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*Nucleotide*Protein
Medline Plus Health Information
*Premature Babies
The Journal of Immunology, 2007, 178: 1180-1188.
Copyright © 2007 by The American Association of Immunologists, Inc.

The Postnatal Maturation of the Immunoglobulin Heavy Chain IgG Repertoire in Human Preterm Neonates Is Slower than in Term Neonates1

Michael Zemlin2,*, Gabriele Hoersch*, Cosima Zemlin{dagger}, Anja Pohl-Schickinger{ddagger}, Michael Hummel§, Claudia Berek, Rolf F. Maier* and Karl Bauer||

* Department of Pediatrics and {dagger} Department of Gynecology, Philipps University Marburg, Marburg, Germany; {ddagger} Department of Pediatrics, Charité Berlin, Campus Virchow, Berlin, Germany; § Department of Pathology, Charité Berlin, Campus Benjamin Franklin, Berlin, Germany; Deutsches Rheumaforschungszentrum, Berlin, Germany; and || Department of Pediatrics, Johann Wolfgang Goethe University, Frankfurt, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During the perinatal period the development of the IgH chain CDR3 (CDR-H3) repertoire of IgM transcripts is maturity-dependent and not influenced by premature exposure to Ag. To study whether maturity-dependent restrictions also predominate in the perinatal IgG repertoire we compared 1000 IgG transcripts from cord blood and venous blood of extremely preterm neonates (24–28 wk of gestation) and of term neonates from birth until early infancy with those of adults. We found the following. First, premature contact with the extrauterine environment induced the premature development of an IgG repertoire. However after preterm birth the diversification of the IgG repertoire was slower than that after term birth. Second, the IgG repertoire of preterm neonates retained immature characteristics such as short CDR-H3 regions and overrepresentation of DH7–27. Third, despite premature exposure to the extrauterine environment, somatic mutation frequency in IgG transcripts of preterm infants remained low until they reached a postconceptional age corresponding to the end of term gestation. Thereafter, somatic mutations accumulated with age at similar rates in preterm and term neonates and reached 30% of the adult level after 6 mo. In conclusion, class switch was inducible already at the beginning of the third trimester of gestation, but the developing IgG repertoire was characterized by similar restrictions as those of the developing IgM repertoire. Those B cells expressing more "mature" H chain sequences were not preferentially selected into the IgG repertoire. Therefore, the postnatal IgG repertoire of preterm infants until the expected date of delivery differs from the postnatal repertoire of term neonates.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the primary B cell repertoire, the diversification of the third CDR of the Ig H chain gene (CDR-H3) is strictly regulated during ontogeny. Early restraints of the fetal IgM repertoire caused by reduced N insertion, biased VH, DH, and JH gene use, and low somatic mutation frequency undergo a slow programmed release during intrauterine and postnatal maturation (1, 2, 3, 4, 5, 6, 7, 8). A recent comparison of nonfunctional and functional CDR-H3 sequences demonstrated differences between the age-related changes in CDR-H3 composition and those changes that occur due to the expression of a functional CDR-H3 (9). In term neonates and adults, functional CDR-H3 sequences were shorter than nonfunctional sequences, whereas in preterm neonates this selection appeared to be inactive (9). With few exceptions, the existing studies focused on IgM transcripts (2, 4, 5, 6, 9, 10), genomic rearrangements of IgM-positive cells (9, 11), or unselected total peripheral blood B cells (12). This raises the question of whether the secondary repertoire of class-switched IgG follows the same age-related schedule as IgM during the perinatal period.

In the cord blood of term neonates only a few IgG transcripts were found (3, 13, 14). In a preliminary analysis including 67 unique IgG transcripts from preterm neonate postnatal blood samples, we found indications that preterm birth triggers the premature class switch to IgG (13). This finding is in agreement with in vitro experiments showing successful stimulation of neonatal B and T cells (15). Therefore, we have now systematically studied the postnatal diversity of IgG transcripts in preterm and term neonates and in adults to test the hypothesis that the age-related changes observed during the diversification of the IgM repertoire are accelerated in the IgG repertoire after the premature transition from the intrauterine to the extrauterine environment.

