Distinct targeting and uptake of platelet and red blood cell-derived extracellular vesicles into immune cells

Abstract

Blood-derived extracellular vesicles (EVs) hold great therapeutic potential. As blood contains mixed EV populations, it is challenging to study EVs originating from different cells separately. Blood cell concentrates manufactured in blood banks offer an excellent non-invasive source of blood cell-specific EV populations. To study blood cell-specific EVs, we isolated EVs from platelet (TREVs) and red blood cell (EryEVs) concentrates and characterized them using nanoparticle tracking analysis, imaging flow cytometry, electron microscopy and western blot analysis and co-cultured them with peripheral blood mononuclear cells (PBMCs). Our aim was to use imaging flow cytometry to investigate EV interaction with PBMCs as well as study their effects on T-lymphocyte populations to better understand their possible biological functions. As a conclusion, TREVs interacted with PBMCs more than EryEVs. Distinctively, TREVs were uptaken into CD11c+ monocytes rapidly and into CD19+ B-lymphocytes in 24 h. EryEVs were not uptaken into CD11c+ monocytes before the 24-h time point, and they were only seen on the surface of lymphocytes. Neither TREVs nor EryEV were uptaken into CD3+ T-lymphocytes and no effect on T-cell populations was detected. We have previously seen similar differences in targeting PC-3 cancer cells. Further studies are needed to address the functional properties of blood cell concentrate-derived EVs. This study demonstrates that imaging flow cytometry can be used to study the distinctive differences in the interaction and uptake of EVs. Considering our current and previous results, EVs present a new valuable component for the future development of blood-derived therapeutics.

Keywords: blood cell; extracellular vesicle; imaging flow cytometry; platelet; red.

FIGURE 1

Schematic design of the study illustrating the materials and methods used and highlighting the most significant steps in the workflow of this study. CFSE, ((5(6)‐CFDA, SE; CFSE (5‐(and‐6)‐carboxyfluorescein diacetate, succinimidyl ester; CryoEM, cryogenic electron microscopy; EV, extracellular vesicle; Far‐Red, Far‐Red CellMask labelling; NTA, nanoparticle tracking analysis; SEM, scanning electron microscopy. Figure created in Biorender.

FIGURE 2

EV characterization using nanoparticle tracking analysis, western blotting, scanning electron microscopy (SEM), cryogenic electron microscopy (cryoEM) and imaging flow cytometry. (a) Particle size distribution measured with the Zetaview PMX‐120 (ParticleMetrix). The mean particle size in TREV samples was 126 nm (SD ± 9.9 nm, n = 6 biological replicates) and in EryEVs 185 nm (SD ± 11.3 nm, n = 6 biological replicates). There is a significant difference in particle size distribution between TREVs and EryEVs. (b–e) Scanning electron microscopy images illustrate the size and shape difference between EryEVs (b and d) and TREVs (c and e), the tubular shape of EryEVs is highlighted. Original magnifications are ×50,000 in (b), ×100,000 in (C) (scale bar 1 µm), ×200,000 in (d) (scale bar 400 nm) and ×250,000 in (e) (scale bar 200 nm). (f–g)) Cryogenic electron microscopy images that show the lipid bilayer composition on single vesicles in (f) EryEV and (g) in TREV samples. (h, i) Representative panels from imaging flow cytometry images of (h) CD41‐labelled (AF647, Biolegend) TREVs and (i) CD235a‐labelled (APC, BD Bioscience) EryEVs and their corresponding brightfield images. (j) Western blot analysis shows that ApoB48, ApoB100 (both bands), CD9, CD41 and CD63 were present in TREV samples while ApoA1 and CD235a were absent and were detected only in EryEV samples.

FIGURE 3

EV interaction and uptake with peripheral blood mononuclear cells (PBMCs). EVs labelled with Far‐Red CellTrace. Most interaction was seen between TREVs and CD11c+ monocytes (a). 0.5 × 10⁶ PBMCs were administered with 1 × 10¹⁰ Far‐Red‐labelled TREVs or EryEVs. Cells were identified using CD11c FITC (Invitrogen), CD19 Bright Violet 510 (BioLegend) and CD3 PE (Invitrogen) and analysed with Amnis® ImageStreamX Mark II imaging flow cytometer (Luminex). (a) Shows EV interaction with CD11c+ cells, (b) interaction with CD19+ B‐lymphocytes and (c) interaction with CD3+ T‐lymphocytes. (a–c) show the percentage of cells that have EVs either only on the membrane or also uptaken into cells. Data are presented as mean ± SD, n = 4. Representative image panels from Amnis® ImageStreamX Mark II imaging flow cytometry, showcasing a brightfield image on Ch01, fluorescently labelled cells from Ch02, Ch03 and Ch08, Far‐Red‐labelled EVs on Ch11 and a merged image to illustrate EV interactions in different cells after either 30 min, 2 h or 24 h of co‐culture.

FIGURE 4

EVs have no effects on main T‐cell populations under 4‐day co‐culture. Percentage of CD4+, CD8+ or CD25+ cells of CD3+ T‐lymphocytes presented as mean ± SD, (n = 6 for TREVs and EryEV treated samples and n = 3 for DPBS control and 0‐h sample). Measured with Amnis® ImageStreamX Mark II imaging flow cytometry. No significant changes were seen, determined with multiple Whitney–Mann U tests, ns = p‐value over 0.05.

FIGURE 5

EVs have no effects on lymphocyte proliferation. After a 5‐day co‐culture, proliferation of lymphocytes was determined by Far‐Red CellMask proliferation dye intensity. Measured with Amnis® ImageStreamX Mark II imaging flow cytometry. In theory, the intensity of each generation is half of the previous generation and the proliferation rate was studied by gating each generation. CD3 antibody‐coated plates were used to see if EVs had inhibitory effects, CD3 antibody‐coated wells as such and with CD28 antibody were used as positive controls, and DPBS‐treated wells as negative control. TREV or EryEV treatment does not affect proliferation by either activating or inhibiting it.