PROJECT SUMMARY
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.