Functional dosing of mesenchymal stromal cell-derived extracellular vesicles for the prevention of acute graft-versus-host-disease

Abstract

Graft‐vs‐host‐disease (GvHD) is currently the main complication of allogeneic hematopoietic stem cell transplantation. Mortality and morbidity rates are particularly high, especially in steroid‐refractory acute GvHD (aGvHD). Immune regulatory human bone marrow mesenchymal stromal cells (hMB‐MSCs) represent a therapeutic approach to address this issue. Unfortunately, their effect is hardly predictable in vivo due to several variables, that is, MSC tissue origin, concentration, dose number, administration route and timing, and inflammatory status of the recipient. Interestingly, human bone marrow MSC‐derived extracellular vesicles (hBM‐MSC‐EVs) display many of the hBM‐MSC immunoregulatory properties due to their content in paracrine factors that greatly varies according to the collection method. In this study, we focused on the immunological characterization of hBM‐MSC‐EVs on their capability of inducing regulatory T‐cells (T‐regs) both in vitro and in a xenograft mouse model of aGvHD. We correlated these data with the aGvHD incidence and degree following hBM‐MSC‐EV intravenous administration. Thus, we first quantified the EV immunomodulation in vitro in terms of EV immunomodulatory functional unit (EV‐IFU), that is, the lowest concentration of EVs leading in vitro to at least threefold increase of the T‐regs compared with controls. Second, we established the EV therapeutic dose in vivo (EV‐TD) corresponding to 10‐fold the in vitro EV‐IFU. According to this approach, we observed a significant improvement of both mouse survival and control of aGvHD onset and progression. This study confirms that EVs may represent an alternative to whole MSCs for aGvHD prevention, once the effective dose is reproducibly identified according to EV‐IFU and EV‐TD definition.

HS-5 and HS-27A Stromal Cell Lines to Study Bone Marrow Mesenchymal Stromal Cell-Mediated Support to Cancer Development

“…In order to validate our meta-analysis, we applied standardized assays to evaluate the immunological properties of immortalized cell lines. As previously reported by our group, the presence of inflammatory cytokines makes primary MSCs acquire an inflammatory phenotype characterized by increased expression of CD54 (I-CAM), CD106 (V-CAM), HLA-ABC, and HLA-DR (MHC-II), and CD274 (PD-L1) (Dal Collo et al, 2020). Furthermore, the inflammatory microenvironment induces a strong inhibitory effect on primary MSCs (primed MSCs, pMSCs), leading to the inhibition of immune responses mediated by different IECs (Di Trapani et al, 2016).…”

“…This phenomenon, called "MSCs licensing, " induces MSCs to become strongly inhibitory towards different immune effector cells (IECs) of both innate immunity, such as neutrophils, monocytes and natural killer (NK) cells, and adaptive immunity, such as T cells, B cells and dendritic cells (Krampera, 2011;Di Trapani et al, 2016). MSC-mediated immunosuppression has been confirmed by several preclinical and clinical studies related to a large spectrum of inflammatory and autoimmune diseases, such as Graft-versus-Host Disease, Crohn's disease, sepsis, colitis, acute kidney injury, autoimmune encephalomyelitis, and other disorders (García-Olmo et al, 2005;Le Blanc et al, 2008;Gonzalez-Rey et al, 2009;Patel and Genovese, 2011;Ciccocioppo et al, 2012;Ciccocioppo and Corazza, 2016;Dal Collo et al, 2020). The wellknown molecular mechanisms involved in MSC-mediated immunosuppression are represented by the up-regulation of several immunosuppressive molecules, including IDO1 and PD-L1 (Krampera, 2011;Di Trapani et al, 2016).…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles: Opportunities and Challenges for Clinical Translation

“…However, the protein or RNA content may quantitatively and qualitatively vary in function of MSC culture conditions or activation status and EV production method. In general, a dose-dependent effect of MSC-EVs has been observed using different functional assays (Cosenza et al, 2018;Bari et al, 2019a;Dal Collo et al, 2020). For instance, 50 µg of MSC-EVs are needed to induce the proliferation and differentiation of neural stem cells to oligodendrocytes (Otero-Ortega et al, 2020), while 10 µg of placenta-derived MSC-EVs are sufficient to increase the migration and tube formation of placental microvascular endothelial cells (Salomon et al, 2013).…”

Extracellular Vesicle-Dependent Communication Between Mesenchymal Stromal Cells and Immune Effector Cells

“…MSC-EVs may prevent aGvHD onset, attenuate symptoms, and prolong animal survival through several mechanisms. For instance, MSC-EVs is capable of reducing CD8 + T cell number, leading to the increase of CD4 + /CD8 + T cell ratio; in addition, they block CD4 + T cell migration and activation inside target organs, promote Treg expansion, downregulate IL-2, CCR6, TNF-α, and IFN-γ expression while increasing IL-10, reduce Th17 cell recruitment while lowering RORγτ, STAT3, IL-17, IL-21, IL-22 expression (Wang et al, 2016;Fujii et al, 2018;Lai et al, 2018;Zhang et al, 2018a;Dal Collo et al, 2020). Other potential MSC-EV immunomodulatory mechanisms on T cells involve miR-223 and the adenosine metabolism.…”

“…Recently, a functional in vitro assay was suggested to assess the MSC-EV therapeutic dose (EV-TD) in vivo in a mouse model of aGvHD; EV-TD, associated with the improvement of mouse overall survival, corresponded to 10-fold the EV immunomodulatory functional unit (EV-IFU), i.e. the lowest concentration in vitro of resting MSC-EV-pool leading to at least threefold increase of Tregs compared to control (Dal Collo et al, 2020). Nevertheless, all these assays need to be validated in a large cohort of patients before being accepted as predictive methods of MSC-EV therapeutic efficacy.…”