Project Summary/Abstract Protective humoral immunity is mediated by both long-lived memory B cells (MBC) and plasma cells. MBC are uniquely important because they are multipotent and can rapidly diversify their BCR repertoire compared to both terminally differentiated plasma cells and naïve B cells. MBC represent a heterogenous population of cells with different subsets primed to either rapidly generate plasma cells or form secondary germinal centers. Additionally, MBC can arise in response to a diverse array of stimuli including independently of a germinal center through a potential extrafollicular pathway. While all MBC seem to have enhanced recall properties, it is not known how MBC phenotypes are programed in different subsets or what MBC programming is dependent on GC? Epigenetic mechanisms are heritable programs that act to guide cell fate decisions and determine potential phenotypes. Given the cell intrinsic nature of MBC properties, we hypothesize that MBC harbor a distinct epigenetic programming that serves as a molecular memory of prior states and instructs cell fate decisions and enhanced function during recall. Recent work from our group has shown that atypical memory B cells, which are expanded in patients with autoimmune diseases such as Lupus, show an epigenetic signature of extrafollicular activation pathways. Thus, a full understanding of MBC properties is essential to design vaccine strategies that maximize MBC potential and develop therapies that target diseases where MBC are a component. To elucidate the epigenetic mechanisms governing MBC phenotypes, we propose two aims designed to 1) define the cis-regulatory landscape, transcription factor networks, and metabolism of influenza specific MBC subsets that are derived from or independently of a germinal center; and 2) understand how the epigenetic repressor EZH2 controls the formation of MBC subsets and recall responses. To accomplish these aims we have established a series epigenetic and transcriptional profiling protocols and bioinformatic approaches; assembled a series of genetic mouse strains that allow the conditional deletion of EZH2; and developed an ex vivo MBC assay to fine map the molecular changes during recall responses. Ultimately our studies will provide an epigenetic road map to MBC formation and function and a platform that could aid in the manipulation of immune memory and therapeutic targets for MBC mediated diseases.