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Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions.

Di Trapani M, Bassi G, Midolo M, Gatti A, Kamga PT, Cassaro A, Carusone R, Adamo A, Krampera M - Sci Rep (2016)

Bottom Line: Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease.We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division.Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Italy.

ABSTRACT
Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus

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.
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f3: 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.

Mentions: First, EVs were isolated by using differential centrifugation (Fig. 3a) and quantified by FACS analysis to confirm the effects of each inhibitor. Beads of different size (0.1 μm, 0.2 μm, 0.5 μm and 1.0 μm) were used for instrument calibration, defining the upper and the lower limits of EV gate with 1.0 μm and 0.1 μm beads, respectively (Fig. 3b). EV concentration was calculated by using Trucount Tubes containing a known number of 4.3 μm beads. GW4869 significantly reduced the EV release by more than 50%. By contrast, imipramine and DEVD (Fig. 3c) showed a lower inhibition, probably due to the EV isolation strategy (Fig. 3a).


Differential and transferable modulatory effects of mesenchymal stromal cell-derived extracellular vesicles on T, B and NK cell functions.

Di Trapani M, Bassi G, Midolo M, Gatti A, Kamga PT, Cassaro A, Carusone R, Adamo A, Krampera M - Sci Rep (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4829861&req=5

f3: 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.
Mentions: First, EVs were isolated by using differential centrifugation (Fig. 3a) and quantified by FACS analysis to confirm the effects of each inhibitor. Beads of different size (0.1 μm, 0.2 μm, 0.5 μm and 1.0 μm) were used for instrument calibration, defining the upper and the lower limits of EV gate with 1.0 μm and 0.1 μm beads, respectively (Fig. 3b). EV concentration was calculated by using Trucount Tubes containing a known number of 4.3 μm beads. GW4869 significantly reduced the EV release by more than 50%. By contrast, imipramine and DEVD (Fig. 3c) showed a lower inhibition, probably due to the EV isolation strategy (Fig. 3a).

Bottom Line: Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease.We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division.Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Italy.

ABSTRACT
Mesenchymal stromal cells (MSCs) are multipotent cells, immunomodulatory stem cells that are currently used for regenerative medicine and treatment of a number of inflammatory diseases, thanks to their ability to significantly influence tissue microenvironments through the secretion of large variety of soluble factors. Recently, several groups have reported the presence of extracellular vesicles (EVs) within MSC secretoma, showing their beneficial effect in different animal models of disease. Here, we used a standardized methodological approach to dissect the immunomodulatory effects exerted by MSC-derived EVs on unfractionated peripheral blood mononuclear cells and purified T, B and NK cells. We describe here for the first time: i. direct correlation between the degree of EV-mediated immunosuppression and EV uptake by immune effector cells, a phenomenon further amplified following MSC priming with inflammatory cytokines; ii. induction in resting MSCs of immunosuppressive properties towards T cell proliferation through EVs obtained from primed MSCs, without any direct inhibitory effect towards T cell division. Our conclusion is that the use of reproducible and validated assays is not only useful to characterize the mechanisms of action of MSC-derived EVs, but is also capable of justifying EV potential use as alternative cell-free therapy for the treatment of human inflammatory diseases.

No MeSH data available.


Related in: MedlinePlus