Memory B Cells of Mice and Humans. of functional anergy characteristic of autoreactive B cells. Fourth, ABCs express memory B cell surface markers consistent with being poised for quick plasma cell differentiation during recall responses. Finally, in a murine model of viral contamination, adoptively transferred CD11c+ B cells rapidly differentiated into class-switched antibody-secreting cells (ASCs) upon antigen rechallenge. In summary, we phenotypically and functionally characterize ABCs as IgM-expressing memory B cells, findings that together implicate ABCs in the pathogenesis of systemic autoimmunity. INTRODUCTION Immunologic memory is a defining feature of the adaptive immune system. During a humoral immune response, the activation of antigen-specific B cells results in the generation of plasma cells and memory B cells (MBCs). While plasma cells provide long-term protection via the production of specific antibodies, MBCs persist in a quiescent state for prolonged periods. Relative to na?ve B cells, MBCs exhibit a lower threshold for antigen stimulation resulting in rapid cell cycle entry, and differentiation into antibody-secreting plasma cells or seeding of secondary germinal centers (GCs). In this manner, the generation of Sodium Channel inhibitor 1 long-lived MBCs allows efficient recall responses to Sodium Channel inhibitor 1 secondary antigen challenge (1, 2). In addition to protective functions during contamination, B cells promote the pathogenesis of systemic autoimmunity. In this context, the presence of autoreactive MBC likely contributes to long-term disease persistence and represents an important barrier to immunologic remedy. However, the study of MBCs in autoimmunity is usually hampered by the lack of uniform surface markers to identify MBC subsets. Whereas MBCs in infectious and candidate antigen models can be recognized by antigen-specificity and efficient secondary responses, the diversity of disease-associated autoantigen epitopes and ongoing nature of autoimmune inflammation prevents the ready identification of autoreactive MBCs. In 2011, impartial groups recognized a novel B cell subset, now termed age-associated B cells (ABCs), characterized by lack Sodium Channel inhibitor 1 of surface CD21 and CD23, or expression Sodium Channel inhibitor 1 of integrins CD11b and CD11c (3, 4). Importantly, several lines of evidence linked this B cell subpopulation to the pathogenesis of systemic autoimmunity, including ABC accumulation in diverse murine lupus models and human subjects with autoimmunity (4C10), and the production of anti-nuclear antibodies by Toll-like receptor (TLR)-stimulated ABCs (4). Since ABC do not spontaneously secrete antibodies but increase in number with age, ABCs have been hypothesized to represent a new MBC subset (11C13). However, a definitive functional characterization of this B cell subset is usually lacking. In the current study, we present functional and phenotypic evidence that ABCs are a populace of IgM+ MBCs. Using a surface marker agnostic definition of B cell memory, we demonstrate that ABCs are antigen-experienced B cells with an extensive replicative history, CORO1A that persist in a resting state but Sodium Channel inhibitor 1 can rapidly differentiate into antibody-secreting plasma cells following secondary antigen challenge. MATERIALS AND METHODS Mice Wild-type (WT), MT (14), expression vectors, transfected into HEK293T cells, and monoclonal antibodies purified from culture supernatants using protein ACagarose beads. Measurement of autoantibodies ELISAs were performed using 96 well Nunc-Immuno MaxiSorp plates (Thermo Fisher) coated with dsDNA (Sigma-Aldrich), phosphorylcholine (PC)-10 (Sigma-Aldrich), Sm/RNP (Arotec Diagnostic), or Q-VLP (1g/ml in PBS) (23). Plates were blocked with 1% BSA in PBS prior to incubation with diluted serum or supernatant. Specific antibodies were detected using goat anti-mouse IgM-, IgG-, or IgG2c -HRP (SouthernBiotech) and peroxidase reactions were developed using OptEIA TMB substrate (BD Biosciences) and halted with 2N H2SO4. Absorbance at 450nm was.