Supplementary Materials1. In Brief Plasma cell survival and the consequent duration of immunity vary widely with contamination or vaccination. Lam et al. demonstrate that short- and long-lived plasma cells are distinguished by metabolic properties such as nutrient uptake. In contrast, very few conserved transcriptional changes are observed between plasma cells of varying longevity. Graphical Abstract Open in a separate window INTRODUCTION Upon contamination or vaccination, naive B cells become activated by foreign antigens, and a subset of these cells differentiate into antibody-secreting plasma cells. Once formed, plasma cells secrete antibodies constitutively as long as they live (Manz et al., 1998; RGS7 Slifka et al., 1998). Because these antibodies preexist subsequent exposures to pathogens, plasma cells have the ability to provide sterilizing immunity and prevent re-infection. As a result, plasma cells and the antibodies they produce are the primary determinants of humoral immunity following vaccination (Zinkernagel and Hengartner, 2006). The transience of plasma cell persistence and consequent antibody production is the major reason for the loss of immunity against infectious diseases such as malaria (Weiss et al., 2010; White et al., 2015). Reciprocally, long-lived plasma cells pose a major problem in certain autoimmune disorders and are the cell of origin in multiple myeloma (Winter et al., 2012). A mechanistic understanding of plasma cell survival may provide additional targets for the above disorders. In T cell-dependent reactions, an initial wave of extrafollicular plasma cells tends to be relatively short-lived and produces germline-encoded antibodies (Sze et al., 2000). These cells form an early response to provide partial control of buy Z-FL-COCHO the infection until plasma cells encoding higher affinity antibodies emerge later from the germinal center reaction. As the germinal center progresses, there is a concomitant increase in both the affinity of the encoded antibodies as well as in the lifespans of the selected plasma cells (Weisel et al., 2016). Yet germinal centers are not required per se for the formation of long-lived plasma cells. T cell-independent responses, which yield neither germinal centers nor robust immunological memory, can also yield plasma cells of extended lifespans, as well as a proliferative subset of antibody-secreting cells that together maintain serum antibodies long after immunization (Bortnick et al., 2012; Reynolds et al., 2015; Savage et al., 2017). These and other data demonstrate substantial functional heterogeneity in ontogeny and lifespan within the plasma cell compartment (Amanna et al., 2007), but the underlying molecular basis is unclear. We reasoned that coupling specific metabolic and transcriptional properties in conjunction with other markers would allow for prospective separation of new plasma cell subsets with a range of lifespans. This in turn would allow for an assessment of how metabolic, buy Z-FL-COCHO transcriptional, and buy Z-FL-COCHO endoplasmic reticulum (ER) stress pathways integrate to regulate plasma cell lifespan and antibody secretion. Using this strategy, we found a very limited correlation between transcriptional changes, ER stress responses, and plasma cell lifespan. Instead, nutrient uptake and catabolism consistently distinguished plasma cell subsets with differing lifespans and antibody secretion rates. RESULTS Prospective Separation of Developmentally Distinct Plasma Cell Subsets with Varying Lifespans We reasoned that prospectively separating plasma cells into functionally distinct groups would provide a cellular foothold to define pathways that regulate lifespan. Intracellular staining for immunoglobulin (Ig) demonstrated very high levels of antibodies in almost all CD138high cells (Figure S1A). We further buy Z-FL-COCHO separated polyclonal CD138+ plasma cells in the spleen and bone marrow, formed in response to natural infections in the colony, based on uptake of 2-(cultures found minimal phosphorylation of eIF2 (Ma et al., 2010), we observed clear activation of this pathway in all plasma cell subsets (Figure 4C). B220+ plasma cells.