No abstract

To gain insight into the mechanisms that regulate the development of the H chain CDR3 (CDR-H3), we used the scheme of Hardy to sort mouse bone marrow B lineage cells into progenitor, immature, and mature B cell fractions, and then performed sequence analysis on VH7183-containing Cμ transcripts. The essential architecture of the CDR-H3 repertoire observed in the mature B cell fraction F was already established in the early pre-B cell fraction C. These architectural features include VH gene segment use preference, DH family usage, JH rank order, predicted structures of the CDR-H3 base and loop, and the amino acid composition and average hydrophobicity of the CDR-H3 loop. With development, the repertoire was focused by eliminating outliers to what appears to be a preferred repertoire in terms of length, amino acid composition, and average hydrophobicity. Unlike humans, the average length of CDR-H3 increased during development. The majority of this increase came from enhanced preservation of JH sequence. This was associated with an increase in the prevalence of tyrosine. With an accompanying increase in glycine, a shift in hydrophobicity was observed in the CDR-H3 loop from near neutral in fraction C (−0.08 ± 0.03) to mild hydrophilic in fraction F (−0.17 ± 0.02). Fundamental constraints on the sequence and structure of CDR-H3 are thus established before surface IgM expression.

V(H) replacement has been proposed as one way to modify unwanted antibody specificities, but analysis of this mechanism has been limited without a dynamic cellular model. We describe a human cell line that spontaneously undergoes serial V(H) gene replacement mediated by cryptic recombination signal sequences (cRSS) located near the 3' end of V(H) genes. Recombination-activating gene products, RAG-1 and RAG-2, bind and cleave the cRSS to generate DNA deletion circles during the V(H) replacement process. A V(H) replacement contribution to normal repertoire development is revealed by the identification of V(H) replacement "footprints" in IgH sequences and double-stranded DNA breaks at V(H) cRSS sites in immature B cells. Surprisingly, the residual 3' sequences of replaced V(H) genes contribute charged amino acids to the CDR3 region, a hallmark of autoreactive antibodies.