We found that in preterm neonates the class switch to IgG was induced and a secondary repertoire of IgG transcripts developed prematurely. Thus, at the expected date of delivery preterm infants had a more diverse IgG repertoire than term neonates. Yet, variable region maturation was not accelerated and the number of somatic mutations remained as low during extrauterine development as it did during intrauterine development until the expected date of delivery. The somatic mutation frequency of IgG transcripts was still restricted to approximately one-third of the adult level 6 mo after birth in term and preterm infants.

We speculate that these limitations in the neonatal Ig H chain repertoire may in part be responsible for the characteristically low affinity and increased polyreactivity and autoreactivity of the neonatal IgG repertoire as compared with the adult one (16).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Patients

From both extremely preterm neonates (24–28 wk of gestation) and near term neonates (36–42 wk of gestation) cord blood was collected at birth and venous blood was collected at postnatal ages of 1–28 wk. In addition, venous blood was collected from healthy adults. The numbers of the neonates and the sequences of IgG transcripts in each group are given in Table I.


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  		Table I. Somatic diversity and mutational frequency of IgG transcripts

 
All preterm neonates studied were delivered by caesarian section. After birth, most of the preterm neonates developed between one and four episodes of bacterial infection that were treated with antibiotics for 5 or more days. An infection was defined as either positive blood cultures combined with clinical and/or laboratory signs of infection or as clinical signs of infection and elevation of C-reactive protein >15 mg/L and/or >20% immature granulocytes in the differential blood count. Antibiotics were not given prophylactically but only when clinical and/or laboratory signs of infection were present. The term neonates were born spontaneously. Because most of the preterm neonates developed one or more infection during postnatal life, we included term neonates with and without a known history of infection in the study. Enteral nutrition with preterm formula or mother’s milk was started on the first day of life. 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. The Institutional Review Boards of University of Berlin and University Marburg approved the study protocol and the written consent of parents and adult donors was obtained.

RNA extraction and RT-PCR amplification

RNA was extracted from 0.2 ml of heparinized blood with a commercial kit (QIAamp RNA blood mini kit; Qiagen). RT-PCR was performed with 0.1–0.2 µg RNA using a 30-cycle, one-step RT-PCR kit (Qiagen) with a mixture of family-specific consensus primers for framework region (FR)3 1 (14) and two antisense primers binding to the CH1 domain of the {gamma} (5'-CACGTCGCAGATGTAGGTCTGG-3') C-region gene, as previously described (13). The Taq error of this RT-PCR protocol, measured by analyzing the sequences of the IgH chain constant region that was part of the sequences analyzed in this study, was 0.065% and thus similar to other published protocols (17).

Cloning and sequencing

PCR products were separated by PAGE, eluted from the gel, and cloned using the TOPO TA cloning kit (Invitrogen Life Technologies). After the transformed cells had grown on agar plates, 20–35 clones from each subject were randomly selected. The plasmid DNA was isolated, linearized, and sequenced on an ABI 377A automated sequencer (Applied Biosystems). A total of 1000 sequences was analyzed in this study, including 165 sequences that were previously reported (13). GenBank accession numbers are listed in Table I.

Spectratyping

Spectratyping was performed as previously described (13). Briefly, RNA was used for RT-PCR using a mixture of family-specific FR3 primers in conjunction with a FAM-labeled primer for the IgG constant region (Qiagen one-step RT-PCR kit). Amplificates were separated on a sequencing gel (ABI 310C; Applied Biosystems) and analyzed with the GeneScan software (ABI 672, version 3.1). We have previously shown that randomly chosen sequences were representative in length and clonal diversity of the total amplificate by comparing spectra types (GeneScan) and sequencing results (13). In the present study, 23 of the samples were studied by GeneScan, confirming diversity and length distribution of the sequencing results in each of the samples studied. This is in harmony with reports that analysis of VH gene repertoires by bulk PCR and random sequencing is similarly quantitative to single cell PCR (17, 18).

