nature publishing group Articles Clinical Investigation Preterm neonates show marked leukopenia and lymphopenia that are associated with increased regulatory T-cell values and diminished IL-7 Rafael Correa-Rocha1, Alicia Pérez1, Raquel Lorente1, Sara Ferrando-Martínez1, Manuel Leal2, Dolores Gurbindo3, and M. Ángeles Muñoz-Fernández1 INTRODUCTiON: Current advances in neonatology have In fact, the most important factors that ­predispose to infection improved survival among preterm and low-birth-weight are prematurity and/or low birth weight. infants. However, the risk of neonatal death in preterm infants Little is known about innate immunity in premature neo- is much greater than in full-term neonates and is frequently nates and the capacity of their immune systems to fight against associated with infections. infections. Several authors have reported differences in pheno- METHODS: Little is known about the immune status of pre- type and function of lymphocytes between healthy ­newborns, term neonates; therefore, we analyzed the frequency and abso- children, and adults (5). The immune response of preterm lute counts of different immune populations in 211 cord blood infants is assumed to be immature, although quantitative samples taken from very-preterm to full-term neonates. data on differences in immune capacity between preterm and RESULTS: We found that absolute counts of all the immune ­full-term infants are scant. subsets analyzed (i.e., monocytes, granulocytes, B cells, natu- The objective of this study was to investigate whether the ral killer (NK) cells, CD4+, and CD8+ T cells) were markedly high incidence of infections in preterm infants could be due lower in preterm infants than in full-term infants. Surprisingly, to an immunodeficiency and, if so, to identify which immune we observed that regulatory T cells (Tregs) were the only cell cell populations are implicated in the absence of an adequate subset that did not decrease in preterm infants, and their fre- immune response. We performed a comprehensive analysis of quency was even higher than in full-term infants. the frequency and absolute counts of several cell populations DiSCUSSiON: Tregs are crucial to maternal–fetal tolerance, in preterm and full-term infants to establish immune status but their suppressive role could be also implicated in the leu- according to GA and weight. We also studied the presence of kopenia observed in preterm infants. We did not observe dif- regulatory T cells (Tregs) in preterm infants; this cell population ferences in thymic function, but we found that plasma levels of may play a key role in preventing immune deregulation result- interleukin (IL)-7 and the frequency of its receptor were signifi- ing in acute infections or chronic immune modulation. Tregs cantly decreased in preterm infants. Our results could help to play a crucial role in maternal–fetal tolerance, and their levels are identify leukopenia and to implement immune therapies that increased during pregnancy (6,7). However, little information is significantly diminish mortality in preterm neonates. available about the frequency of these cells in neonates, particu- larly in preterm infants, or about their potential role in immune ecent developments in neonatology and improvements in homeostasis during this period of life. Finally, establishing refer- Rtechnology to manage preterm neonates have significantly ence ranges for immune cell subpopulations in preterm infants increased the survival of very-preterm neonates (less than 28 wk could help the neonatologist to identify severe leukopenia and of gestation) and very-low-birth-weight infants (less than 1,500 g) thus prevent infections. In addition, it could constitute a tool for (1). However, the relative risk of neonatal death is much greater implementing immunostimulatory treatments that could sig- for preterm infants than for full-term infants, and prematurity nificantly decrease mortality in preterm infants. has become the leading cause of perinatal morbidity and mortal- ity (2,3). Along with decreasing gestational age (GA), low birth RESULTS weight is also associated with increased perinatal morbidity and We analyzed the percentage and absolute counts of different mortality (4). Among the causes responsible for this increased immune populations in fresh cord blood from 211 healthy mortality, neonatal sepsis and bronchopulmonary dysplasia are neonates. Infants were stratified into two groups according to the most frequent