Human Bone Marrow Mesenchymal Stromal/Stem Cell-Derived Extracellular Vesicles Ameliorate Graft-Versus-Host Disease-Related Manifestations

A substantial proportion of patients who receive allogeneic hematopoietic stem cell transplantation develop an immunologic adverse event of acute graft-versus-host disease (GVHD). Cell therapy with bone marrow mesenchymal stromal/stem cells (BM-MSC) is one of the effective treatments for such patients with GVHD. Lines of evidence have suggested that BM-MSC inhibit the function of immune cells mainly through secretion of soluble immune modulators and partially through cell-to-cell contact-dependent mechanism. However, further understanding of how they work has been an important issue. Recently, extracellular vesicles (EV) gain interests as mediators based on their capacity of transmitting signals to sites of action regardless of physiological barriers to reach. In this study, we examined the effects of human BM-MSC-derived EV on clinical, histological and immunologic manifestations of the acute GVHD model mice. We also analyzed whether biological processes mediated by microRNA (miRNA) in human BM-MSC-derived EV are involved in such immunologic effects.

Human MSC were isolated from BM samples purchased from AllCells (Alameda, CA) based on our previously published method (Biochem Biophys Res Commun 2016;469:823). EV were isolated from the culture supernatant of BM-MSC by using the Total Exosome Isolation kit (Invitrogen, Carlsbad, CA). We confirmed that the isolated EV expressed CD63 and CD81 by immunoblot analysis and showed morphology of saucer-like shape by transmission electron microscopy. For in vitro experiments, peripheral blood mononuclear cells (PBMC) were activated by anti-CD3/CD28 antibodies with a Dynabeads Human T-Activator (Life Technologies AS, Oslo, Norway). For in vivo experiments, we used a mouse model where GVHD was induced by intravenously injecting spleen cells (1 x 10⁸ per mouse) from C57BL/6 mice (H-2^b) into irradiated (8 Gy) BDF1 mice (H-2^{b x d}). The severity of GVHD was assessed histologically and clinically as previously described method (Blood 1996;88:3230, J Clin Invest 1998;102:115).

We first tested the effects of BM-MSC-derived EV on T cell subsets in the expansion culture of anti-CD3/CD28-stimulated PBMC. In culture with human BM-MSC-derived EV, the expansion of PBMC was suppressed. Flow cytometric analysis showed that the expansion of CD3+ T cells including CD4+ and CD8+ T cells was suppressed. The frequency of CD45RA+Foxp3^low cells (naive Treg cells) was higher and that of CD45RA-Foxp3^high cells (effector Treg cells) was lower in culture with EV than those in a single culture of PBMC. In addition, the ratio of CD8/naive Treg cells was lower in culture with EV than that in a single culture of PBMC. Thus, BM-MSC-derived EV suppressed the T cell expansion and preserved naive Treg cells.

We then investigated the effects of BM-MSC-derived EV on acute GVHD in mouse model. Systemic infusion of human BM-MSC-derived EV significantly prolonged the survival of GVHD mice with the median survival period of 16 days while that was 10 days in control untreated GVHD mice. The clinical scores of BM-MSC-derived EV-treated mice were lower than those of the control mice, indicating that EV ameliorated systemic symptoms of GVHD. In the histological assessment of GVHD-targeted organs, EV treatment significantly mitigated GVHD-associated abnormalities in specimens particularly from large bowel. Flow cytometric analysis of PB from the BM-MSC-derived EV-treated GVHD mice indicated that both CD4+ and CD8+ T cells were suppressed, and CD62L-CD44+ T cells was decreased and CD62L+CD44- T cells was increased compared with the control untreated GVHD mice. In addition, EV-treated mice still contained substantial Treg population. Thus, EV suppressed functional differentiation of peripheral T cells from naïve phenotype to effector phenotype and did not enhance depletion of Treg cells. Collectively, BM-MSC-derived EV showed immunomodulatory effects in murine GVHD model.

Finally, we performed microarray analysis of microRNAs in BM-MSC-derived EV and found down-regulated cell proliferative processes as identified by Gene Ontology enrichment analysis.

This study revealed that BM-MSC-derived EV captured the therapeutic effects of cell-of-origin against GVHD in association with immunologic mechanism of T cell suppression and peripheral naïve T cell preservation. Our findings provide the basis of BM-MSC-mediated anti-GVHD effect with the EV-mediated mechanism.