Preterm neonates are exposed to extrauterine environmental Ags during the time period that corresponds to the last trimester of normal intrauterine development. To study whether this precocious exposure to Ags accelerates the Ig repertoire diversification, we compared IgH chain genes of preterm neonates (gestational age, 25–29 wk) during their first postnatal months with those of term neonates. Preterm infants approaching their expected date of delivery after 8–13 wk of extrauterine life used a similar VH, DH, and JH gene segment repertoire as term neonates born after intrauterine development. Furthermore, the length increase of the NDN region between VH and JH by 0.25 nt per gestational week (r = 0.556, p < 0.0001) was not accelerated. Thus, the generation of the VH region gene repertoire is developmentally controlled and independent of environmental influences. However, exposure to extrauterine Ags induced class switch and somatic mutations in IgH chain genes within 2 wk after premature birth and IgG transcript diversity and mutational frequency increased with the duration of extrauterine life. Three-month-old preterm infants expressed a heterogeneous IgG repertoire at their expected date of delivery with VH region genes carrying significant numbers of somatic mutations with evidence for Ag selection. Term neonates, however, had no such IgG repertoire. We conclude that restrictions in the neonatal Ig VH region gene repertoire persist until term despite exposure to environmental Ags. Yet, many weeks before term the immune system of the preterm neonate can already support germinal center reactions in response to environmental Ags.
The specificity of the B cell Ag receptor is determined by the combined V regions of the Ig H and L chains. The diversity of the V regions allows the recognition of a broad range of antigenic determinants and thus ensures an efficient, specific immune response. In particular, the V region of the IgH chain (VH region), is essential for Ag recognition (1). The VH region is encoded by the VH region gene which is generated during B cell development by the combination of the VH, DH, and JH gene segments. The rearrangement of the gene segments occurs in two steps. First, at the pro-B cell stage, a DH segment is joined with a JH segment. During this recombination, the enzyme TdT is activated and randomly inserts nucleotides (N nucleotides) between the rearranged DH and JH segments. Second, the DH-N-JH rearrangement is joined with a VH gene segment. Again, TdT is active and inserts N-nucleotides during joining (2). Thus, the NDN region is the most diverse part of the rearranged VH gene because it contains the randomly inserted N nucleotides and most of the DH segments can be translated in more than one reading frame (3).
Diversity of the rearranged VH region genes is limited during the early development of the immune system and evolves during ontogeny. In fetal mice, restrictions due to overrepresentation of a few gene segments and lack of N regions persist throughout gestation (4, 5). In human fetal liver lymphocytes, a considerably restricted VH region gene repertoire with overrepresentation of certain gene segments, like VH6–1 and DH7–27, and length restrictions in the NDN region has been described in the first and second trimester of pregnancy (6, 7, 8). We recently demonstrated that the third trimester of gestation is a period of intensive development of the B cell receptor repertoire in peripheral blood, but restrictions nevertheless persist until term (9). Constraints in the VH gene repertoire may be of functional significance because in preterm and term neonates the humoral immune responses to infection or vaccination are weaker and less protective than later in life (10).
Little is known about the effect of early antigenic exposure on the evolution of the VH region gene repertoire (11). In this study, we present a first analysis of the IgG response of extremely premature infants during their first postnatal weeks. To compare premature infants at their expected date of delivery with term neonates offered the unique opportunity to compare human subjects at the same developmental stage, but after different Ag exposure.
Rearranged VH region genes were amplified and sequenced from B cells of term neonates (gestational age, 36–40 wk) and of preterm neonates (gestational age, 25–29 wk) at different time points after birth until they reached term. We found that the exposure to Ag had no influence on the VH, DH, and JH diversity and did not accelerate the NDN length increase. However, the environmental Ags activated the immune system of preterm neonates and induced both class switch and somatic mutations. The complexity of the resulting IgG repertoire correlated with the time of Ag exposure.
