V-H Replacement In Primary Immunoglobulin Repertoire Diversification

The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D) J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. V-H replacement represents a RAG-mediated secondary rearrangement in which an upstream V-H element recombines with a rearranged V(H)D(H)J(H) joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original V-H element and the conventional RSS of the invading V-H gene, leaving behind a footprint of up to five base pairs (bps) of the original V-H gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that V-H replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive V(H)D(H)J(H) allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive V(H)D(H)J(H) rearrangements. Our results indicate that V-H replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.