Class switch recombination regulation by extracellular vesicles mediated intercellular epigenomic signaling

B cell maturation is crucial for effective adaptive immunity and requires a complex signaling network to control gene expression and protein function to mediate antibody diversification. B cells also rely on extracellular signaling queues from other cells within the germinal center. Recently, a novel class of intercellular signaling mediated by extracellular vesicles (EVs) has emerged. Studies have shown B cell EVs mediated signaling is involved in immune response regulation, infection control, and tumorigenesis. However, the mechanistic role of B cell EVs in B cell maturation is not yet established. We herein study the biological properties and physiological function of B cell EVs in the context of B cell maturation. We use novel technologies to characterize B cell EVs surface marker signatures, molecular cargo and physiological roles in B cell maturation. A new specialized nanoparticle analyzer was used to profile B cell EVs at the single particle level and characterize their surface markers and sub-populations at various maturation stages. EV ncRNA cargo was characterized by RNA-seq and bioinformatic analyses identified an EVs mediated regulatory network for B cell maturation. A previously uncharacterized micro-RNA (miR-5099) in combination with a set of long non-coding RNA carried within B cell EVs is shown to be important for antibody diversification signaling. The physiological role of EVs in B cell maturation is investigated using EV blockade assays and complementation studies using diverse EV sources further confirmed the physiological role and mode of action of EVs in B cell maturation. To investigate how EV-RNA regulate CSR, we profiled RNA from unstimulated and stimulated primary B cells and CH12F3 and paired EVs. Differential RNA expression between cells and secreted EVs; unstimulated and stimulated were analyzed to identify responsible RNA