Materials and Methods
Cord blood or peripheral blood was collected from three groups: extremely preterm neonates at birth, extremely preterm infants at a postnatal age of 12–14 wk corresponding to their expected date of delivery, and healthy term neonates. The numbers of the neonates and the sequences in each group subdivided for analysis of genomic DNA rearrangements, IgM transcripts, and IgG transcripts are given in Table I⇓. Additional peripheral blood samples were taken from the preterm neonates between 2 and 14 wk after birth (Table II⇓).
All extremely preterm neonates studied were delivered by cesarean section without clinical or laboratory signs of intrauterine infection. During extrauterine life, each of the preterm neonates developed between one and four episodes of bacterial infection. Blood culture-proven sepsis was defined as positive blood cultures combined with clinical and/or laboratory symptoms of infection. Clinically suspected sepsis was defined as clinical signs of infection and elevation of C-reactive protein >15 mg/L, and/or >20% immature granulocytes in the differential blood count. The healthy term neonates were born spontaneously after uncomplicated normal pregnancies with no history or clinical signs of prenatal or perinatal infection. Enteral nutrition with preterm formula or mother’s milk was started on the first day of life. At 3 wk of age, all preterm infants received exclusively enteral nutrition. Gestational age was calculated from the first day of the last menstrual period and confirmed by early ultrasound and by clinical examination. Postconceptional age was calculated as gestational age plus postnatal age. Neonates of the same postconceptional age are at the same developmental stage. The institutional review board had approved the study protocol and written consent of parents was obtained.
DNA extraction and nested primer PCR amplification
DNA was extracted from 0.2 ml of heparinized blood using a commercial kit (QIAamp DNA Blood Mini kit; Qiagen, Hilden, Germany). Briefly, 0.5–1.0 μg of DNA were used for the amplification of the VH region gene with a nested primer PCR procedure previously established by our group (12). Briefly, for the first-round PCR, a mixture of family-specific primers for a conserved region of the framework region (FR)3 1 was combined with a JH consensus primer. For the second PCR, a mixture of family-specific primers for FR2 was combined with a nested consensus JH primer. The amplification products of the second PCR were ∼230–280 nt long. Positive (B cell line Raji) and negative (water) controls were run with each PCR. The Taq error rate measured by repeated sequence reactions of the B cell line Raji was 0.08%. This was similar to previous estimates of the Taq polymerase error rate for the amplification of VH region genes using nested PCR (13).
RNA extraction, RT-PCR amplification
RNA was extracted from 0.2 ml of heparinized blood with a commercial kit (QIAamp RNA Blood Mini kit). RT-PCR was performed with 0.1–0.2 μg of RNA using a 30-cycle Qiagen One-Step RT-PCR kit (Qiagen) with a mixture of family-specific consensus primers for the FR1 (14) and two antisense primers binding to the CH1 domain of the γ (5-CACGTCGCAGATGTAGGTCTGG) or μ (ACGGGGAATTCTCACAGGAGAC) C region gene. The Taq error of the RT-PCR measured by analyzing sequences of the IgH chain constant region was 0.07%.
Cloning and sequencing
Only functional rearrangements, defined as in-frame rearrangements without stop codons, were analyzed using the VBASE directory (15). We first identified VH and JH gene segments. The NDN region length was defined as the number of nucleotides between the last nucleotide matching with the 3′ end of the VH gene segment and the first nucleotide matching with the 5′ end of the JH gene segment.
Assignment of a DH segment to the NDN region required at least six consecutive nucleotides sequence identity with a known germline DH segment or seven nucleotides of sequence identity interrupted by no more than one mismatch and with at least two identical nucleotides both at the 3′ and 5′ ends. We accepted only conventional VHDHJH recombinations without DIR segments, inverted DH segments, or DH-DH recombinations as proposed by Corbett et al. (3). To estimate the diversity of the respective B cell population, the percentage of different VHDHJH rearrangements among all sequences was calculated. The numbers of nucleotide exchanges were determined in CDR2 and FR3 by comparison with the germline sequence of the respective VH gene segment. The frequency of nucleotide exchanges was not corrected for Taq polymerase error.
