No abstract

Tyrosine and glycine constitute 40% of complementarity determining region 3 of the immunoglobulin heavy chain (CDR-H3), the center of the classic antigen-binding site. To assess the role of DH RF1-encoded tyrosine and glycine in regulating CDR-H3 content and potentially influencing B cell function, we created mice limited to a single DH encoding asparagine, histidine, and arginines in RF1. Tyrosine and glycine content in CDR-H3 was halved. Bone marrow and spleen mature B cell and peritoneal cavity B-1 cell numbers were also halved, whereas marginal zone B cell numbers increased. Serum immunoglobulin G subclass levels and antibody titers to T-dependent and T-independent antigens all declined. Thus, violation of the conserved preference for tyrosine and glycine in DH RF1 alters CDR-H3 content and impairs B cell development and antibody production.

Heterosubtypic immunity (HSI) is defined as cross-protection to infection with an influenza A virus serotype other than the one used for primary infection. Although HSI has been thought to be mediated by serotype cross-reactive cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural proteins, recent studies suggest that antibodies (Abs) may make a significant contribution. In this study, we provide further evidence for the role of Abs in HSI using transgenic mice lacking terminal deoxyribonucleotidyltransferase (TdT), which adds N nucleotides to V-D and D-J junctions of the complementary determining region 3 (CDR3) (TdT ؊/؊ ) and mice with altered Ab repertoires due to replacement of the complete locus of heavy chain diversity segments (D H ) with an altered D H segment (namely, ⌬D-iD). Both types of mice failed to generate complete HSI, although they were able to mount protective immunity to a homologous challenge. Lower levels of virus-specific antibodies along with more severely impaired HSI were observed in TdT ؊/؊ mice compared to those in ⌬D-iD mice, while CTL activity remained unchanged in both types of mice. These findings indicate that a properly diversified antibody repertoire is required for HSI and that N addition by TdT is a more effective mechanism in the induction of a properly diversified antibody repertoire and, therefore, complete HSI. The results suggest that the diversity of the antibody repertoire as determined by the composition of the D region of HCDR3 and by N addition are among the mechanisms selected for in evolution to create a favorable environment to resolve infections with mutated viruses.

In jawed vertebrates most expressed Ig H chains use only one of six possible DH reading frames. Reading frame (RF)1, the preferred reading frame, tends to encode tyrosine and glycine, whereas the other five RFs tend to be enriched for either hydrophobic or charged amino acids. Mechanisms proposed to favor use of RF1 include a preference for deletion over inversion that discourages use of inverted RF1, RF2, and RF3; sequence homology between the 5′ terminus of the JH and the 3′ terminus of the DH that promotes rearrangement into RF1; an ATG start site upstream of RF2 that permits production of a truncated Dμ protein; stop codons in RF3; and, following surface expression of IgM, somatic, presumably Ag receptor-based selection favoring B cells expressing Igs with tyrosine- and glycine-enriched CDR-H3s. By creating an IgH allele limited to the use of a single, frameshifted DFL16.1 DH gene segment, we tested the relative contribution of these mechanisms in determining reading frame preference. Dμ-mediated suppression via an allelic exclusion-like mechanism dominated over somatic selection in determining the composition of the CDR-H3 repertoire. Evidence of somatic selection for RF1-encoded tyrosine in CDR-H3 was observed, but only among the minority of recirculating, mature B cells that use DH in RF1. These observations underscore the extent to which the sequence of the DH acts to delimit the diversity of the Ab repertoire.

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...

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.

Germline DH sequences are required for the generation of natural antibodies reactive to bacterial phosphorylcholine but not for those reactive to self-antigen.

After birth, contact to environmental antigens induces the production of IgA, which represents a first line of defense for the neonate. We sought to characterize the maturation of the repertoire of IgA H chain transcripts in circulating blood B cells during human ontogeny. We found that IgA H chain transcripts were present in cord blood as early as 27 weeks of gestation and that the restrictions of the primary antibody repertoire (IgM) persisted in the IgA repertoire. Thus, B cells harboring more “mature” VH regions were not preferred for class switch to IgA. Preterm and term neonates expressed a unique IgA repertoire, which was characterized by short CDR-H3 regions, preference of the JH proximal DH7-27 gene segment, and very few somatic mutations. During the first postnatal months, these restrictions were slowly released. Preterm birth did not measurably accelerate the maturation of the IgA repertoire. At a postconceptional age of 60 weeks, somatic mutation frequency of IgA H chain transcripts reached 25% of the adult values but still showed little evidence of antigen-driven selection. These results indicate that similar to IgG, the IgA repertoire expands in a controlled manner after birth. Thus, the IgA repertoire of the newborn has distinctive characteristics that differ from the adult IgA repertoire. These observations might explain the lower affinity and specificity of neonatal IgA antibodies, which could contribute to a higher susceptibility to infections and altered responses to vaccinations, but might also prevent the development of autoimmune and allergic diseases.