. However, the effector memory and central memory T cell subsets were not distinguished in those reports [6]. Effector memory T cells preferentially migrate to peripheral tissues, do not express CD45RA or CCR7 and may or may not express CD27/CD28, depending on their state of differentiation [44]. Effector T cells are identified by the expression of various markers, including CD69 and CD25, and low levels of CD62L [47]. Because these cells have been already activated upon previous contact with antigen, the higher frequencies found in adults in this study would be expected.

Most B lymphocytes consist mainly of naïve cells in full-term and preterm newborns, and our group and others have observed significantly reduced frequencies of memory B cells in neonates compared with adults [8, 48]. More recently, it has been shown that memory B cells constitutively express TLR-2 to some degree [50]. In the present work, we observed extremely low frequencies of B lymphocytes expressing TLR-2 and TLR-4 in all groups, including adults (data not shown).

A premature birth is generally caused by negative perinatal factors such as maternal pre-eclampsia or intrauterine infection, and antenatal steroids are commonly used in preterm infants. These factors can influence lymphocyte subpopulations in neonatal cord blood [26, 28, 51, 52]. However, studies examining these factors reported relative values and not absolute values, which may lead to misinterpretation, as we and others have shown significantly increased numbers of leucocytes in preterm and term infants [26-29].

A growing body of evidence suggests that the incidence of medical problems, either short term or long term, is higher among late preterm infants than among term infants [53]. During the birth hospitalization, late preterm infants experience more difficulties with feeding (32% versus 7%), hypoglycaemia (16% versus 5%), jaundice (54% versus 38%), temperature instability (10% versus 0%), apnoea (~6% versus <0.1%) and respiratory distress (29% versus 4%) compared with term infants. Late preterm infants also receive intravenous fluids (27% versus 5%), evaluations for sepsis (37% versus 13%) and mechanical ventilation (3.4% versus 0.9%) more often than their term counterparts [16]. In a large healthcare system, 88% of infants born at 34 weeks' gestation, 12% born at 37 weeks' gestation and 2.6% born at 38–40 weeks' gestation were admitted to an intensive care unit, showing that admission for intensive care is inversely proportional to gestational age [54]. Regarding infections, it was reported significantly more sepsis evaluations and culture-proven sepsis in the late preterm infants compared with term infants, with suspected or proven infections detected five times as frequently [55].

The heightened risk of bacterial infection in preterm infants has a number of determinants: first, extremely preterm infants require prolonged intensive care, including mechanical ventilation, parenteral feeding and intravenous access, which breach physical barriers against infection and facilitate invasion by nosocomial pathogens. Second, neonates are rapidly colonized by microbes in the intensive care environment. Third, many elements of the acquired immune system function less well in the neonate, with gestational and post-natal age among the most important determinants of immune function [56]. Here, in addition to the already well-described differences in T and B lymphocyte subpopulations compared with adults, we found lower numbers of monocytes and dendritic cells expressing the activation markers CD80 and CD86 and TLR-2 and TLR-4 in very preterm newborns from Group 1 compared with term newborns. Moreover, we showed lower numbers of mDC CD80+, mDC TLR-2+ and monocytes TLR-4+ in late preterm newborns compared with term ones.

Several studies have suggested that some innate immune mechanisms are impaired in neonates. However, it has been discussed that neonates are immunocompetent to mount mature innate as well as adult-level T cell responses [57]. In this work, we showed that neonates, particularly late preterm neonates, frequently have adequate tools to combat extracellular pathogens, as evidenced by the relative numbers of the majority of innate cells and the expression of pattern recognition and activation molecules. However, an appropriate number of cells do not indicate proper function; thus, it is imperative to clarify defective mechanisms that may underlie the immune responses in newborns and contribute to the overall risk of infection by common neonatal pathogens.

Our study provides a more thorough characterization of leucocyte subsets in cord blood from preterm and full-term newborns that may facilitate the identification of immunological deficiencies in the protection against extracellular pathogens. These data may also permit the development of clinical strategies to limit invasive medical interventions, particularly in the most vulnerable groups, such as extreme preterm newborns.

Acknowledgment

Financial support was provided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) Grants 2009/54246-5, 2009/52515-9, 2009/54400-4 and 2009/53864-7. The authors are grateful to the mothers who kindly agreed to participate in this study and Dr. Ulysses Doria Filho for statistical analysis.

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