Summary Antibody based humoral immunity arises following the initial activation of naïve B cells and their subsequent differentiation to antibody secreting cells (ASC), as well as memory B cells (MBC). Antigen-experienced MBC provide a more rapid and robust immune response through their differentiation to ASC. The current pandemic has focused the world’s attention on the generation of antibodies and B cell protective humoral immunity, which is derived from MBC and ASC. Yet, there are significant gaps in our basic knowledge of the cell fate heterogeneity and epigenetic choices that B cells take to become ASC. For example, how do MBC respond more quickly to recall challenge, are there underlying pathways in MBC that do not exist in naïve cells; and how do these processes define the heterogeneity observed in the transcriptomes and epigenomes of MBC? Our recent work and preliminary data suggest that: 1) epigenetic and transcriptional programming of multiple pathways, including the use of iron/heme pathways is important as heme-treated ex vivo differentiation cultures results in sharp increases in the number of ASCs formed; 2) MBC are epigenetically primed to respond to secondary challenges; and 3) isotype specific plasma cell and MBC display heterogeneous programming at the transcriptional and also at the epigenetic level for MBC. How this programming is established is not fully understood. To address these gaps in our knowledge, Aim 1 will determine how iron/heme dependent pathways modulate ASC formation and test the hypothesis that heme content alters the kinetics and programming of ASC differentiation through multiple mechanisms and pathways, including mitochondrial metabolism, modulation of transcription factor activity, and the activity of epigenetic modifiers that require iron. We will take advantage of a lentigenic CRISRP/Cas9 gene editing system that we established to introduce mutations and probe the roles of factors influencing iron/heme metabolism. In conjunction with Projects 3 and 4, we will examine human B cell subsets from healthy and SLE subjects to characterize their responses to heme and heterogeneity. Aim 2 will determine the epigenetic bases and plasticity for distinct transcriptomes associated with IgM, IgG, and IgA MBC and ASC using an influenza-infection model developed with Project 1. We hypothesize that distinct isotype- specific epigenetic programs are initiated during MBC formation that are maintained as the cells differentiate to ASC of the same isotype, and that these programs may be plastic as cells undergo additional class switch recombination. To test these hypotheses, we will determine the epigenetic basis of isotype-switched ASC transcriptomes; the heterogeneity of the ASC response to influenza; and determine if the epigenome is plastic when MBC and ASC are further class switched from IgG to IgA. Understanding the molecular and epigenetic underpinnings of how MBC differentiate would ultimately aid our ability to deve...