Sequence analysis

Only functional rearrangements, defined as in-frame rearrangements without stop codons, were analyzed using the VQUEST software (http://imgt.cines.fr/textes/vquest/) (19) and IgBlast (http://www.ncbi.nlm.nih.gov/igblast/) (20). The International Immunogenetics Information System (IMGT) numbering and definitions of CDRs and FRs were always applied (19). The N(D)N region length was defined as the number of nucleotides between the last nucleotide matching the 3'-end of the VH gene segment and the first nucleotide matching with the 5'-end of the JH gene segment. For DH gene assignment, a minimum of six consecutive nucleotides having sequence identity with a germline DH segment or seven matches interrupted by no more than one mismatch and at least two identical nucleotides both at the 3' and 5' ends was required. We accepted only conventional VHDHJH recombinations without DIR segments, inverted DH segments, or DH-DH recombinations as proposed by Corbett et al. (21).

To estimate the diversity of the B cell population, the percentage of unique VHDHJH junctions among all functional sequences (including duplicates) was calculated. Unique H chain sequences were defined as sequences that had at least one nucleotide difference to any other sequence analyzed. Because each unique sequence is likely to represent one individual B lymphocyte, the unique sequences were used for the statistical analysis of gene segment use, CDR-H3 composition, and somatic mutation frequency. Sequences with identical CDR-H3 regions but differing nucleotide exchanges within their VH gene segments were regarded as clonally related sequences.

The numbers of nucleotide exchanges were determined in CDR-H2 and FR-H3 by comparison with the germline sequence of the most homologous VH gene segment. If more than one nucleotide exchange occurred within one codon, each exchange was counted as one separate event. The frequency of nucleotide exchanges was not corrected for Taq polymerase error.

Statistical analysis

Statistical calculations were done with the SPSS statistical software version 12.0 and with Microsoft Excel. Normally distributed data like N(D)N lengths 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. Frequency of VH, DH, and JH gene segment use was compared as appropriate using the Chi (2) test or two- tailed Fisher exact test if one of the expected frequencies was 5 or less.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
To compare the age-related and environmental influences on the postnatal development of the IgG repertoire in preterm and term neonates, we analyzed a total of 1000 IgG transcripts (Table I). Of these, 688 (69%) had different CDR-H3 regions, including 19 IgG transcripts that were derived from extremely preterm neonate cord blood, 185 from preterm neonate postnatal blood samples, 45 from term cord blood, 358 from term neonate postnatal samples, and 81 from adult venous blood.

Premature exposure to the extrauterine environment induced the premature development of an IgG repertoire

To address the question of whether exposure to environmental Ags induces a diversification of the IgG repertoire, we calculated the sequence diversity of an individual blood sample as the percentage of sequences with different CDR-H3 regions of all functional sequences from this sample, a method used already in our previous article (13) (Fig. 1). In a number of samples we simultaneously performed an analysis of the CDR-H3 length distribution (spectratyping). Samples with a low percentage of different CDR-H3 regions always had oligoclonal spectratypes. Thus both methods confirmed a low diversity in such a sample. Samples with a high percentage of different CDR-H3 regions had polyclonal spectratypes (Fig. 2).


Figure 1
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  		FIGURE 1. Sequence diversity of IgG transcripts from preterm and term neonates vs postconceptional age. Sequence diversity of each blood sample (see Table I) was defined as the percentage of different VHDHJH junctions among all sequences analyzed from one blood sample. Diversity in preterm infants increased with postconceptional age between 24 and 41 wk (r = 0.799, p = 0.005) and reached a median diversity of 85% near term. In term infants, diversity reached 80–00% 1 wk after birth. Horizontal bars represent the median for preterm infant cord blood at the expected date of delivery (postconceptional ages 36–41 wk), and the broken bar represents the median for term infant cord blood (postconceptional age 36–41 wk). Symbols connected by dotted lines represent repeated blood samples taken from the same individual.

