Mesenchymal stromal cells (MSC) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 hours to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 hours post-irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500 cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% DMSO at −80°C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells.

Aims/hypothesis Mesenchymal stem cells (MSCs) can exert an immunosuppressive effect on any component of the immune system, including dendritic cells (DCs), by direct contact, the release of soluble markers and extracellular vesicles (EVs). We evaluated whether MSCs and MSC-derived EVs have an immunomodulatory effect on monocyte-derived DCs in type 1 diabetes. Methods Bone marrow derived MSCs were characterised and EVs were obtained by ultracentrifugation. DCs were differentiated from CD14 + cells, obtained from nine type 1 diabetic patients at disease onset, pulsed with antigen GAD65 and cultured with MSCs or EVs. Levels of DC maturation and activation markers were evaluated by flow cytometry. GAD65-pulsed DCs and autologous CD14− cell were cocultured and IFN-γ enzyme-linked immunosorbent spot responses were assayed. Secreted cytokine levels were measured and Th17 and regulatory T cells were analysed.Results MSC-and EV-conditioned DCs acquired an immature phenotype with reduced levels of activation markers and increased IL-10 and IL-6 production. Conditioned DC plus T cell co-cultures showed significantly decreased IFN-γ spots and secretion levels. Moreover, higher levels of TGF-β, IL-10 and IL-6 were detected compared with unconditioned DC plus T cell co-cultures. Conditioned DCs decreased Th17 cell numbers and IL-17 levels, and increased FOXP3 + regulatory T cell numbers. EVs were internalised by DCs and EV-conditioned DCs exhibited a similar effect. Conclusions/interpretation In type 1 diabetes, MSCs induce immature IL-10-secreting DCs in vitro, thus potentially intercepting the priming and amplification of autoreactive T cells in tissue inflammation. These DCs can contribute to the inhibition of inflammatory T cell responses to islet antigens and the promotion of the anti-inflammatory, regulatory responses exerted by MSCs.

These results provide evidence that MSC-derived MVs can inhibit in vitro a proinflammatory response to an islet antigenic stimulus in type 1 diabetes. The action of MVs involves PGE2 and TGF-β signalling pathways and IL-10 secretion, suggesting a switch to an anti-inflammatory response of T cells.

Acute graft-vs-host disease (GVHD) is a serious complication after allografting. We carried out an exploratory study to investigate a potential correlation of surface antigens on extracellular vesicles (EVs) and acute GVHD. EVs were extracted from serum samples from 41 multiple myeloma patients who underwent allografting. EVs were characterized by flow cytometry using a panel of 13 antibodies against specific membrane proteins that were reported to be predictive of acute GVHD. We observed a correlation between three potential biomarkers expressed on EV surface and acute GVHD onset by both logistic regression analysis and Cox proportional hazard model. In our study, CD146 (MCAM-1) was correlated with an increased risk-by almost 60%-of developing GVHD, whereas CD31 and CD140-α (PECAM-1 and PDGFR-α) with a decreased risk-by almost 40 and 60%, respectively. These biomarkers also showed a significant change in signal level from baseline to the onset of acute GVHD. Our novel study encourages future investigations into the potential correlation between EVs and acute GVHD. Larger prospective multicenter studies are currently in progress.

HRP express miR-126, and this expression is down-regulated in diabetic-like conditions. Exposure of HRP to EV obtained in diabetic-like conditions is able to decrease miR-126 expression, consistently with previous observations of its involvement in DR and providing further insights into the role of EV in vessel destabilization. In contrast, PDGF and Ang-2 signalling pathways do not seem to be involved in these mechanisms.

