The diversity of rearranged immunoglobulin heavy chain variable region genes in peripheral blood B cells of preterm infants is restricted by short third complementarity-determining regions but not by limited gene segment usage

Abstract

The immunoglobulin diversity is restricted in fetal liver B cells. This study examined whether peripheral blood B cells of extremely preterm infants show similar restrictions (overrepresentation of some gene segments, short third complementarity-determining regions [CDR3]). DNA of rearranged immunoglobulin heavy chain genes was amplified by polymerase chain reaction, cloned, and sequenced. A total of 417 sequences were analyzed from 6 preterm infants (25-28 weeks of gestation), 6 term infants, and 6 adults. Gene segments from the entire V H and D H gene locus were rearranged in preterm infants, even though the D H 7-27 segment was overrepresented (17% of rearrangements) compared to term infants (7%) and adults (2%). CDR3 was shorter in preterm infants (40 ؎ 10 nucleotides) than in term infants (44 ؎ 12) and adults (48 ؎ 14) (P < .001) due to shorter N regions. Somatic mutations were exclusively found in term neonates and adults (mutational frequency 0.8% and 1.8%). We conclude that preterm infants have no limitations in gene segment usage, whereas the diversity of CDR3 is restricted throughout gestation. ( IntroductionThe huge variety of immunoglobulin specificities is generated during B-cell development by rearrangements of the variable (V H ), diversity (D H ), and joining (J H ) gene segments (combinatorial diversity), by insertion and deletion of random nucleotides during joining (junctional diversity), 1 and by the introduction of somatic mutations. 2 In B cells of fetal liver the diversity of rearranged immunoglobulin heavy (IgH) chain variable region genes is restricted by a marked overrepresentation of some V H and D H gene segments 3 and by short CDR3. 4 It is unknown whether these restrictions are limited to the immature B cells of the fetal liver 5 or are also present in peripheral B cells, and if restrictions persist only during the first trimester of pregnancy 6 or until early infancy. 4 To test the hypothesis that marked restrictions in immunoglobulin diversity persist in preterm infants and throughout gestation, we studied rearranged IgH chain variable region genes of peripheral blood B cells from extremely preterm infants, term infants, and adults. Study design PatientsWe collected cord blood of 6 preterm infants (25-28 weeks of gestation, birth weight 470-1120 g), 6 term infants (39-42 weeks), and peripheral blood of 6 adults, aged 26 to 43 years. Infections were ruled out in all individuals (normal clinical examination, blood count, and C-reactive protein). The study protocol had been approved by the institutional review board and written consent was obtained. Amplification and sequencing of VDJ rearrangementsDNA (0.5-1.0 g) extracted from heparinized blood samples was used for the polymerase chain reaction (PCR) amplification of the rearranged IgH chain variable region with a nested primer PCR previously established by our group. 7,8 For the first amplification a mixture of family-specific primers for framework region 1 was used in conjunction with a consensus J H primer; for the reamplificat...

Relatively Normal Human Lymphopoiesis but Rapid Turnover of Newly Formed B Cells in Transplanted Nonobese Diabetic/SCID Mice

“…These observations suggest that the lymphocyte population in these chimeric mice is in a dynamic state, with cells being selected at certain points. 3) HCDR3 lengths as well as the presence of N nucleotides are similar to those described for human cord blood and term infants (40,42,43). This shows that the TdT expressed by lymphocyte precursors was functional in the murine environment.…”

“…The J H 4 segment is most commonly used by normal fetal (40) and cord blood B cells (41), as was the case for 58% of the lymphocytes analyzed from transplanted mice. The average of the mean HCDR3 lengths for the population was 15.2 Ϯ 0.7 codons and falls within limits previously described for cord blood and term infants (42,43). Each junction represented a unique sequence and there was clear evidence for N nucleotide insertion, i.e., TdT activity.…”

Diversification of Ig Heavy Chain Genes in Human Preterm Neonates Prematurely Exposed to Environmental Antigens

“…With increasing fetal maturity, the importance of proximity decreases but still plays a role in neonates at term who favor the D H 6, D H 7 families and J H 3, J H 4, whereas in adults D H 2, D H 3 families and J H 6 are preferably used (9,21). We found that Ag exposure did not accelerate these developmental trends in the repertoire.…”

“…In neonates, V H 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 V H gene segment to the D H 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 V H gene segment usage.…”

“…In human fetal liver lymphocytes, a considerably restricted V H region gene repertoire with overrepresentation of certain gene segments, like V H 6 -1 and D H 7-27, and length restrictions in the NDN region has been described in the first and second trimester of pregnancy (6 -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 V H 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).…”