Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy(VH)chain regions with ultralong third complementarity determining regions(CDR3)which,compared to other species,can provide a potent response to challenging antigens like HIV env.These unusual CDR3 can range to over seventy highly diverse amino acids in length and form uniqueβ-ribbon‘stalk’and disulfide bonded‘knob’structures,far from the typical antigen binding site.The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood.Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene,IGHV1-7(VHBUL)rearranged to the longest diversity gene,IGHD8-2.An eight nucleotide duplication at the 3′end of IGHV1-7 encodes a longer V-region producing an extended Fβ-strand that contributes to the stalk in a rearranged CDR3.A low amino acid variability was observed in CDR1 and CDR2,suggesting that antigen binding for this subset most likely only depends on the CDR3.Importantly a novel,potentially AID mediated,deletional diversification mechanism of the B.taurus VH ultralong CDR3 knob was discovered,in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place.These deletions serve to further diversify cysteine positions,and thus disulfide bonded loops.Hence,both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.

Development of the expressed immunoglobulinμchain repertoire during maturation of mice B cells

In the bone marrow and spleen,the developing B cell populations undergo both negative and positive selections to shape their B cell receptor repertoire.To gain insight into the shift of the immunoglobulin heavy(IgH)chain repertoire during B cell development,we undertook large scale Igμchain repertoire analysis of pre-B,immature B and spleen B cell populations.We found that the majority of VH gene segments,VH families,JH and D gene segments,were observed to have significantly different usage frequencies when three B cell populations were compared,but the usage profile of the VH,D,and JH genes between different B cell populations showed high correlations.In both productive and nonproductive rearrangements,the length of CDRH3 shortened significantly on average when B cells entered the periphery.However,the CDRH3 length distribution of nonproductive rearrangements did not follow a Gaussian distribution,but decreased successively in the order 3n–2,3n–1 and 3n,suggesting a direct correlation between mRNA stability and CDRH3 length patterns of nonproductive rearrangements.Further analysis of the individual components comprising CDRH3 of productive rearrangements indicated that the decrease in CDRH3 length was largely due to the reduction of N addition at the 5′and 3′junctions.Moreover,with development,the amino acid content of CDRH3 progressed toward fewer positively charged and nonpolar residues but more polar residues.All these data indicated that the expressed Igμchain repertoire,especially the repertoire of CDRH3,was fine-tuned when B cells passed through several checkpoints of selection during the process of maturation.