Hydroxychloroquine modulates extracellular vesicle release and LC3-positive EV profiles in activated human B cells

B cells play a critical role in the pathogenesis of autoimmune diseases. Activated B cells upregulate autophagy to evade apoptotic checkpoints, and our previous studies demonstrated that B-cell activation enhances extracellular vesicle (EV) secretion. However, how HCQ affects EV release and LC3-associated EV features in activated B cells remains unclear. In this study, we investigated how HCQ affects EV release and LC3-associated EV features in activated human B cells and examined whether HCQ use is associated with alterations in circulating serum EVs in patients with SLE. Blood samples were collected from five healthy controls and fifteen patients with systemic lupus erythematosus (SLE). EVs were isolated from plasma or serum by differential centrifugation. Purified B cells from healthy controls were stimulated with CpG ODN 2006 and anti-IgG + M F(ab′)2 in the presence or absence of hydroxychloroquine (HCQ). B cells and EVs were analyzed using light microscopy, transmission electron microscopy (TEM), flow cytometry, nanoparticle tracking analysis, ELISA, and western blotting. LC3-positive EVs were directly visualized in human plasma by immunogold TEM, and EV-associated LC3 was also detected by ELISA and western blotting. In patients with SLE, HCQ use was associated with lower serum EV concentrations and a higher LC3/CD63 signal ratio, whereas the absolute LC3-associated ELISA signal did not significantly differ between patients receiving and not receiving HCQ. Double-immunogold TEM detected CD19-associated labeling on LC3-positive EVs in patients receiving prednisolone plus HCQ, whereas such labeling was not observed in the examined EVs from patients receiving prednisolone alone. In activated human B cells, NTA showed that HCQ increased small-EV release, whereas LC3 immunogold TEM demonstrated an increased proportion of LC3-positive EVs in culture supernatants. Immunofluorescence microscopy further showed increased intracellular LC3 signal and LC3/CD63 co-localization, while TEM revealed HCQ-associated ultrastructural changes in intracellular vesicular structures. These findings indicate that HCQ modulates EV release and LC3-positive EV profiles in activated human B cells, while HCQ use is associated with altered serum EV profiles in SLE. Further studies are needed to determine the mechanisms and clinical significance of these changes.