causes of death in premature newborns (3). GA, and differences between these groups were studied. The 1Laboratorio de Inmunobiología Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain; 2Laboratorio de Inmunovirología, Instituto de Biomedicina de Sevilla, Hospital Virgen del Rocío, Sevilla, Spain; 3Sección de Inmunopediatría, Hospital General Universitario Gregorio Marañón, Madrid, Spain. Correspondence: M. Ángeles Muñoz-Fernández ([email protected]) Received 08 July 2011; accepted 22 December 2011; advance online publication 7 March 2012. doi:10.1038/pr.2012.6 590 Pediatric RESEArCH Volume 71 | Number 5 | May 2012 Copyright © 2012 International Pediatric Research Foundation, Inc. Leukopenia in preterm infants and Tregs Articles preterm group comprised 117 infants with a GA <37 wk; the and increased mortality) in 97% of the studied samples (8). full-term group comprised 94 infants with a GA ≥ 37 wk. Therefore, no differences were established in the incidence of acidosis between preterm and full-term infants, and no Clinical Parameters correlation was established between prematurity and mater- The analysis of clinical parameters after delivery revealed nal age (P = 0.390) (Table 1). that prematurity was associated with lower weight and length. Preterm neonates showed significantly lower weight Preterm Infants Showed Marked Leukopenia (P < 0.001) and length (P = 0.044) than full-term neonates We compared the percentages of different immune subsets (Table 1), and weight and length values were directly cor- between the groups and observed that preterm infants had a related with GA (P < 0.001). Arterial pH values were higher higher percentage of total lymphocytes than full-term neo- in preterm infants (Table 1), although values were higher nates. We found that there were no significant differences in than 7.15 (which has been defined as the cutoff for acidosis the percentage of monocytes. In addition, the percentage of Table 1. Clinical data and percentages of immune cells in cord blood samples from preterm and full-term infants n Preterm n Term P value Infant characteristics Wk 117 33 (31–35) 94 39.6 (38–40.9) 0.000 Weight (g) 110 2,050 (1,539–2,540) 91 3,300 (3,070–3,570) 0.000 Length (cm) 16 47.7 (44.2–49) 19 49.5 (48–51) 0.044 Arterial pH 107 7.34 (7.29–7.38) 81 7.28 (7.22–7.33) 0.000 Maternal age 108 31 (26.3–35) 85 31 (28–35) NS % Total lymphocytes 21 45.93 (36.3–59.4) 17 31.09 (26.96–35.3) 0.000 % CD4+ Ta 103 47.8 (42.4–54.5) 83 44.38 (38.01–51.1) 0.011 % CD8+ Ta 103 17.96 (14.5–22.2) 83 16.15 (13.56–19.6) 0.048 % NK cellsa 105 7.09 (3.8–11.8) 84 10.62 (4.98–18.4) 0.004 % B cellsa 103 15.84 (11.9–20.8) 83 17.91 (14.1–23.07) 0.029 % T cells CD127+a 29 90.9 (89.8–92.2) 18 92.3 (90.9–93) 0.029 % Monocytesb 23 9.73 (7.2–11.1) 18 8.69 (7.48–10.11) NS % Basophilsb 23 1.14 (0.89–1.44) 18 0.84 (0.61–1) NS % Granulocytesb 23 40.03 (26.1–51.5) 18 56.49 (53.03–59.9) 0.000 % Neutrophilsc 23 84.12 (73.3–87.2) 18 89.42 (84.2–92.85) 0.002 % Eosinophilsc 23 9.66 (5.19–13.53) 18 4.04 (2.85–6.67) 0.020 % Immature Grns.c 23 8.15 (3.72–9.75) 18 6.06 (3.22–8.48) NS % CD4+ T celld % Naive 80 92.3 (71.4–95.6) 77 90.08 (70.3–95.9) NS % Memory 78 2.77 (1.6–4.6) 77 3.03 (1.8–4.79) NS % Activated 15 0.99 (0.7–2.4) 16 1.48 (0.98–1.92) NS % Effector 80 0.10 (0.03–0.2) 77 0.05 (0.02–0.17) NS % RTEs 23 51.7 (46.5–60.6) 17 64.79 (49.11–69.7) NS % Tregs 29 8.97 (7.25–10.07) 19 6.91 (6.12–7.91) 0.001 % CD4+CD127+ 29 89.2 (88–90.7) 18 90.9 (89.9–92.1) 0.013 % CD8+ T cellsd % Naive 78 88.5 (81.4–93) 77 89.43 (85.61–93.2) NS % Memory 78 1.85 (0.35–4.68) 77 2.32 (0.63–4.2) NS % Activated 15 0.31 (0.23–0.39) 16 0.26 (0.21–0.38) NS % Effector 78 1.06 (0.29–3.12) 77 0.89 (0.24–2.77) NS % RTEs 23 77.06 (70.8–84.8) 17 80.31 (76.01–84.2) NS Values are given as median (25th–75th percentiles). P value ≥ 0.05 by two-sided test was considered nonsignificant. Grns., granulocytes; NK, natural killer; NS, nonsignificant; RTEs, recent thymic emigrants; Tregs, regulatory T cells. aPercentages of cells in the total lymphocyte population. bPercentages of cells in the total of live cells. cPercentages of cells in the total granulocyte population. dPercentages of subpopulations in the CD4+ or CD8+ T cells. Copyright © 2012 International Pediatric Research Foundation, Inc. Volume 71 | Number 5 | May 2012 Pediatric RESEARCH 591 Articles Correa-Rocha et al. total granulocytes was lower, probably due to the significantly more premature the infant, the lower the lymphocyte values.