Large extracellular vesicles derived from human regulatory macrophages (L-EV_Mreg) attenuate CD3/CD28-induced T-cell...

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 × 10⁶ 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 × 10⁶ 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 × 10⁶ L-EV_Mreg/ml; P < 0.01 for 3.2 × 10⁶ 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 × 10⁶ L-EV_Mreg/ml and 3.2 × 10⁶ 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.