 

Figure 2
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  		FIGURE 2. Spectratype analyses from serial blood samples. Shown are serial spectratyping results from the following: a preterm neonate (gestational age 27 wk) at birth at postnatal ages of 2 and 14 wk (A); a preterm neonate (gestational age 27 wk) at birth and at postnatal ages of 3 and 10 wk (B); a preterm neonate (gestational age 25 wk) at birth and at a postnatal age of 10 wk (C); and cord blood from a term neonate (gestational age 39 wk) and peripheral blood from another term neonate at a postnatal age of 7 wk (D). Spectratypes indicated a postnatal diversification of the IgG transcripts and were in agreement with the estimated sequence diversity, defined as the percentage of sequences with differing VHDHJH junctions of all sequences analyzed within the blood sample and given on the right side of each plot under the postnatal age of blood sampling (see Table I). Peaks of the size standard occur at 160, 200, and 240 nucleotides. The spectratypes from patient 1040 have been previously shown (13 ).

 
In preterm neonates born at 24–28 wk postconception, IgG diversity in cord blood was low (median 33% with different CDR-H3 regions; range 11–50%). After preterm birth, IgG diversity increased slowly during the time period corresponding to the last trimester of gestation (r = 0.799; p = 0.005) (Fig. 1). After 10–14 wk of exposure to the extrauterine environment when preterm infants reached their expected date of delivery (postconceptional ages of 36–41 wk), the diversity of the IgG repertoire was greater than in cord blood B cells of term neonates, who were at the same developmental stage (postconceptional age 36–41 wk) but had no previous exposure to the extrauterine environment (median 92%, range 73–100% vs median 32%, range 16–100%, p = 0.042; Mann-Whitney U test) (Table II).


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  		Table II. Development of the IgG repertoire during ontogeny

 
Yet, after term birth, the diversity of the IgG repertoire increased almost instantaneously and was >80% already after 1 wk of postnatal life (Fig. 1). Thus, at a postconceptional age of >41 wk the IgG diversity was comparable in preterm and term neonates. In adults, IgG diversity ranged between 40 and 95% (median 90%).

In six preterm neonates we longitudinally followed IgG diversity by taking repeated blood samples at different postconceptional ages. In all six preterm neonates the IgG diversity increased with postconceptional age and thus supported the results of the cross-sectional analysis (Fig. 1).

The IgG repertoire of preterm infants retained characteristics of immature variable regions like short N(D)N regions and overrepresentation of DH7–27

In preterm neonates, the N(D)N length increased during the time period corresponding to the third trimester of gestation by 0.27 nucleotides per week (r2 = 0.23; p < 0.001). At the expected date of delivery, the N(D)N region in preterm neonates was shorter than in cord blood of term neonates (22.3 ± 1.16 (mean ± SEM) vs 27.2 ± 1.14; p < 0.01 per two-sided t test; Table II). Thus, premature birth did not accelerate but rather delayed the N(D)N length increase compared with intrauterine development. At a postconceptional age of >40 wk the N(D)N length was similar in preterm and term neonates, had reached adult N(D)N length, and showed no further increase during the postnatal period studied (Fig. 3).


Figure 3
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  		FIGURE 3. N(D)N length of IgG transcripts from preterm and term neonates. Each data point represents the mean of one blood sample (see Table I). N(D)N length in preterm infants increased with postconceptional age between 24 and 41 wk (r = 0.39, p = 0.01) and remained within similar ranges in preterm and term neonates after 41 wk. Horizontal bars represent the mean for preterm infant cord blood at the expected date of delivery (postconceptional ages 36–41 wk), and the broken bar represents the mean for term infants cord blood (postconceptional ages 36–41 wk).

