VH 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_HD_HJ_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_HD_HJ_H allele. Using this mouse model, we characterized the role of V_H replacement in the diversification of the primary Ig repertoire through the modification of productive V_HD_HJ_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.A hallmark of the adaptive immune system is its ability to generate a large antibody repertoire despite a limited genome through the rearrangement of variable (V), diversity (D), and joining (J) genes during B-cell development in a process called V(D)J recombination (1). Each antibody-producing cell expresses a single pair of heavy and light chains generated by this process during the pro–B-cell and pre–B-cell stages of development, respectively. V(D)J recombination is mediated by the recombination activating proteins RAG1 and RAG2, which bind and cleave recombination recognition sequences (RSSs) flanking V, D, and J genes. Because there are several members of V, D (for the heavy chain), and J segments, the combinatorial nature of V(D)J rearrangement allows for the generation of a vastly diverse antibody repertoire from a relatively modest amount of genetic information present in the germline DNA.Ig gene rearrangement progresses in an ordered stepwise manner during B-cell development, with Ig heavy (IgH) chain assembly before Ig light (IgL) chain assembly. In-frame rearrangement of a V_HD_HJ_H joint leads to expression of an IgH chain that pairs with an invariant surrogate light chain, and, in association with the Igα and Igβ signal-transducing subunits, these proteins form the pre–B-cell receptor (pre-BCR) (2, 3). Signaling through a functional pre-BCR allows the cell to progress from the pro–B-cell stage to the pre–B-cell stage, where IgL rearrangement can occur. This progression is accompanied by a burst of proliferation, which ensures that the functional IgH rearrangement is not lost in the event of unsuccessful IgL recombination and increases diversity by allowing a given IgH chain to pair with multiple IgL chains. Only cells that acquire a functional B-cell receptor (BCR), with an in-frame rearranged IgH chain that successfully pairs with a productively rearranged IgL chain, progress to become mature B cells.The imprecise nature of the joining process in V(D)J recombination contributes to the diversity of the antibody repertoire, but also leads to a significant number of nonfunctional rearrangements, with approximately two-thirds of the joints being out-of-frame. At the IgH locus, V_H replacement can rescue “dead-end” pro-B cells that have acquired nonproductive IgH joints on both alleles by rearranging an upstream V_H gene with a nonfunctional V_HD_HJ_H (4–7).Ordered rearrangement of the IgH and IgL loci is mediated by the RAG1/2 proteins that recognize RSSs flanking V, D, and J segments. The consensus RSS is composed of a heptamer (CACTGTG) and a nonamer (GGTTTTTGT) separated by either a 12-bp or 23-bp spacer (8). V(D)J recombination occurs preferentially between gene segments flanked by RSSs of dissimilar lengths, thus directing the order of recombination; this is known as the 12/23 rule. Because the V_HD_HJ_H recombination process eliminates any remaining D_H genes and their flanking 12-bp RSSs, further editing of this locus requires a recombination event that violates the 12/23 rule. V_H replacement occurs between a 23-bp RSS of an upstream invading V_H gene and a highly conserved 7-bp cryptic RSS (cRSS; TACTGTG) in the body of the recipient V_H gene (6, 7). Because the cRSS is located near the 3′ terminus of the recipient V_H gene, this process leaves behind only 5 bps proximal to the highly variable CDR3 region of the original V_HD_HJ_H joint, a minimal footprint that can be modified by exonuclease “chewback” and N-nucleotide addition (9, 10).V_H replacement was initially observed in mouse B-cell lines in which a small fraction of cells regularly acquired alternate IgH rearrangements, thus “editing” their original specificity (6, 7). Since then, V_H replacement has been studied in transgenic mouse models, human cell lines, and human blood. Recent studies that bioinformatically examined the frequency of 4- to 5-bp V_H footprints at the V_H-D_H vs. D_H-J_H junctions have suggested evidence of V_H replacement in at least 5% of the human Ig repertoire (9). Analysis of V_H footprints in mouse IgH rearrangements revealed a similar frequency of V_H replacement in wild-type (WT) mice, and also demonstrated a significantly higher frequency of V_H replacement footprints in IgH sequences from patients with autoimmune diseases and animals of autoimmune-prone backgrounds (11, 12). However, the random nature of exonuclease and terminal deoxynucleotidyl transferase (TdT) activity at the CDR3 region of IgH joints makes it difficult to assess the contribution of V_H replacement to the repertoire by sequence analysis, owing to loss of the V_H footprint. Furthermore, our earlier work with a mouse model carrying a predefined nonproductive IgH rearrangement in the IgH locus has demonstrated that up to one-third of V_H replacement events are mediated by sequence microhomology of the highly conserved 3′ bases of V_H genes (4). Replacement reactions of this nature lack a detectable footprint and thus escape detection by sequence analysis.Although the nonproductive V_HD_HJ_H mouse model allowed us to study how V_H replacement rescues nonproductive V_HD_HJ_H rearrangements, it did not address the contribution of V_H replacement to the diversification of the primary immune repertoire or its role in editing self-reactive antibody specificities. Thus, we generated a knock-in mouse with a productive V_HD_HJ_H rearrangement knocked into its physiological position within the IgH locus. With this mouse model, we assessed the role of V_H replacement in modifying productive IgH rearrangements in nonautoreactive and autoreactive settings.