Identification of enhancers that facilitate genetic manipulation of specific B-cell subsets

About This Grant

Project Summary/Abstract Protective humoral immunity is mediated by both long-lived memory B cells (MBC) and antibody secreting plasma cells (ASC). Recently it has become increasingly clear that MBC and ASC are in fact composed of functionally diverse subpopulations that can be distinguished based on unique surface marker expression, tissue localization, and the B-cell receptor (BCR) isotype expressed. During a humoral immune response, naïve B cells (nB) give rise to differentiated subsets, with the ultimate composition of the MBC and ASC pool primarily influenced by the antigen, the involvement of T cells, and the cytokine environment. Given the evidence for increased heterogeneity, it is surprising that we currently lack the genetic tools to further interrogate the complexity, molecular properties, and immunological importance of the known B cell fates. A precise phenotypic analysis of MBC and ASC subsets cannot currently be performed with current Cre-lox mouse models. Cis- regulatory elements (CEs), or enhancers, are DNA sequences that act to promote cell-type and context-specific gene expression programs. These sequences are regulated by epigenetic mechanisms, which act to control the accessibility of CEs to DNA-binding transcription factors. Over the past several years, we have characterized the epigenetic architecture that controls primary humoral immune responses to T cell dependent and independent antigens, integrated newly published datasets defining MBC subsets, and generated new preliminary data defining the CEs of ASC expressing distinct BCR isotypes. These data have revealed specific CEs that are active in defined stages of B cell differentiation, including IgA ASC and extrafollicular (EF)-MBC that arise independently of a germinal center reaction. Therefore, we hypothesize that cell-type specific CEs can be co-opted to provide precise genetic manipulation that enables functional exploration of B-cell subsets. To address this, we propose two aims designed to 1) develop new Cre recombinase tools for genetic manipulation of IgA ASC and 2) map the CEs for EF-MBC that arise during influenza infection and integrate CEs specific for EF-MBC to allow precise genetic editing of this MBC subset. These aims will utilize novel hematopoietic stem cell engineering to rapidly generate chimeric mice expressing genes of interest; therefore, bypassing the need to generate transgenic animals. Completion of these aims will provide a novel set of DNA sequences that allow for MBC and ASC specific genetic manipulation. These tools are critically important begin to derive the biology, molecular properties, and importance of the entire spectrum of B-cell differentiation to humoral immunity. If successful, it also has the potential to redefine how Cre recombinase vectors are engineered and further our understanding of CE biology in B cells and the immune system.