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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

Immunosuppressive properties of MSC-derived EVs.EVs were purified from resting and primed MSCs and added to unfractionated PBMCs or sorted-T, B and NK cells that were activated by specific stimuli (1 × 104:3 × 106 IEC:EV ratio). At the end of co-cultures, the cells were harvested and IEC proliferation was assessed by FACS analysis. CFSE fluorescence was analyzed after 4 days for PBMCs, T and B cells (a–c, respectively), while for NK cells (d) the fluorescence was analyzed after 6 days of co-culture. The results are expressed as relative proliferation percentage of IECs, normalized to IEC cultured alone (100%). Error bars represented mean ± SEM of six for PBMCs, and five for sorted-T, NK cells and B cells independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
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f5: Immunosuppressive properties of MSC-derived EVs.EVs were purified from resting and primed MSCs and added to unfractionated PBMCs or sorted-T, B and NK cells that were activated by specific stimuli (1 × 104:3 × 106 IEC:EV ratio). At the end of co-cultures, the cells were harvested and IEC proliferation was assessed by FACS analysis. CFSE fluorescence was analyzed after 4 days for PBMCs, T and B cells (a–c, respectively), while for NK cells (d) the fluorescence was analyzed after 6 days of co-culture. The results are expressed as relative proliferation percentage of IECs, normalized to IEC cultured alone (100%). Error bars represented mean ± SEM of six for PBMCs, and five for sorted-T, NK cells and B cells independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: PKH26pos EVs were cultured in presence of PBMCs to verify the ability of IECs to internalize purified EVs (Supplemental Fig. S4a). We observed a trend of EV internalization by the different IEC subpopulations, similarly to that previously shown for MSC:IEC co-culture (Fig. 2). Thereafter, to validate the immunomodulatory effects of MSC-derived EVs, purified resting and primed EVs were added to IEC cultures. First, IECs were exposed to increasing quantities of EVs (data not shown) to identify the most suitable EV:IEC ratio for the standardization of the immunological assays. In the following experiments all IECs were treated with the same number of EVs (3 × 106 particles/1 × 104 IECs) obtained from resting or primed MSCs (Fig. 5). When assessing EV-dependent immunomodulation on PHA-stimulated PBMCs, no inhibitory effect on CD45pos cell proliferation was observed (Fig. 5a); similar results were obtained by analyzing only CD3pos cells by gating strategy on PBMCs (Supplemental Fig. S4b).


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)

Immunosuppressive properties of MSC-derived EVs.EVs were purified from resting and primed MSCs and added to unfractionated PBMCs or sorted-T, B and NK cells that were activated by specific stimuli (1 × 104:3 × 106 IEC:EV ratio). At the end of co-cultures, the cells were harvested and IEC proliferation was assessed by FACS analysis. CFSE fluorescence was analyzed after 4 days for PBMCs, T and B cells (a–c, respectively), while for NK cells (d) the fluorescence was analyzed after 6 days of co-culture. The results are expressed as relative proliferation percentage of IECs, normalized to IEC cultured alone (100%). Error bars represented mean ± SEM of six for PBMCs, and five for sorted-T, NK cells and B cells independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Immunosuppressive properties of MSC-derived EVs.EVs were purified from resting and primed MSCs and added to unfractionated PBMCs or sorted-T, B and NK cells that were activated by specific stimuli (1 × 104:3 × 106 IEC:EV ratio). At the end of co-cultures, the cells were harvested and IEC proliferation was assessed by FACS analysis. CFSE fluorescence was analyzed after 4 days for PBMCs, T and B cells (a–c, respectively), while for NK cells (d) the fluorescence was analyzed after 6 days of co-culture. The results are expressed as relative proliferation percentage of IECs, normalized to IEC cultured alone (100%). Error bars represented mean ± SEM of six for PBMCs, and five for sorted-T, NK cells and B cells independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: PKH26pos EVs were cultured in presence of PBMCs to verify the ability of IECs to internalize purified EVs (Supplemental Fig. S4a). We observed a trend of EV internalization by the different IEC subpopulations, similarly to that previously shown for MSC:IEC co-culture (Fig. 2). Thereafter, to validate the immunomodulatory effects of MSC-derived EVs, purified resting and primed EVs were added to IEC cultures. First, IECs were exposed to increasing quantities of EVs (data not shown) to identify the most suitable EV:IEC ratio for the standardization of the immunological assays. In the following experiments all IECs were treated with the same number of EVs (3 × 106 particles/1 × 104 IECs) obtained from resting or primed MSCs (Fig. 5). When assessing EV-dependent immunomodulation on PHA-stimulated PBMCs, no inhibitory effect on CD45pos cell proliferation was observed (Fig. 5a); similar results were obtained by analyzing only CD3pos cells by gating strategy on PBMCs (Supplemental Fig. S4b).

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