Development Of Lymphocyte Subsets And Its Clinical Significance In Very Preterm Infants

BackgroundVery preterm infants with gestation age (GA) less than 32 weeks or very low birth weight (VLBW) infants less than 1500g are the most vulnerable population with high mortality and morbidity. High incidence of infectious diseases such as late onset sepsis, necrotizing enterocolitis (NEC), pneumonia is one of the main causes of mortality. Other inflammatory diseases such as bronchopulmonary disease (BPD), severe intra-ventricular hemorrhage (FVH) affect preterm infants more frequently than term infants. These infants are also prone to re-hospitalization in the first year. Allergic diseases such as atopic dermatitis or asthma have higher incidence in very preterm infants than in term infants. These infectious or allergic diseases, which affect their survival rate and life quality, increase financial burden of family and the whole society, indicate the imbalanced immune status of the very premature infants.Immune cells are those immune components executing immunological function, whereas lymphocytes are the primary immune cells including CD3+T cell、 CD3+CD4+T helper (Th) cell、CD3+CD8+cytotoxic or suppressor T cell (Tc or Ts cell), CD 16+CD56+natural killer (NK) cell and CD 19+B cell subsets. T cells account for about 60%, B cells account for 10%～15%, NK cells account for 10%-15% of the total lymphocyte in adult peripheral blood. Compared with adults, the neonates bear much lower level of innate immune cells at birth, and thejr adaptive immunity develops only later in the early years of life, which is one reason of neonates’vulnerability to infection. This is far more severe in the preterm infants, especially those very or extremely preterm infants, because they lack maternal antibodies transferred through placenta, which largely occurs during the third trimester of gestation. Preterm neonates rely heavily on their innate immune system to fight against infection. Several studies have been tried to investigate the innate immune status of lymphocyte subsets in preterm infants and found that preterm neonates have different immune components compared to term neonates and children. Generally speaking, the proportion and absolute number of innate immune cells such as NK cell are deficient in preterm infants compared with term infants. However, detailed information related to immune status at birth and after born in the early life of preterm infants, especially those born very or extremely immaturely has not been fully described. There are several gaps needed to be determined with respect of the lymphocyte subset studies in preterm infants including larger sample size, follow-up study of longer term, association between lymphocyte subsets and perinatal factors or diseases.Here we designed a cohort study, targeting very preterm infants as main subjects, using moderate preterm, term neonates and children less than 6 years as control to observe the development of lymphocyte subsets and their association with perinatal and postnatal factors in very preterm infants. The primary outcomes were absolute count and percentage of lymphocyte subsets including total lymphocyte, T cell, B cell, Th cell Ts cell and NK cell in cord blood or peripheral blood in neonates or children. For the prospective cohort study part, the above subsets were repeatedly checked on the first day,7th day,14th day,28th day and 6th month in very preterm infants. The associations between lymphocyte subsets and perinatal factors or diseases were carried out. Our overall aim was to investigate immune status of lymphocyte subsets at birth and in the first 6 months in very preterm infants and its association with perinatal factors or diseases. Specific objectives were as below.1. Fully describe lymphocyte subsets absolute count and percentage in neonates with different GA at birth and children with different ages, which might be also as the references for neonates, infants and children.2. Investigate the influence of perinatal factors on lymphocyte subsets level at birth and in the first 6 months.3. Investigate association of lymphocyte subsets at birth with late onset infection in very preterm infants.4. Investigate the association of lymphocyte subsets in the first 6 months with late onset sepsis, BPD, severe IVH and cholestasis. We hypothesize that lymphocyte subsets level at birth in very preterm infants are much lower than in moderate preterm, term neonates and children, and might be influenced by a variety of perinatal factors, lymphocyte subsets in very preterm infants develop soon after birth and get higher and higher in the first 6 months, which might be associated with many diseases.Based on fully described lymphocyte subsets status in very preterm infants and its association with influencing factors and diseases, this study will help to understand immunological function in preterm infants and prevent associated diseases.Part 1 Lymphocyte Subsets Status in Preterm Infants at Birth and Its Association with Perinatal FactorsObjective To fully describe lymphocyte subsets status at birth in very preterm infants by comparing the subsets absolute count and percentage levels in neonates with different GA and children with different ages, and to investigate the influence of perinatal factors on lymphocyte subsets level at birth in preterm infants.Methods One hundred and fifty six preterm neonates who had detailed perinatal histories were divided into 3 groups according to their GA:group 23-28w (GA<28w, n=27), group 28-32w(28w^GA<32w, n=68)and group 32-37w(32w≤GA<37w, n=61). Twenty three healthy term neonates with GA of 37-42w,20 healthy infants of 0-1y,17 healthy children of 1-3y and 26 children of 3-6y were as control. Absolute counts and percentages of lymphocyte subsets including CD3+T cell、CD3+CD4+T helper (Th) cell、CD3+CD8+cytotoxic or suppressor T cell (Tc or Ts cell), CD16+ CD56+natural killer (NK) cell and CD 19+B cell were measured in cord or peripheral blood samples in term and pretenn neonates and peripheral blood samples in healthy infants and children, and compared among the above groups. The associations between lymphocyte subsets level at birth in preterm infants with their perinatal factors were carried out by association analysis and linear regression.Results Most lymphocyte subsets (except B cell) absolute counts in preterm infants with different GA were significantly reduced compared with healthy term infants and children (P<0.05). Percentages of lymphocyte and B cell in 23-32w group were higher than in term neonates, whereas percentages of T cell, Th cell and Ts cell were comparable to term neonates. Absolute count and percentage of NK cell in extremely preterm neonates<28w had the lowest level. Absolute counts of all the lymphocyte subsets at birth in all the preterm infants were not significantly different. Except lymphocyte and B cell, percentages of other subsets were not significantly different either in all the preterm infants.Absolute count and percentage of NK cell at birth demonstrated negative correlation with both GA and birth weight, whereas absolute count and percentage of B cell, percentage of lymphocyte showed positive correlation with GA and birth weight. Multivariate linear regression analysis showed negative effects of GA on lymphocyte count and percentage, positive effects of birth weight on T and Th cell percentage and negative on B cell percentage, Male preterm neonates had higher level of Ts cell percentage and B cell count than female preterm neonates. Prenatal steroid increased T cell and Ts cell percentage and reduced NK cell count and percentage. Intrauterine infection increased B cell percentage and decreased T cell percentage. Higher Apgar score at 1 minute was associated with higher B cell count.Conclusion Lymphocyte subsets levels in preterm infants were significantly lower than in term neonates and healthy children, whereas there were no significant difference among preterm neonates with different GA. Lymphocyte subsets levels in preterm infants-were influenced by a variety of perinatal factors including-gender, Apgar score, prenatal steroid and intrauterine infection. Our results indicate that high susceptibility to infection of preterm infants might be ascribed to the combination of many factor besides their reduced innate immune status at birth. Higher percentage of B cell and lower percentage of T cell might suggestive of intrauterine infection. On the other hand, the lymphocyte subsets levels in control group could be as the reference for their corresponding gestational or postnatal age.Part 2 The Association of Lymphocyte Subsets Levels at Birth with Late Onset InfectionObjective Immune status in the early life of preterm infants and its association with late onset infection has not been fully described. The objective of this study was to investigate the association of lymphocyte subsets status at birth with late onset infection in very preterm infants.Methods Eighty four preterm neonates with gestational age less than 32 weeks who survived beyond 7 days were recruited and divided into 2 groups according to presence or absence of late onset infection.Late onset infections comprise culture proven or suspected late onset sepsis, hospital acquired pneumonia or other infectious diseases presenting later than 72h after birth. Absolute counts and percentages of lymphocyte subsets were measured by flow cytometry in umbilical cord or venous blood in the first 3 hours after born.Results Late onset infections were documented in 33 of 84 very preterm infants at the age of 10.5 (7.25-13.0) days during hospitalization. Percentages and absolute counts of NK cell showed significant reduction in infants with late onset infection, as well as both GA and birth weight. In order to control the confounding of GA and birth weight, stratified analysis was done in those preterm infants with GA<30w. Percentages instead of absolute counts of NK cell were significantly reduced in infants with late onset infection compared with none infection group.Conclusion Our results showed that very preterm infants with reduced NK cell percentages at birth had higher incidence of late onset infection, suggesting that measurement of NK cells at birth could be used to identify a group of preterm neonates who might be particularly at risk for infection and maybe suitable for potential immune modulation or prophylactic antibiotics.Part 3 Postnatal Development of Lymphocyte Subsets in Very Preterm Infants and its Clinical SignificanceObjective To observe the postnatal development trend of lymphocyte subsets in very preterm infants in the first 6 months and investigate the influence of perinatal factors on postnatal lymphocyte subsets level and the association of postnatal lymphocyte subsets level with postnatal diseases.Methods This was a prospective cohort study with very pretenn:infants as the study population. Sixty one very preterm infants were recruited who complied with the following inclusion criteria including GA<32w and birth weight<1500g, postnatal age<24 hours at recruiting and hospitalized for at least 28 days at the end of, study. Primary outcomes were absolute count and percentage of lymphocyte subsets on the first,7th,14th,28th day and 6th month. Lymphocyte subsets levels were compared among the above time point and with those in healthy infants and children. The association of postnatal lymphocyte subsets level at each time point with perinatal factors (GA, birth weight, gender, delivery mode, premature rupture of membrane, intrauterine infection, prenatal steroid, ureaplasma urealyticum colonization) and postnatal diseases (late onset sepsis, severe IVH, cholestasis, BPD) were performed.Results All the lymphocyte subset counts were at the lowest level except NK cell and increased significantly in first week, then last