1. DC-EV induce GM-CSF signaling and convey a cornucopia of effector molecules.
DC-EV induce GM-CSF signaling and convey a cornucopia of effector molecules. (A) DC-EV induce Stat5 phosphorylation. Peripheral monocytes (2 × 105) were incubated with EV (10 μg) derived from imDC and maDC and 293T cells (control EV) or stimulated with GM-CSF/IL-4 (each for 15 min) or left untreated. Subsequently, cells were fixed and analyzed for Stat5 phosphorylation by intracellular FACS. FACS blots depict one representative experiment. Three healthy donors were analyzed to calculate the mean and SEM. (B) Anti-GM-CSF blocks DC-EV–induced Stat5 phosphorylation. Same experimental setup as in (A); however, one cell aliquot of each culture was left untreated or was supplemented with anti-GM-CSF. Triplicate cultures were performed for each donor (three donors) to calculate the mean and SEM. (C) DC-EV–derived GM-CSF is derived from the producer DC. Lysates of purified DC-EV and control EV (from 293T cells) was blotted for endogenous (endg.) GM-CSF using lysates of maDC (maDC lys.) and recombinant (recomb.) GM-CSF as control. (D, E) DC-derived EV contain multiple CCF. (D) EV were collected from monocytes and monocyte-derived imDC and maDC (50 μg) and subsequently analyzed for the indicated factors using commercially available protein arrays (RayBiotech). The pixel intensity of each dot was determined by ImageJ, and the value was adjusted in relation to the internal positive control, which was set to 1. Shown is one representative analysis performed with four different donors (see also Fig S4C). (E) Same experimental setup as in (D); however, the EV contents were analyzed using bead-based quantitative immunoassays (BioLegend). imDC-EV were analyzed from six and eight different donors for cytokine and chemokine content, respectively (gray columns). maDC-EV from six different donors were analyzed (black columns). Heat maps depict the common logarithm (log(10)) of the cytokine and chemokine concentrations of each individual sample. Bar graphs indicate mean values ± SEM. Statistical significance was analyzed by the t test: *P < 0.05, **P < 0.01, and ***P < Source data are available for this figure.
Stefan Schierer et al. LSA 2018;1:e
© 2018 Schierer et al.