The NIH estimates that up to 23.5 million Americans suffer from autoimmune disease, driven in large part by germinal center (GC)-derived pathologic autoantibodies. GC B cells undergo iterative rounds of somatic hypermutation of B cell receptor (BCR) genes, and selection based on BCR-antigen affinity. The random nature of somatic hypermutation allows emergence of autoreactive B cells, but these cells die because they have lost antigen specificity, and fail to be selected. Without a stringent selection threshold, autoreactive B cells may survive and become long-lived plasma cells. Regulation of surface BCR turnover is critical for faithful antigen- driven selection. During each round of somatic hypermutation and selection, the pre-mutated BCR must be removed and replaced with the newly-mutated BCR to ensure that the genetically encoded BCR dictates selection. Failure to remove previous ?versions? of the BCR could allow selection of a BCR that has not been tested for antigen affinity. BCR turnover is an integral component of the current model of GC selection, yet this has never been fully explored. The ubiquitin ligase Itch prevents humoral autoimmunity in humans and mice, but how Itch limits antibody production is unclear. I have recently determined that Itch functions in B cells to limit GC B cells, plasma cells, and antibody production, aligning with its role in preventing autoimmunity. Through analysis of somatic mutations in GC B cells, I now show that Itch supports stringent selection of GC B cells. After immunization, I identified Itch deficient GC B cells bearing antigen-specific surface BCRs, yet containing nonproductive BCR genes. These data support the idea that Itch prevents survival of B cells acquiring detrimental BCR mutations and promotes pre-mutated BCR removal after mutation. Itch has been shown to regulate BCR internalization and trafficking to lysosomes in nave B cells. It is unknown how Itch controls BCR turnover in GCs, and how this impacts GC selection. In this proposal I will test the hypothesis that Itch regulates surface BCR turnover in GC B cells to ensure stringent selection and prevent the emergence of autoantibody. I will use B cell specific Itch knockout mice and analysis of antigen-specific GC B cell somatic mutations to model GC dynamics and determine how Itch regulates selection. Additionally, I will define the role of Itch in GC BCR removal and replacement using cell biology approaches to probe surface BCR removal/replacement in GC B cells. To complete these studies, I have formed collaborations with experts in immunoreceptor biology, intracellular trafficking, next generation sequencing, and computational modeling. These studies will advance the understanding of mechanisms regulating selection in GCs, and provide a unique system in which to study the link between BCR turnover, GC B cell selection, and antibody responses.
Germinal center-derived antibody responses contribute to pathology in autoimmunity and protection after immunization. The E3 ubiquitin ligase Itch limits antibody responses and prevents humoral autoimmunity, but the mechanisms by which Itch alters B cell biology to regulate antibody production are unclear. This project aims to determine how Itch shapes antibody responses through control of germinal center B cell selection.
