Background/Purpose: Mesenchymal stromal cells (MSCs) are progenitor cells with suppressive capacities that have fueled ample translation for the treatment of immune mediated diseases including Graft versus Host Disease (GVHD), one of the human indications with regulatory approval. Paracrine effects of the MSC secretome contribute to these actions, but recent evidence suggests that cell-to-cell transfer of MSC mitochondria to different tissues (MitoT) exerts significant regenerative and restoring effects. We hypothesized that MitoT would also encompass immunocompetent cells, and provide the first evidence herein of MitoT to T cells, relating it to a novel immune reprogramming mechanism of MSC therapy, tested both in-vitro and in-vivo, in an animal model of GVHD.

Methods: MitoT was assessed by flow cytometry, qPCR and confocal microscopy of human mononuclear cells (PBMCs) previously co-cultured in vitro with mitotracker (mitochondrial stained) umbilical cord MSCs at different ratios and conditions. To assess the direct functional impact of MitoT we employed the artificial transfer of MSC-derived mitochondria (“Mitoception”)¹ to PBMCs. Mitotracker-positive (Mito+) acceptor cells were sorted for further testing and compared to Mitotracker-negative (Mito-) sorted cells. Experimental readouts included target cell phenotype and function, immune-related gene expression by RNA sequencing and qPCR, and metabolic “Seahorse” analysis of OXPHOS and glycolytic functions. In vivo effects were tested in a humanized xenogenic model of GVHD in NSG immunodeficient mice.

Results: Dose-dependent MitoT was observed from MSCs to T and B lymphocytes, NK and Dendritic cells (100%), reaching 60%-90% in CD3+CD4+ cells according to their state of activation. Transfer was independent of proinflammatory MSC pre-treatment, inhibition of gap junctions, hemi-channels or nanotubule formation. The supression of MitoT at 4°C and the detection of human-specific mitochondrial genes (qPCR) in murine MSC-cocultured lymphoid cells confirmed the mediation of an energy dependent process, not attributable to cell fusion.

Genetic analysis showed most significant changes of mRNA expression in Treg activation pathways, including FOXP3, IL2RA, CTLA4, TGFβ and Runx1. Indeed, naïve Mito+ sorted T cells were driven towards a CD127low, CD25+ FoxP3+ (Treg) functionally proven suppressor phenotype as opposed to Mito- cells (fold change of 14) (p<0.01). Preliminary metabolic analysis of MitoT+ PBMCs displayed a 50% increase in the Glycolytic/OXPHOS ratio, which is a hallmark of T cell activation.

In vivo testing of xeno-GVHD induced by human Mito+ cells as compared to non-treated cells, showed significant improvement in weight loss (p<0.05), gut (p<0.05) and liver (p<0.01) tissue damage, organ infiltrating CD4+, CD8+ (p<0.05) and IFNγ+ cells (p<0.0001) as well as survival (p<0.05).

Conclusion: These findings present the first evidence of MitoT from MSC to T-lymphocytes and immune cell subpopulations, pointing to a new level of complexity of the immunoregulatory role of MSCs, widening the horizon of their clinical applications.

¹Patent application PCT/EP2015/066190. Fondecyt 1170852.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/mesenchymal-stromal-cell-transferred-mitochondria-elicit-an-immune-function-reprogramming-that-entails-t-regulatory-cell-commitment-and-clinical-improvement-of-graft-vs-host-disease/