 
The frequency of the DH7–27, the most JH proximal DH gene segment, undergoes greater changes during ontogeny than any other VH, DH, or JH gene segment in humans and in mice (reviewed in Ref. 22). We found that DH7–27 was more frequently used in IgG transcripts from preterm (13 of 29 sequences with identifiable DH gene) than in term neonate cord blood B cells (1 of 60 sequences, p = 5 x 10–5; Fisher exact test vs preterm cord blood) or adults (1 of 91 sequences, p = 9 x 10–9; Fisher exact test) (Table II). In preterm neonates, DH7–27 use in IgG transcripts decreased during the time period corresponding to the third trimester of gestation as previously reported for IgM transcripts (11). At their expected date of delivery the frequency of DH7–27 was still higher in preterm neonates (11%) than in term neonate cord blood (2%, p = 0.038; two-tailed Fisher exact test) (Table II).

Use of the other VH, DH, and JH genes in IgG transcripts was similar to the one previously reported for DNA rearrangements (12) and IgM transcripts from peripheral blood (4, 9, 13).

Briefly, in comparison to the frequency expected from the number of germline genes, the DH3, VH3, and VH4 gene families and the JH4 and JH6 genes were over-represented, whereas the VH2, DH4, and DH1 families and the JH1 and JH5 genes were underrepresented in all groups of IgG transcripts studied (not shown).

Despite premature exposure to the extrauterine environment, somatic mutation frequency in IgG transcripts increased only when preterm infants passed a postconceptional age corresponding to the end of term gestation

The somatic mutation frequency within CDR-H2 and FR-H3 (number of mismatches to the most homologous VH gene segment per 100 nucleotides) was between 0.0 and 1.1% in preterm cord blood IgG sequences (Table II and Fig. 4). The somatic mutation frequencies in IgG sequences from preterm and term cord blood B cells were ~10 times higher than the Taq error rate (0.065%). Thus, somatic mutations are truly present in our sequences but are probably slightly overestimated.


Figure 4
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  		FIGURE 4. Average somatic mutation frequency of IgG transcripts from preterm and term neonates. The somatic mutation frequency of each blood sample (see Table I), defined as the number of somatic mutations per 100 nucleotides of CDR2 and FR3, was plotted against postconceptional age (weeks). Somatic mutation frequency in preterm infants between the postconceptional ages of 25 and 41 wk remained unchanged despite Ag exposure. Beyond the end of term gestation, the somatic mutation frequency increased with postconceptional age both in preterm (r = 0.785, p = 0.031) and term neonates (r = 0.787, p < 0.001). Horizontal bars represent the median for preterm infants cord blood at the expected date of delivery (postconceptional ages 36–41 wk), and the broken bar represents the median for term infant cord blood (postconceptional ages 36–41 wk).

 
Until preterm neonates reached their expected date of delivery there was no discernible increase in somatic mutation frequency (Fig. 4). Although the exposure of preterm neonates to environmental Ags induced class switch in B cells, the somatic mutation frequency was not higher than in IgG transcripts isolated from the cord blood B cells of term neonates (median of 0.67% and range of 0.0–0.81% vs median of 0.22% and range of 0.0–0.60%, p > 0.05; Mann-Whitney U test, preterm vs term neonates both at postconceptional ages of 36–41 wk). Thus, the extrauterine environment did not induce significantly more somatic mutations in IgG transcripts than intrauterine development.

