Ezrin promotes antigen receptor diversity during B cell development by supporting immunoglobulin heavy chain variable gene recombination

Abstract

Abstract Genome-level rearrangements of immunoglobulin genes during B cell development are critical for generation of a diverse repertoire of B cell antigen receptors (BCRs) that bind to a multitude of foreign antigens and some self-antigens. 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 towards 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 if Ezrin plays a role in immunoglobulin heavy chain 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 CDR3s in the BCRs and reduced Shannon entropy. Ezrin-deficient pro-, pre- and immature B cells utilized similar number of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length, nucleotide additions and deletions, was also altered in Ezrin-deficient pro-, pre- and immature B cells. Mechanistically, Ezrin-deficient bone marrow B cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate a novel role for Ezrin in shaping the BCR repertoire through combinatorial and junctional diversification. Supported by grants from NIH (R01 AR067705) to N.G.