B and T lymphocytes form the foundation of our adaptive immune system, which is based on specific recognition of foreign molecules by structurally diverse surface antigen receptors. Structural diversity in these receptors originates through site-specific rearrangement of antigen receptor genes during lymphocyte development. This rearrangement process, called V(D)J recombination, is initiated when the RAG1/2 proteins cleave antigen receptor gene segments at recombination signal sequences (RSS) through a nick-hairpin mechanism, and is completed when the DNA breaks are sensed and repaired by non-homologous end-joining. Classically, B cell repertoire diversity is considered restricted by the number of functional V, D, and J gene segments in the immunoglobulin (Ig) heavy and light chain gene loci. However, forms of secondary V(D)J rearrangement have been reported in which an unrearranged Ig heavy chain V (VH) gene segment replaces a rearranged VH gene via recombination with a cryptic RSS embedded in the 3? end of the VH gene (called VH gene replacement). Mechanistic insight into this process has been hampered by the low frequency of these events and, typically, the use of germline pre-rearranged VH alleles. Whether similar events occur in the light chain loci remains unclear. We recently performed bulk light chain repertoire sequencing of B cells from transgenic mice expressing the Ig VH12 heavy chain, sorted based on immunoreactivity to phosphatidylcholine. Interestingly, our analysis uncovered infrequent, but recurrent, endogenous hybrid rearranged Ig kappa V (KV) gene sequences, in which the 3? end of the highly selected KV4-91 gene was replaced by another KV gene. Based on this preliminary data, our working hypothesis is that the RAG proteins mediate KV gene replacement in VH12 mice via cleavage of a cryptic RSS identified in framework region 3 of the KV gene (KV FR3 cRSS). The proposed project will extend our preliminary findings to confirm KV gene replacement leads to productive antibody generation in single B cells, test whether the KV FR3 cRSS supports RAG-mediated cleavage and rearrangement in vitro and in cells, and also exclude activation-induced cytidine deaminase (AID) as an alternative mechanism for KV gene replacement in VH12 mice. This project will challenge our current understanding of the theoretical constraints on antibody structural diversity, and lead to considering KV gene chimeras for rational design of therapeutic antibodies. This work will also highlight an important potential caveat of automated KV sequence analysis, because such events (regardless of origin) may be missed in KV gene calls or be mistaken for somatic hypermutation. The potential of this project to shift existing paradigms places the project in the ?high risk-high reward? category for which the R21 was designed.
V(D)J recombination is a DNA rearrangement process that is responsible for generating a diverse population of B and T cells that together provide highly specific immunity to pathogenic microorganisms. The proposed research will investigate whether an atypical form of V(D)J recombination mediates the generation of chimeric light chain genes to produce novel, yet function, antibody structures. This project will challenge our current understanding of the theoretical constraints on antibody structural diversity, raise the potential for using hybrid light chains to generate therapeutic antibodies, and promote reassessment of automated programs that identify light chain repertoires and gene assignments to account for hybrid light chain genes.