Large extracellular vesicles derived from human regulatory macrophages (L-EV Mreg ) attenuate CD3/CD28-induced T-cell activation in vitro.

Macrophages belong to the innate immune system, and we have recently shown that in vitro differentiated human regulatory macrophages (Mreg) release large extracellular vesicles (L-EV Mreg ) with an average size of 7.5 μm which regulate wound healing and angiogenesis in vitro. The aim of this study was to investigate whether L-EV Mreg also affect the CD3/CD28-mediated activation of T-cells. Mreg were differentiated using blood monocytes and L-EV Mreg were isolated from culture supernatants by differential centrifugation. Activation of human T-cells was induced by CD3/CD28-coated beads in the absence or presence of Mreg or different concentrations of L-EV Mreg . Inhibition of T-cell activation was quantified by flow cytometry and antibodies directed against the T-cell marker granzyme B. Phosphatidylserine (PS) exposure on the surface of Mreg and L-EV Mreg was analyzed by fluorescence microscopy. Incubation of human lymphocytes with CD3/CD28 beads resulted in an increase of cell size, cell granularity, and number of granzyme B-positive cells (P < 0.05) which is indicative of T-cell activation. The presence of Mreg (0.5 × 10 6  Mreg/ml) led to a reduction of T-cell activation (number of granzyme B-positive cells; P < 0.001), and a similar but less pronounced effect was also observed when incubating activated T-cells with L-EV Mreg (P < 0.05 for 3.2 × 10 6 L-EV Mreg /ml). A differential analysis of the effects of Mreg and L-EV Mreg on CD4 + and CD8 + T-cells showed an inhibition of CD4 + T-cells by Mreg (P < 0.01) and L-EV Mreg (P < 0.05 for 1.6 × 10 6 L-EV Mreg /ml; P < 0.01 for 3.2 × 10 6 L-EV Mreg /ml). A moderate inhibition of CD8 + T-cells was observed by Mreg (P < 0.05) and by L-EV Mreg (P < 0.01 for 1.6 × 10 6 L-EV Mreg /ml and 3.2 × 10 6 L-EV Mreg /ml). PS was restricted to confined regions of the Mreg surface, while L-EV Mreg showed strong signals for PS in the exoplasmic leaflet. L-EV Mreg attenuate CD3/CD28-mediated activation of CD4 + and CD8 + T-cells. L-EV Mreg may have clinical relevance, particularly in the treatment of diseases associated with increased T-cell activity. KEY MESSAGES: Mreg release large extracellular vesicles (L-EV Mreg ) with an average size of 7.5 µm L-EV Mreg exhibit phosphatidylserine positivity L-EV Mreg suppress CD4 + and CD8 + T-cells L-EV Mreg hold clinical potential in T-cell-related diseases.