GeneScan analysis of IgG amplificates
Statistical calculations were done with the SPSS statistical software version 9.0 (SPSS, Chicago, IL). Normally distributed data are presented as mean ± SD and comparisons between groups were made using ANOVA. Data that were not normally distributed are presented as median and range. Correlations between parameters were determined by regression analysis.
The frequencies of rearranged VH, DH, and JH gene segments after extrauterine development were similar to those after intrauterine development
To address the question whether exposure to environmental Ags alters the VH gene repertoire, VH, DH, and JH gene segment usage was determined for lymphocytes isolated from the cord blood of term neonates and from the peripheral blood of premature infants at their expected date of delivery. In both groups rearrangements using VH gene segments of the VH3 family were dominant (Fig. 1⇓A). There was no overrepresentation of VH 6–1, the most DH proximal VH gene segment. Overall, the VH family usage in neonates resembled that seen in adults. Other similarities between the Ag-exposed and nonexposed neonates emerged, when individual gene segments were analyzed: 1) A similar number of different gene segments was used in each group: 30 different VH gene segments in term neonates compared with 33 in preterm infants at their expected date of delivery. 2) The rearranged VH gene segments were taken from across the entire gene locus. 3) VH gene segment usage was nonrandom, i.e., some gene segments were overrepresented, while others were not used at all. DH gene segments could be identified in 81% of functional sequences from preterm infants and in 73% of functional sequences from term neonates. In both groups, all seven DH families were found. Extrauterine development had not reduced the overrepresentation of the DH6 and DH7 family in neonates compared with adults (Fig. 1⇓B). Furthermore, there was a clear bias toward the JH3 gene segment in both groups of neonates. This occurred at the expense of the JH6 gene segment which was used considerably less often in neonates than in adults (Fig. 1⇓C).
In conclusion, exposure to extrauterine Ags during the last trimester of gestation did not shift the frequencies of the expressed gene segments toward a more adult-like distribution.
The increase in NDN region length was not accelerated by extrauterine development
DNA rearrangements from cord blood lymphocytes of preterm and term neonates showed a slow increase in the length of the NDN region by 0.25 nt/wk (r = 0.556; p < 0.001; Fig. 2⇓A). Consequently, NDN regions in cord blood lymphocytes of term neonates were significantly longer than in cord blood lymphocytes of preterm neonates (22 ± 3 vs 19 ± 2 nt; p < 0.001). Within the NDN region the number of nucleotides inserted during joining of DH and JH was most closely related to gestational age (r = 0.693; p < 0.001) and 48% of sequences from preterm neonates (gestational age, 25–29 wk) had no N nucleotides between DH and JH. This percentage decreased to 30% after normal intrauterine development until term.
After extrauterine development, NDN region length of preterm infants at their expected date of delivery (22 ± 2 nt) was the same as in term neonates (Fig. 2⇑B) and the percentage of sequences without N addition between DH and JH (34%) was not reduced. There was no difference in the NDN region length when IgG transcripts were compared with DNA rearrangements (Fig. 2⇑B). In conclusion, the increase in NDN region length until term was not influenced by Ag exposure but rather was developmentally controlled.
The IgM and IgG repertoire in cord blood lymphocytes
To study the IgM and IgG repertoires, RNA was isolated from cord blood. Cord blood lymphocytes of preterm neonates (gestational age, 25–27 wk) expressed a diverse IgM repertoire and 78% of the IgM transcripts were different VHDHJH rearrangements (Table I⇑). In contrast, among 23 IgG sequences isolated from the cord blood of three preterm neonates, only four different VHDHJH rearrangements were found (Table III⇓): in neonate a34a, one VH 5–51 rearrangement was found 3 times, in neonate a42a one VHDHJH rearrangement was isolated 12 times, a second one 6 times, and in neonate a41a, a VH 4–30.4 rearrangement was found 2 times. The repeated finding of identical VHDHJH rearrangements suggests that only a few B cells that switched to IgG were present in the cord blood of preterm neonates.
