1. DC-derived EV differentiate monocytes.
DC-derived EV differentiate monocytes. (A) Monocytes ingest EV. Monocytes were incubated with DC-derived and GFP-labeled EV for 3 h, washed, and subsequently analyzed by confocal microscopy using Z-stack imaging. DIC: differential interference contrast. (B) maDC-EV differentiate a DC-like morphology in monocytes. Monocytes were incubated with the dose of EV (30 μg for 106 cells) derived from imDC and maDC or stimulated with GM-CSF/IL-4 for 6 d. Subsequently, images were taken from representative cells. (C) DC-EV induce proliferation in monocytes. PBMCs were labeled with CFSE and treated either with a single dose of imDC or maDC-derived EV (50 μg) or cytokines or LPS as indicated. CFSE dilution in CD11b+ cells was determined at day 1 and day 10 by flow cytometry, and the number of cell divisions is indicated. The graph summarizes the results from four different donors; one representative result is shown on the left side of the panel. Results are presented as mean ± SEM; statistical significance was analyzed by one-way ANOVA: *P < 0.05, **P < 0.01, and ***P < (D) maDC-EV–treated monocytes maintain a DC-like morphology upon exposure to maturation stimuli. Same experimental setup as in (B). Subsequently, cells were incubated for 24 h with a MC (IL-1β, IL-6, TNF-α, and PGE2) or LPS and images were taken from representative cells. (E) maDC-EV–treated monocytes that received a maturation stimulus induce T-cell proliferation. Monocytes incubated with imDC and maDC-derived EV (10 μg), or stimulated with GM-CSF/IL-4 (serving as positive control) for 6 d, either received a maturation stimulus (MC) or were left untreated. Subsequently, CFSE-labeled T cells were co-incubated at a defined ratio as indicated and proliferation of cells was determined by radiolabeled thymidine incorporation. Shown is one representative experiment of five performed with different donors (see Fig S3A). Scale bars represent 7.5 μm.
Stefan Schierer et al. LSA 2018;1:e
© 2018 Schierer et al.