IMMUNOLOGICAL EFFECTS OF EXTRACELLULAR VESICLES FROM BONE MARROW-DERIVED MESENCHYMAL STEM CELLS ON GLIADIN-SPECIFIC T-CELLS

Background & Aim: Mesenchymal stem cells (MSCs) are multipotent progenitor cells endowed with strong immunomodulant effects potentially useful to treat immune-mediated diseases. However, their therapeutic application carries several hurdles that may be overcome by using their extracellular vesicles (EVs) that contain an array of bioactive molecules. The aim is to evaluate the immunoregulatory effects of EVs from bone marrow MSCs on pathogenic gliadin-specific T-cell lines (TCLs) obtained from intestinal biopsies of celiac disease (CD) patients. Methods, Results & Conclusion: A pure population of MSCs was obtained following standard procedure from bone marrow blood samples harvested from four adult donors and expanded until passage 3rd to 4th and phenotypically characterized. EVs were obtained by two rounds of ultracentrifugation of supernatants collected at 80% confluence of MSCs cultured in medium deprived of FBS. The size and surface profile of EVs were assessed by cytofluorimetric analyses. For generation of TCLs, mononuclear cells were isolated from endoscopic duodenal biopsies of 6 CD patients (F/M 4/2, mean age: 53 years, range 35-64) by enzymatic digestion, and expanded through weekly stimulation with deamidated peptic-tryptic digested gliadin (PT-gliadin, 50 ?g/ml), interleukin(IL)-2 (40 U/ml) and IL-15 (10 ng/ml). In functional assays, TCLs were re-stimulated with PT-gliadin in the absence (ctr-TLCs) and presence of EVs obtained from MSCs unlicensed or licensed with interferon (IFN)-g (25 ng/mL/daily) for 3 to 7 days. Both EV samples were used at 2, 4, 8 ?g/ml concentration. EVs obtained from fibroblasts were used as controls at the same doses. After 48 hours of incubation, IFN-g production was assessed by ELISA. A significant and dose-dependent reduction of IFN-g production in response to gliadin stimulation was observed in all analyzed TCLs using EVs from both unlicensed (p=0.05 at the EV concentration of 8 ?g/ml) and licensed MSCs (p=0.04 at the EV concentration of 8 ?g/ml). No alteration of T-cell response was observed with EVs from fibroblasts at any tested dose. Moreover, the incubation with EV batches from both kind of MSCs did not alter the T-cell viability. It appears clear that EVs from bone marrow-derived MSC are able to dampen antigen-specific inflammatory response in CD that may be exploited for therapeutic purpose.