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

Abstract

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.

Keywords: Large extracellular vesicles; Macrophages; Phosphatidylserine; T-cell activation.

Fig. 1

Isolation and basic characterization of Mreg and L-EV_Mreg. A Schematic representation of the differentiation of Mreg from human monocytes and subsequent purification of L-EV_Mreg. B Results of the basic characterization of one representative Mreg and L-EV_Mreg fraction by flow cytometry and Coulter counter–based analysis. Mreg and LEV_Mreg can be distinguished as two distinct populations based on their granularity and size

Fig. 2

Schematic illustration of CD3/CD28-induced T-cell activation. A Administration of CD3/CD28 Dynabeads and subsequent binding to the corresponding surface receptors on resting T-cells lead to their activation. Using brightfield microscopy, activated T-cells dominate as clusters of large cells and are also detectable in the FSC/SSC flow cytometry blots. B Addition of Mreg or increasing concentrations of L-EV_Mreg to CD3/CD28-induced T-cells leads to inhibition of T-cell activation and lack of T-cell clustering

Fig. 3

Effects of Mreg and L-EV_Mreg on the T-cell activation (cell size and cell granularity). Relative number of activated T-cells. A representative flow cytometry dot plot is shown below the respective columns. *P < 0.05; ****P < 0.0001 vs. activated T-cells (group: T-cells + CD3/CD28 beads)

Fig. 4

Effects of Mreg and L-EV_Mreg on the T-cell activation (intracellular granzyme B). A Relative number of activated T-cells. B Activated ${\mathrm{CD}4}^{+}$ T-cells. C Activated ${\mathrm{CD}8}^{+}$ T-cells. A representative flow cytometry dot plot is shown below the respective columns in B and C. *P < 0.05; **P < 0.01; ***P < 0.001 vs. activated T-cells (group: T-cells + CD3/CD28 beads)

Fig. 5

Phosphatidylserine exposure on the membrane of Mreg and L-EV_Mreg. A Cells and vesicles were stained with Hoechst33342 (violet) and PSVue480 (green) to label DNA and PS, respectively. Signals were merged on the brightfield background to distinguish morphological features. Yellow arrows show L-EV_Mreg. Bar denotes 50 µm. B Left: magnified views of two selected areas from A. Right: same area as on the left showing only the nuclei staining. Yellow arrows denote L-EV_Mreg. Note that L-EV_Mreg lack nuclei and are positive for PS, while Mreg contain nuclei and are either negative for PS or reveal PS to be restricted to only a confined region of the cell surface. Bar denotes 20 µm. C Typical PS staining of a Mreg cell and one L-EV_Mreg. Yellow arrows denote L-EV_Mreg. Bar denotes 15 µm. D Left: Flow cytometry and gating (red box) of L-EV_Mreg. Right: Gated L-EV_Mreg are positive for PS (annexin V staining) and negative for nucleic acids (propidium iodide staining)