All IgG transcripts were unmutated and had short NDN regions with little N region diversity (Table III⇑). In particular, N nucleotides were missing between the DH segment and JH gene segment. Sequences without N addition often had short overlapping sequences between DH and JH. For example, from PBL of neonate a42, nine different IgG rearrangements were sequenced (sample a42b). In six of the seven sequences without N addition, an overlap of one to three nucleotides was observed. These characteristics indicate that the IgG transcripts were from neonatal cells and not a contamination with maternal cells.
Similarly, a diverse IgM repertoire, but only a few IgG transcripts were isolated from cord blood lymphocytes of term neonates (gestational age, 36–39 wk). From each of two neonates (c38a and c39a), eight sequences were analyzed and in both cases only two different VHDHJH rearrangements were seen (Table III⇑). From a third neonate (c43a), 35 IgG transcripts were sequenced, but only 13 different VHDHJH rearrangements were found (Table II⇑). In the IgG transcripts of the term neonates, the NDN regions were longer and N insertions between DH and JH occurred more frequently than in the sequences from the preterm neonates. Nevertheless, their overall characteristics were those of a neonatal repertoire.
Taken together, these results demonstrated that a diverse IgM repertoire already existed at the end of the second trimester of gestation. However, there were only a few IgG transcripts in cord blood, suggesting that class switch occurred only sporadically during intrauterine development.
Ag exposure induced class switch
The exposure to extrauterine Ags induced an immune response with class switch in preterm neonates and their IgG repertoire increased rapidly with postnatal age. Whereas in the cord blood of the infant a42 (gestational age, 27 wk), only 2 (11%) among 18 IgG transcripts were different VHDHJH rearrangements, already 15 days after birth (sample a42b) a much more heterogeneous IgG repertoire was detectable with 8 different rearrangements among a total of 13 sequences (61%). When another blood sample (a42 day) of the same baby was analyzed 14 wk after birth, a further increase in the diversity of the IgG repertoire was seen with 23 (92%) different sequences of 25 (Table II⇑).
This rapid increase in the diversity of the IgG repertoire estimated from the percentage of different sequences was confirmed by gene scan analysis of RNA from these three postnatal blood samples of neonate a42. In the cord blood gene scan, there was one single peak. Already in the sample 15 days after birth a oligoclonal length distribution was seen and in the sample 14 wk after birth further diversification was detectable with a polyclonal length spectrum that was normally distributed (Fig. 3⇓). Comparable results were found when the RNA samples of baby a41 were spectratyped (data not shown).
The close relationship between IgG diversification and the duration of exposure to environmental Ags was also confirmed when the percentages of different rearrangement in the postnatal blood samples were plotted against postnatal age: this percentage increased linearly with postnatal age from 35% at birth (gestational age, 27 wk) to 80% at 14 wk of postnatal age, corresponding to a postconceptional age of 40 wk (r = 0.768, p < 0.016; Fig. 4⇓).
Ag exposure induced somatic mutations with evidence of Ag selection
The mutational frequency in VH rearrangements isolated from cord blood lymphocytes of both preterm and term neonates was low (Table I⇑). IgG transcripts in cord blood of term neonates were also practically unmutated, 65% had no nucleotide exchange, 5 sequences had single nucleotide exchanges, and no sequence had >1 exchange. Thus, it is most likely that these substitutions are errors introduced by the Taq polymerase or polymorphisms, rather than somatic mutations.
A different result was found in PBL of preterm neonates after birth. The mutational frequency in IgG transcripts was closely related to the duration of exposure to extrauterine Ags (Fig. 5⇓). It increased from 0% at birth (gestational age, 27 wk) to 0.6–0.9% after 12–14 wk postnatal age, corresponding to a postconceptional age of 39–41 wk (r = 0.897; p = 0.002).