steadily for about 7-28 days, rose again and reached the highest level at 6th month comparable to those healthy 0-1 year infants. Lymphocyte subsets counts were decreased after 1 year in healthy children. NK cell count remained in the low level until 6 months. With respect of lymphocyte subset percentages, lymphocyte and B cell showed the trend of increase and reached the highest level at 6th month similar with those healthy 0-l year infants. Percentages of T and Th cell showed trends of decrease after born, the former got to the lowest level similar with those healthy 0-l year infants. Percentages of NK cell in very preterm infants decreased after birth, reached to the lowest level at 1st week and rose hereafter. Children of 3-6 years had the highest level of NK cell percentage.According to GA, the 61 preterm infants were divided into very preterm and extremely preterm infants groups. On the first day, all the subsets count and percentages were not different significantly. While from and after the first week, the increase of T, Th and NK cell counts were slower in extremely preterm infant group than in very preterm infant group and maintained to the 4th week. By contrast, B cell counts were higher in extremely preterm group than in very preterm group. Lymphocyte and T cell percentages were lower at 7th and 14th day in extremely preterm group than in very preterm group as well as lymphocyte subset counts at 28th day. Thereafter, no matter absolute count or percentage, there were no significant different in the two groups.T and Th cell subset percentages were lower in the first month in male preterm infants than in female, which was contrary to B cell. T cell percentages at 6th month was higher in infants with cesarean section than by vaginally born. Prenatal steroid exposure increased all the subset absolute counts at 14th day compared with none exposure. Premature rupture of membrane only affected subsets level in the 1st day. Intrauterine infection had inhibiting effects on T and Th cell levels from day 7 to 28, whereas promoting effects on B cell from day 7 to 14. Ureaplasma urealyticum colonization increased lymphocyte, T, Th,Ts cell counts on the 1st day and B cell counts on day 14.When controlling for confounding factors, there were no significant differences in preterm infants with or without late onset sepsis. T and Th cell percentages on the 14th day were lower in BPD infants than in non-BPD infants, whereas B cell counts and percentages were higher in BPD infants than in non-BPD infants. From day 7 to 28, absolute counts and percentages of T, Th and NK cells were significantly reduced in severe IVH infants compared with control group. T cell counts were lower in infants with cholestasis than controls.Conclusion Very preterm infants showed deficient innate immune cells at birth, however they will develop and expand after born and reach the healthy infant level at about 6th month. Very preterm infants demonstrated different developmental velocity after born, although they had similar lymphocyte subset levels at birth. A variety of preterm birth related perinatal factors affect postnatal lymphocyte subsets in the first month. Postnatal lymphocyte subsets were associated with several diseases. Generally, reduced T, Th and NK cell levels and increased B cell level were associated with adverse events.Part 4 Postnatal Development of Th1,Th2,Th17 Cell in Very Preterm InfantsObjective To observe the postnatal development trend of Thl, Th2 and Th17 cell percentage in very preterm infants in the first 6 months and investigate the influence of perinatal and other clinical factors on their changes.Methods This was a prospective cohort study with very preterm infants as the study population. Sixty one very preterm infants were recruited who complied with the following inclusion criteria including GA<32w and birth weight<1500g, postnatal age<24 hours at recruiting and hospitalized for at least 28 days at the end of study. Primary outcomes were Thl,Th2 and Thl7 cell percentage on the first,7th, 14th,28th day and 6th month. Th1,Th2 and Thl/Th2 ratio were compared with those in reported full term infant cord blood. The association of postnatal Th1,Th2 and Th17 cell percentage level at each time point with perinatal factors (GA, birth weight, gender, delivery mode, premature rupture of membrane, intrauterine infection, prenatal steroid, ureaplasma urealyticum colonization) and postnatal clinical factors (late onset sepsis, eosinophilia, probiotic use) were performed.Results Percentages of Th1,Th2 and Th17 cell were very low after born in very preterm infants.Th17 cell percentage was not increased until the 28th day and decreased again after that, with low level never more than 0.2%. Percentages of Th1,Th2 were not significantly different from full term infants, while Thl/Th2 ratio in the first day was lower than in full term infant cord blood. The percentages of Thl and Th2 cell were not changed in the first week. Thl cell level increased slowly after one week and significantly increased after 4 weeks, reached to the highest level at 6 month. Percentage of Th2 cell on day 14th and 28th were higher than on day 1st, but always less than 1%. Th1/Th2 ratio was about 1.0 in the first month and increased to about 2.5 at 6 month. Most perinatal and postnatal factors had no influence on Th1,Th2 and Th17 cell change. Th17 cell percentage on day 14th in extremely low birth weight infant was higher than in control. Th17 cell percentage on day 14th in very preterm infants with PROM was lower than those without PROM. Th17 cell levels on day 7 and day 14 in very preterm infants with late onset sepsis were higher than in controls.Conclusion Th cell subsets level of Thl,Th2 and Th17 were low in the early life of very preterm infants after born. Thl/Th2 ratio was significantly lower than in full term infant cord blood. Late onset sepsis might be associated with Th17 activation.