Expansion of the Preimmune Antibody Repertoire by Junctional Diversity in Bos taurus

Abstract

Cattle have a limited range of immunoglobulin genes which are further diversified by antigen independent somatic hypermutation in fetuses. Junctional diversity generated during somatic recombination contributes to antibody diversity but its relative significance has not been comprehensively studied. We have investigated the importance of terminal deoxynucleotidyl transferase (TdT) -mediated junctional diversity to the bovine immunoglobulin repertoire. We also searched for new bovine heavy chain diversity (IGHD) genes as the information of the germline sequences is essential to define the junctional boundaries between gene segments. New heavy chain variable genes (IGHV) were explored to address the gene usage in the fetal recombinations. Our bioinformatics search revealed five new IGHD genes, which included the longest IGHD reported so far, 154 bp. By genomic sequencing we found 26 new IGHV sequences that represent potentially new IGHV genes or allelic variants. Sequence analysis of immunoglobulin heavy chain cDNA libraries of fetal bone marrow, ileum and spleen showed 0 to 36 nontemplated N-nucleotide additions between variable, diversity and joining genes. A maximum of 8 N nucleotides were also identified in the light chains. The junctional base profile was biased towards A and T nucleotide additions (64% in heavy chain VD, 52% in heavy chain DJ and 61% in light chain VJ junctions) in contrast to the high G/C content which is usually observed in mice. Sequence analysis also revealed extensive exonuclease activity, providing additional diversity. B-lymphocyte specific TdT expression was detected in bovine fetal bone marrow by reverse transcription-qPCR and immunofluorescence. These results suggest that TdT-mediated junctional diversity and exonuclease activity contribute significantly to the size of the cattle preimmune antibody repertoire already in the fetal period.

Internal Duplications of DH, JH, and C Region Genes Create an Unusual IgH Gene Locus in Cattle

“…1). The previously reported 154-and 121-bp-long IGHD segments were not found (25), and they may be allelic variants of IGHD8.…”

“…Several key molecular mechanisms including V(D)J recombination and somatic hypermutation are fundamental to the Ab repertoire diversification in mammals (21)(22)(23). As compared with humans and mice, cattle are characterized by having a single functional IGHV family with a very limited number of IGHV genes (24)(25)(26)(27)(28), which is similar to rabbits (29)(30)(31) and pigs (32,33). The present understanding of the mechanisms involved in bovine Ab diversification is still based on the partial characterization of germline IGHV, IGHD, and IGHJ gene segments.…”

“…The present understanding of the mechanisms involved in bovine Ab diversification is still based on the partial characterization of germline IGHV, IGHD, and IGHJ gene segments. To date, ∼10 functional IGHV genes, 14 IGHD, and 4 functional IGHJ segments have been defined (9,24,25,28,(34)(35)(36). All the functional IGHV genes and some other potentially functional IGHV genes (either additional IGHV genes or allelic variants) belong to a single family (IGHV1) (25,27).…”

“…All members of the two additional IGHV families (IGHV2, IGHV3) identified in cattle are pseudogenes (27). Among the IGHD segments, three IGHD segments with unusual lengths (149, 154, and 121 bp) have been reported and might contribute significantly to the length of H chain CDR 3 (CDRH3) (25). The identification of exceptionally long germline IGHD segments is consistent with the finding that bovine IgH chains possess ultralong CDRH3s (35)(36)(37)(38), which was seen as compensation for the limited diversity generated by VDJ recombination.…”

“…A recent study suggested that the Abs with ultralong CDRH3s can generate structural diversity via combinations of somatically generated disulfides (39). A number of studies have concluded that the bovine Ab repertoire is primarily diversified by somatic hypermutation and junctional flexibility caused by nucleotide deletions and additions during the recombination process (25,26,36,40,41). However, lack of the sufficient information (such as number and physical positions) of the bovine IGHV, IGHD segments has been an obstacle to a complete elucidation of Ab repertoire diversification in cattle.…”