In preterm infants at their expected date of delivery, the mutational frequency in IgG transcripts was clearly higher than in DNA rearrangements or IgM transcripts (0.60 vs 0.27% or 0.30%, respectively; Table I⇑). Fifteen percent of the IgG transcripts had three or more nucleotide exchanges per sequence. Nucleotide exchanges in IgG transcripts of these preterm infants showed the characteristics of Ag-selected somatic mutations (16): they were more frequent in CDR2 (mutational frequency, 0.91%) than in FR3 (mutational frequency, 0.47) and in CDR2 replacement mutations dominated (ratio of replacement:silent mutation, 20:1). In one preterm neonate at term (a34b), five clonally related IgG transcripts with an identical NDN region but a different pattern of three to six somatic mutations were found. Clonal expansion of B cells and Ag-selected somatic mutations are typical results of germinal center reactions.
Comparing the IgH chain gene diversity of preterm neonates exposed to environmental Ags throughout the last trimester of gestation with that of term neonates offered a unique opportunity to study the effects of environmental Ags on the development of the IgH chain gene repertoire in individuals of the same maturational stage. To obtain a representative picture of VH region gene rearrangements without bias from B cells at different activation status, an analysis of genomic DNA was performed. In addition, the expressed mRNA was analyzed to distinguish between the IgM and the IgG repertoire. Otherwise, the small, but relevant subset of IgG-expressing B cells might be overlooked within the predominant IgM-expressing B cell population of neonates (17).
This study presents three novel observations about the evolution of the IgH chain gene repertoire during early human development: 1) Premature exposure to extrauterine environmental Ags did not result in a more adult-like pattern of gene segment usage. 2) During normal intrauterine development class switch occurred only in a few B cells and the observed IgG rearrangements were unmutated. 3) Exposure of preterm neonates to environmental Ags induced class switch and somatic mutations within 2 wk of postnatal life and resulted in a heterogeneous population of IgG-expressing B cells. The pattern of somatic mutations suggested Ag selection.
Restrictions in the neonatal VH, DH, and JH gene segment utilization persisted despite Ag exposure
In neonates, VH gene segments from across the entire locus were used in a frequency pattern similar to the one seen in adults, suggesting that proximity of the VH gene segment to the DH locus seems to play a much more limited role in rearrangement bias in humans than in mice (9, 18, 19). Furthermore, the IgG and the IgM repertoire showed a similar VH gene segment usage. This preponderance of gene locus inherent characteristics over environmental factors on the pattern of VH gene expression is in line with the finding that in mice transgenic for part of the human Ig locus, the VH gene segment usage was similar to that in human adults (20).
Early in gestation proximity between DH and JH gene segments in the genome favors rearrangement of the DH7 family and JH1, JH2, and JH3 (8). With increasing fetal maturity, the importance of proximity decreases but still plays a role in neonates at term who favor the DH6, DH7 families and JH3, JH4, whereas in adults DH2, DH3 families and JH6 are preferably used (9, 21). We found that Ag exposure did not accelerate these developmental trends in the repertoire.
Restrictions in NDN region length persisted despite Ag exposure
The length of the NDN region increased during normal intrauterine development. However, this prolongation was not accelerated by premature exposure to environmental Ags. Instead, the NDN length seemed to be developmentally controlled by a stepwise activation of TdT activity during fetal life.
Sequences without N addition between DH and JH often had short overlapping sequences. Thus, the absence of N regions between DH and JH gene segments decreases diversity not only by the absence of randomly inserted nucleotides, but also because it favors rearrangements between gene segments that share short sequence homologies (22).
The IgM and IgG repertoire during intrauterine development
The analysis of rearrangement length diversity with “spectratyping” demonstrated highly variable IgM transcripts already in fetal liver (23). In support of these spectratyping results, we found that 78–100% of the IgM transcripts and of the DNA rearrangements isolated from cord blood lymphocytes had different NDN region sequences. This indicates that the establishment of a diverse primary repertoire in humans occurs early in gestation independent of exogenous Ags. In agreement with previous results (24, 25, 26), the frequency of somatic mutations in DNA rearrangements and IgM transcripts in cord blood was consistently low and the nucleotide exchanges were randomly distributed.
