Ezrin Promotes Antigen Receptor Diversity during B Cell Development by Supporting Ig H Chain Variable Gene Recombination

Abstract

Abstract Genome-level rearrangements of Ig genes during B cell development are critical for generation of a diverse repertoire of BCRs that bind to a multitude of foreign Ags and some self Ags. Bone marrow B cell development involves a variety of cell–cell interactions, cell migration, and receptor signaling that likely benefit from the activity of membrane-cytoskeletal reorganizing proteins. However, the specific contribution of such proteins toward BCR repertoire diversification is poorly understood. Ezrin is a membrane-cytoskeletal linker protein that regulates mature B cell activation through spatial organization of the BCR. We employed next-generation sequencing to investigate whether Ezrin plays a role in IgH rearrangements and generation of BCR diversity in developing bone marrow B cells. BCR repertoire development occurred stochastically in B cell progenitors from both control and B cell conditional Ezrin-deficient mice. However, the loss of Ezrin resulted in fewer unique CDRs (CDR3s) in the BCRs and reduced Shannon entropy. Ezrin-deficient pre-B cells revealed similar utilization of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length and nucleotide additions and deletions, was not altered in Ezrin-deficient pre-B cells. Mechanistically, Ezrin-deficient cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate that Ezrin shapes the BCR repertoire through combinatorial diversification.