Yet, beyond term, somatic mutation frequency increased with postconceptional age in preterm neonates (r = 0.785; p = 0.031) and in term neonates (r = 0.787; p > 0.001) at comparable frequencies of 0.09 and 0.06% per week so that after a postconceptional age of 50 wk the somatic mutation frequency in IgG sequences had increased in both groups to a range between 1.0 and 3.1%, thus remaining markedly below that of adults, who had a somatic mutation frequency ranging from 5.8 to 9.1% (median 6.8%).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we analyze the development of the IgG repertoire in preterm and term neonates during a postconceptional age of 24 to 68 wk, i.e., during the time period when neonatal immunocompetence gradually increases from the "immunodeficiency of extreme prematurity" to the ability to produce Abs against various groups of Ags (23, 24). By comparing preterm and term neonates at an identical postconceptional age but after different environmental exposures, we could discern changes that are triggered by preterm birth from changes that occur during physiologic intrauterine development. The main results of this study are that premature transition from the intrauterine to the extrauterine environment induced the premature development of an IgG repertoire. The variable regions of this IgG repertoire retained immature characteristics like short CDR-H3 regions and overrepresentation of DH7–27, indicating that there was no preferential selection of sequences with "mature" V regions for class switch from the primary IgM-expressing B cell repertoire. Somatic mutation frequency in IgG transcripts of preterm infants increased only when they had passed a postconceptional age corresponding to the end of term gestation. In conclusion, a secondary IgG repertoire was prematurely induced after preterm birth but was subject to similar age-related restrictions as those of the primary IgM repertoire.

During normal intrauterine development, class switch is a rare event (3, 25). Consequently, we found a low diversity of IgG transcripts in the cord blood of preterm and of term neonates. Also, other researchers using flow cytometry and quantitative PCR could detect only a few surface IgG-positive B cells and IgG transcripts in term cord blood (26). There are three possible explanations for these findings. The first is low exposure to nonself Ags. During the third trimester of intrauterine development the fetus is not completely excluded from environmental Ags, but the concentrations that reach the fetus via the swallowing of amniotic fluid or via the placenta are low (27). It is well known that the absence of antigenic stimuli arrests the development of the humoral immune system. Animals living in a germfree environment and on ultrafiltrated, sterile food show no diversification of the Ig VDJ genes (28). Yet, the human fetus can respond to environmental Ags (29, 30) and maternal Ags can be tolerated (31). The second possible explanation is that the cells and the microenvironment participating in the adaptive immune response are functionally immature until term and cannot sustain an immune reaction (32). The third explanation is that maternal factors (IgG and others) modify the fetal immune reactions during intrauterine development (33). During the third trimester of pregnancy, increasing amounts of maternal IgG Abs are actively transferred to the fetus (25, 34). Maternal IgG represents the mother’s immunologic memory and can imprint the immune system of the offspring (33, 34). The relative importance of these three possible mechanisms is still an open issue.

After birth the exposure to environmental Ags (nonpathogenic microbiota, dietary Ags, and pathogenic microbiota) increases dramatically and begins modulating the neonatal immune system (35, 36, 37, 38, 39). After preterm birth the exposure to environmental Ags occurs much earlier than after term birth, yet the nature of the environmental Ags is similar to those that the term neonate encounters after birth (35, 40). The preterm infants we studied received no antibiotics on 83% of the days between birth and the expected date of delivery, allowing bacterial colonization of their gut by a diverse bacterial flora (41). Skin-to-skin contact with parents was encouraged from the first postnatal week in the preterm infants studied here, allowing bacterial colonization of the neonate’s skin by parental skin commensals. The type of enteral nutrition (breast-milk or cow-milk based formula) was similar between preterm and term neonates. Enteral nutrition was started in preterm and term infants on day 1 of life and by wk 2 to 3 of life all were exclusively fed enterally.

Most preterm neonates had at least one infection during the observation period. Yet, compared with the continuous stimulation by nonpathogenic microbiota or dietary Ags, an episode of infection is a rare and time-limited antigenic exposure. To analyze the influence of infections on the IgG repertoire, we have included eight term infants with a known history of infection (Table I). Postnatal samples of term infants with and without known history of infection did not differ in sequence diversity, somatic mutational frequency, use of the VH, DH, JH gene families, and all other variables tested (Table I and additional data not shown).