IgG transcripts could be detected as early as 27 wk of gestation, but the IgG repertoire seemed to be markedly restricted, as most of the isolated IgG transcripts had identical VHDHJH rearrangements. These IgG transcripts were not due to contamination with maternal cells for two reasons. First, they had no N insertions between DH and JH, which is a characteristic feature of the human fetal VH region gene repertoire, and, second, they were unmutated. The diversity of the IgG repertoire remained low throughout normal intrauterine development.
During the third trimester of gestation, peripheral lymph nodes complete the development of their basic primary structure (27). After normal intrauterine development germinal centers are absent in the human fetus (28). Nevertheless, in the cord blood of preterm neonates, IgG sequences were found, although unmutated. Thus, it seems that in human neonates switch recombination may take place before the development of germinal centers. It is known that class switch and hypermutation are independently regulated (29) and that, in KO mice unable to form germinal centers, class switch can occur although at a lower frequency but that these class-switched rearrangements are unmutated (30).
Studies in Ag-deprived environments have previously only been made in animals, but point in the same direction as our findings during intrauterine development. In mice living in a sterile environment and consuming an ultrafiltered low Ag diet (germfree Ag-free mice), IgM levels were nearly normal and the IgM repertoire showed a normal diversity. But levels of circulating IgG and IgA were profoundly depressed and peripheral lymph nodes and mucosal lymphoid tissue were smaller than in conventionally housed animals (31, 32).
In conclusion, in human neonates class switch during intrauterine development is a sporadic event throughout gestation without evidence of Ag-driven or developmentally driven expansion of class-switched cells.
Evolution of class-switched IgG rearrangements during Ag exposure
The population of class-switched B cells rapidly expanded after preterm birth and hence with exposure to extrauterine environmental Ags from nutrition, from the developing gut microflora, and from infectious pathogens. The finding of heterogeneous, somatically mutated IgG sequences as early as 14 days after premature birth suggests that already early in the third trimester of gestation the immune system of the neonate is sufficiently developed to respond to an external antigenic stimulus. The findings of somatic mutations with evidence of Ag selection and of clonally related sequences are hallmarks of the germinal center reaction (33, 34). The knowledge of the development of the IgG memory B cell population in infancy is still incomplete. We observed an increasing heterogeneity of IgG rearrangements within 12 wk but these rearrangements were very different from IgG later in life because they had short NDN regions and a low frequency of somatic mutations (0.68%). Already in early childhood the frequency of somatic mutations has increased to 3.2% (35).
In conclusion, these data show that VH region gene diversity is mainly developmentally regulated. It evolves without stimulation by non-self-Ags until term and repertoire restrictions persist despite premature contact with exogenous Ags. However, premature exposure to Ag accelerates the development of the functional immune system. Preterm infants exposed to environmental Ags develop a polyclonal population of IgG-expressing memory B cells that are not found after normal intrauterine development. Thus, restrictions in the VH region gene repertoire, in particular in the diversity of the CDR3 region rather than functional defects of the B cells, seem to be responsible for the poor immune responses often described for the neonate.
We thank W. Jekabsons, I. Gläser, H. H. Müller, and H. Lammert for the excellent technical assistance.
↵1 This work was supported by Deutsche Forschungsgemeinschaft (Grant BA1187/6-1) and by a scholarship from the Humboldt Foundation (FLF 1071857). The Deutsche Rheuma ForschungsZentrum is supported by the Berlin Senate of Research and Education.
↵2 Address correspondence and reprint requests to Dr. Karl Bauer, Department of Pediatrics, Freie Universität Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail address:
3 Abbreviation used in this paper: FR, framework region.
- Received January 29, 2002.
- Accepted May 21, 2002.
- Copyright © 2002 by The American Association of Immunologists