Abstract
Phospholipase D4 (PLD4) is an intracellular exonuclease implicated in immune regulation via nucleic acid degradation. While public proteomic databases indicate the presence of PLD4 in human plasma, its mode of extracellular release remains unclear. This study demonstrates that activated B cells secrete PLD4-containing extracellular vesicles (EVs), providing a novel mechanism for its extracellular presence. EVs were purified from human plasma and confirmed to contain PLD4 through immunoelectron microscopy (IEM), Western blotting, and enzyme-linked immunosorbent assay. To further investigate the cellular origin, human B cells were stimulated in vitro with B cell receptor engagement and TLR9 agonist. Light and electron microscopy revealed significant cellular hypertrophy and accumulation of multivesicular bodies following stimulation. Nanoparticle tracking analysis (NTA) confirmed an increase in EV secretion, and IEM demonstrated a higher frequency of PLD4-positive EVs in stimulated B cells. Additionally, immunofluorescence and IEM revealed that PLD4 relocates from the Golgi apparatus to CD63-positive endosomes, where it is incorporated into intraluminal vesicles prior to EV release. These findings establish that activated B cells contribute to the extracellular distribution of PLD4 via EV secretion, highlighting a potential role in intercellular communication and immune regulation.