We demonstrated class-switched sequences in early postnatal samples of preterm infants in a previous study (13). In the present study we expand this observation by including a much larger number of sequences and individuals to describe the postnatal evolution of IgG diversity from extreme prematurity until early infancy and compare the postnatal kinetics of class switch after premature birth and after term birth. We estimated the diversity of class-switched cells by calculating the diversity of the sequences among each blood sample and by spectratype (GeneScan) analysis. Both analyses consistently demonstrated that premature contact to extrauterine Ag induced class switch during the last trimester of gestation, but the diversity of IgG transcripts increased considerably slower in preterm neonates than in term neonates during the first 8 postnatal weeks (Table I and Fig. 1). In vitro, when appropriate T cell help and an appropriate cytokine milieu are artificially provided, cord blood B cells of preterm infants are able to undergo class switch more efficiently than they apparently do in vivo (23, 42).

We found that the IgG repertoire of the premature neonates was characterized by short CDR-H3 regions and frequent DH7–27 use, thereby retaining the characteristics of the immature repertoire found in whole blood DNA extracts (12) and IgM transcripts (4, 9, 13). Thus class switch to IgG did not favor more "mature" VH regions, but instead class-switched sequences were randomly chosen from the immature primary IgM repertoire as for length and DH gene use. These immature structural characteristics might contribute to the well-known polyreactivity, low affinity, and increased autoreactivity of neonatal IgG (16).

The VH gene segment and JH gene segment contribute nucleotides to CDR-H3, but CDR-H3 length is mainly regulated by the length of its core region that is encoded by one of the diversity (D) gene segments and by N nucleotides (N(D)N region) (9, 12, 13, 43, 44, 45, 46, 47). We found that in IgG transcripts there was a slow increase of N(D)N-length during the third trimester of gestation that was not accelerated by premature exposure to the extrauterine environment. At term, the lengths of N(D)N regions were similar to those of adults. N(D)N length increase was predominantly caused by an elongation of DH-JH junctions and less by VH-DH junctions, which apparently underlie differing regulatory mechanisms (48) (data not shown). Thus, the N(D)N-length in IgG transcripts showed very similar kinetics as previously described for IgM transcripts that increased in length during late gestation (13) and during early infancy (4). The findings that human tonsil germinal center IgM and IgG transcripts did not differ in CDR-H3 length, nor did living (annexin V–) vs apoptotic (annexin V+) B cells (49), support the hypothesis that CDR-H3 length is not the primary criterion for the selection of cells during class switch. Yet, the significance of CDR-H3 length on B cell selection is not entirely clear, because functional VHDHJH junctions had shorter CDR-H3 regions than nonfunctional VHDHJH junctions (50, 51), and highly mutated sequences had shorter CDR-H3 regions than nonmutated transcripts (52).

The overrepresentation of DH7–27 in IgG transcripts was another feature of fetal/neonatal IgM transcripts that we rediscovered in class-switched IgG sequences. Premature birth did not accelerate but rather delayed the decrease of the DH7–27 frequency and the increase of N(D)N length in IgG transcripts until term in preterm neonates. This may result from a delayed immunologic maturation and an inability to produce a "mature" H chain repertoire, but somatic selection may also create a bias toward "immature" IgG heavy chains in preterm neonates. The DH segment is often in direct contact with the Ag (44, 45, 46, 53, 54). DH7–27 is the most J-proximal and shortest germline DH gene and, unlike all other DH genes, it does not encode the hydrophilic amino acids serine and glycine. Experimental modification of CDR-H3 amino acid composition alters B cell development and function (55), though the exact functional consequence of the preferential use of DH7–27 early in ontogeny remains to be elucidated.

Somatic mutation is a key event in the humoral immune response and generates high affinity Abs (56). In neonates after normal intrauterine development, somatic mutations are minimal and generally do not show characteristics of selection (Ref. 57 and data not shown). Germinal centers can first be distinguished 1 mo after birth (58, 59, 60). Thus, the low somatic mutation frequency in neonates could represent a spontaneous baseline mutational activity that is independent of exogeneous Ag and potentially, at least in part, independent of germinal centers (58, 60). Nevertheless somatic mutations can be induced in neonates as demonstrated by vaccination experiments in mice under experimental conditions that maximize stimulation of APCs, T cells, and B cells (42).

