Next Generation Sequencing Identifies Biased Light Chain Usage and Evidence of Light Chain Gene Replacement in phosphatidylcholine (PtC)-specific CD5+ B cells from dnRAG1 and VH12 mice.

B cells reactive toward phosphatidylcholine (PtC) are enriched in the B1 B cell subset, and express predominantly one of two VH/Vk combinations to confer this specificity: VH12/Vk4/5H and VH11/Vk9. Studies of transgenic mice expressing the VH12 heavy chain (VH12 mice) suggest two major checkpoints for light chain expression in this system: the first involves selection of V-J rearrangements which encode a “permissive” light chain that can functionally pair with the VH12 heavy chain; the second involves receptor editing to salvage non-PtC reactive B cells to acquire a permissive light chain that confers PtC reactivity. If this model is correct, impairing receptor editing should reduce the frequency of PtC-reactive B1 B cells in VH12 mice. To test this possibility, we bred VH12 mice to transgenic mice expressing a catalytically inactive form of RAG1 (dnRAG1 mice) which show a defect in receptor editing. Interestingly, dnRAG1 expression in VH12 mice enforces development of PtC-reactive B1 B cells, rescuing the loss of splenic B cells observed in VH12 mice. These data suggest receptor editing normally functions to remove a large portion of PtC-specific B cells in VH12 mice. Deep sequencing of the expressed light chain repertoire of PtC-reactive and non-reactive B cells in VH12 mice revealed that PtC-reactive B cells predominantly expressed the Vk4/5H (IGKV4-91) light chain gene, whereas PtC-non-reactive B cells expressed a broader, yet restricted, set of light chain genes. This analysis also revealed a low frequency of in-frame hybrid light chain genes appearing to originate via Type 2 gene replacement, which we show can originate from template switching during PCR. Overall design: The sequencing libraries were generated from total RNA extracted from 60 samples. 24 samples were analyzed in the first round, these were comprised of 3 independent biological replicates for 8 unqiue cell populations from the spleens of 4 geneotypes of mice. For the second round 36 samples were anlysed, comprised of 3 independent biological replicates for 12 unique cell populations from the bone marrow and spleen of 2 genotypes of mice.