INTRODUCTION: Graft-versus-Host-Disease (GVHD) is the main cause of non-relapse mortality after Hematopoietic Stem Cells Transplantation (HSCT). Identyfing potential biomarkers of GVHD could be crucial to define patients at high risk of GVHD development and to better assess GVHD grading. One potential attractive biomarker may be represented by extracellular vesicles (EVs). EVs are membrane-enclosed structures secreted by many cell types and may be represented by exosomes, shedding vesicles (microvesicles; MVs) and apoptotic bodies. EVs are detectable in body fluids, in particular peripheral blood (PB) and urine; EVs play a key role in the regulation of physiological and pathological processes. The aim of this study was to investigate EVs in PB of allotransplanted patients (EVs count, size and phenotype) and analyze a potential correlation with acute and chronic GVHD (aGVHD, cGVHD). METHODS: At our center, between 2000 and 2008, 41 multiple myeloma patients underwent an allograft. Median age of the patients was 53 years (range 34-65). Donors were HLA-identical siblings in 83% of the transplants. Conditioning regimen was mostly non-myeloablative (32/41,78%) and PB stem cells were used as source in all patients. GVHD Prophylaxis consisted of cyclosporine and mycophenolic acid in 34/41 patients (83%). Disease status was partial or less than partial remission in 95% of the patients. For standard policy, serum samples were collected before and monthly after transplant up to 6 months or disease relapse. EVs extraction was feasible due to high EVs stability in frozen serum samples. EVs were extracted using a protamine-based precipitation method and analyzed by flow-cytometry (Guava EasyCyte Flow Cytometer) without using latex-activated beads. We investigated a panel of fluorescence antibodies against specific membrane proteins (CD44, CD138, CD146, KRT18, CD120a, CD8, CD30, CD106, CD25, CD31, CD144, CD86, and CD140a). At each time-point, we determined for each sample: total EVs concentration, fluorescence distribution and percentage of positive EVs for a given marker. Data were computed by logistic regression analysis; Odds Ratio (OR) was calculated as proportional change as compared with pre-transplant baseline level of each marker. RESULTS: In our cohort, the cumulative incidence of aGVHD and cGVHD was 56% (95% 40.7-71.8%) and 71% (95% CI 56.3-85.2%) at day 100 and 24 months, respectively. Four biomarkers (CD146, CD25, CD106, CD31) showed a potential correlation with acute GVHD development. Two biomarkers (CD146 and CD25) were associated with an increased risk of developing aGVHD (CD146 fluorescence, OR 2.94, p=0.040 and CD25 fluorescence, OR 1.61, p<0.001). Furthermore, CD106 positive concentration (OR 0.23, p=0.077) and CD31 fluorescence and positive concentration (OR 0.24, p=0.052; OR 0.50, p=0.067, respectively) were associated with a decreased risk of aGVHD. All the biomarkers associated with aGVHD showed a proportional change from baseline in signal level before GVHD onset (an increase in case of CD146 and CD25, a reduction with CD106 and CD31, respectively). No statistically significant association was observed between our panel of biomarkers and cGVHD. CONCLUSIONS: In this study, we observed a potential association between 4 biomarkers expressed on EVs' surface and aGVHD. CD146, CD31 and CD106 belong to Cell Adhesion Molecule family (MCAM-1, PECAM-1 and VCAM-1, respectively), which are crucial for endothelium and immune cells interaction. CD25 is IL2 Receptor and is a marker of immune activation and inflammation. All these proteins mentioned above, could have a role in acute GVHD pathogenesis. A perspective study is currently ongoing at our Center to validate these data. Disclosures Boccadoro: CELGENE: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Abbivie: Honoraria; Mundipharma: Research Funding; SANOFI: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

Extracellular vesicles (exosomes and microvesicles) have been found to deliver both mRNA and transcriptional modulators to target cells and affect their phenotype. Marrow mesenchymal stem cell derived vesicles (MSC-DV) have also been shown to reverse renal and hepatic injury. We have studied the capacity of MSC-DV to reverse radiation injury to marrow stem cells. B6.SJL mice were exposed to 100 centigrade whole body radiation (WBI), and 4, 24, and 168 hours later marrow is harvested and established in culture for 24-48 hours with either human or murine marrow-derived mesenchymal stem cells (MSV-DV) or vehicle. These cells were then transplanted into 950 cGy exposed C57/BL mice and engraftment evaluated at 3 weeks to 8 months. Alternatively cells were engrafted without competition into 200 centigrade exposed mice. Significant (p<0.05) restoration of engraftment was seen in each setting; restoration of secondary engraftment was also seen. Mice were also subjected to 500 cGy WBI and injected at 6, 24 and 72 hours with human MSC-DV and peripheral blood counts determined. Granulocyte levels were restored to 86% of control at 3 weeks post irradiation and significant restoration seen at multiple other time points. Restoration was seen was seen in both peripheral blood and marrow. Further studies on the murine hematopoietic cell line, FDC-P1 exposed to 500 centigrade showed dramatic recovery on exposure to murine or human MSC-DV. Additional work, employing differential ultracentrifugation as a separative technique for vesicles, showed that a preparation with both exosomes and microvesicles was superior to either microvesicles or exosomes alone. Studies with irradiated FDC-P1 cells indicated that exposure to vesicles decreased apoptosis. These studies indicate that vesicles from marrow-derived mesenchymal stem cells from different sources have the capacity to reverse radiation damage to bone marrow stem cells. Thus administration of MSC-DV to patients exposed to radiation could represent an important new strategy in radiation mitigation. Disclosures No relevant conflicts of interest to declare.

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