In the present expanded data set, which spans a wider range of postnatal ages, the previously observed trend of an increasing somatic mutation frequency during the early postnatal weeks in preterm infants (13) was less pronounced. Instead, somatic mutation frequency remained low for 3 to 4 mo until the expected date of delivery and then started to increase by ~1 mutation per 100 nucleotides per week in preterm as well as term neonates. At the end of the observation period, somatic mutation frequency was still lower than in adults. In unselected peripheral blood B cells, adult levels of somatic mutations were reported at 8 mo of age (10). Interestingly, somatic mutation frequency in our adult IgG transcripts was within a similar range as in human tonsil plasma cell IgA transcripts (61). The enzyme AID catalyzes the introduction of somatic mutations during each cell division at an estimated rate of one mutation per 1000 bp (62). Thus, neonatal peripheral blood B cells may have undergone fewer cell divisions, the AID activity may be reduced, or somatic selection may prefer unmutated sequences in preterm neonates.

The dissociation of class switch, which could be demonstrated shortly after preterm birth, and somatic mutations, which were reduced until infancy, resulted in a population of class-switched unmutated sequences throughout infancy.

The independent regulation of class switch and somatic mutation has been shown in various in vitro systems (63). In an in vitro germinal center model, class switch or somatic mutations could be induced independently in a monoclonal IgM+IgD+ B cell line by exposure to the appropriate cytokines (64). In naive B cells (CD27) from human cord blood, class switch to IgG but not noticeable hypermutation could be induced by B cell receptor engagement and CD40 signaling in the presence of IL-2 and IL-10 (65). Evidence from lymphotoxin-{alpha} knockout mice (66) indicates that class switch can proceed in the absence of germinal center formation.

In summary, premature birth can be regarded as an accidental "experiment of nature" in which human individuals live through the same period of development under markedly different external conditions. The differences between preterm and term neonates we observed are consequences of the premature transition from the intrauterine to the extrauterine environment. Yet, in our observational study we cannot define the specific role of individual mechanisms because in this human system we could not control for individual variables. Similarities in IgG repertoires between preterm and term infants, like the restriction in somatic mutation frequency until term, point to intrinsic immaturities of the immune system that were not modified by the premature transition from the intrauterine to the extrauterine environment. These intrinsic immaturities can act on various levels of the B cell activation, which may include decreased reactivity of APCs, reduced T cell help, or ineffective cell-to-cell communication during the immune reaction. Dissimilarities in the IgG repertoires between preterm and term neonates, like the increased IgG diversity in preterm infants at term, point toward an influence of environmental factors. These may include the selection of B cells by Ags that are encountered only outside the uterus or the absence of factors, like maternal IgG, that are only encountered during a normal intrauterine development until term.

We speculate that, in analogy to murine models where alterations of the fetal Ab repertoire can have life-long consequences for the immunological function (34, 67), the very immature preterm neonate might also offer the opportunity to analyze the influence of premature exposure to environmental Ag on the long-term development of the immune system and immunological diseases such as allergies and autoimmune diseases in humans.


   Acknowledgments
 
We thank Rodica Altmann, Hans-Henning Müller, Sabine Jennemann, and Regina Stoehr for the excellent technical assistance and Hans Versmold for valuable support and advice during the early stages of this work.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Deutsche Forschungsgemeinschaft Grants BA1187/6-1 (to K.B. and M.Z.), SFB/TR22, TPA17 (to M.Z.), and the Humboldt Foundation Fellowship Grant FLF1071857 (to M.Z.). The Deutsches Rheumaforschungszentrum is supported by the Berlin Senate of Research and Education. Back

2 Address correspondence and reprint requests to Dr. Michael Zemlin, Department of Pediatrics, Philipps University Marburg, Baldinger Street, 35033 Marburg, Germany. E-mail address: zemlin{at}staff.uni-marburg.de Back

3 Abbreviation used in this paper: FR, framework region. Back

Received for publication July 14, 2006. Accepted for publication November 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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