Inhibition of EV secretion impairs MSC immunosuppression.
(a) Schematic representation of EV isolation protocol. (b) EV gate was carried out by using size-calibrated fluorescent beads ranging from 0.1 μm to 0.9 μm. The number EVs were calculated using 4.3 μm TruCount beads, which are shown in the upper right corner. The absolute count of EVs was subtracted to background noise events from 0.22 μm-filtered PBS. (c) Count of EVs obtained from MSCs treated for 48 hours with 10 μM GW4869, 10 μM imipramine, 60 μM DEVD and relative control vehicles, including: DMSO for GW4869 and DEVD; H2O for Imipramine. The results are expressed as percentage of relative EV-release inhibition, normalized to number of EVs obtained from untreated MSCs (100%). Resting and primed MSCs treated with GW4869, imipramine and DEVD were cultured in presence of activated CFSE labeled T, B and NK cells (d–f, respectively) in order to assess the effects of the inhibition of EV release on the immunomodulatory properties of MSCs. The results are expressed as relative proliferation percentage of IECs, normalized to IEC cultured alone (100%). Error bars represented mean ± SEM of four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.