Abstract
Background: Mesenchymal Stromal Cells (MSC) possess innate immunomodulatory properties, which can be significantly enhanced through co-culture with peripheral blood mononuclear cells (PBMC), making them attractive tools for the treatment of autoimmune and inflammatory diseases.Methods: Leveraging a multi-omics approach encompassing RNA sequencing, flow and mass cytometry, secretome analysis, completed by functional evaluations, we investigated the mechanisms underpinning PBMC conditioning of MSC in vitro and their benefits in an animal model of Myasthenia gravis. MSC derived from human adipose tissue were left untreated in resting state (rMSC), conditioned by PBMC (cMSC), or activated by the pro-inflammatory molecule interferon (IFN)-γ (γMSC), then compared for their gene expression profiles, phenotypes and functional capacities.Results RNA sequencing identified 244 differentially expressed genes in cMSC compared to rMSC, highlighting key immune mediators such as CCL2, CCL11, DPP4, ICAM1, IL6, PDCD1LG2, TNFRSF11B, TNIP1, TNIP3 and ZC3H12A and pinpointing genes involved in matrix remodeling, paracrine and autocrine communications. Comparatively, 2089 genes were differentially expressed between rMSC and γMSC, highlighting host defense, anti-viral response, NFκB signaling pathways modulated by IFN-γ. Flow and mass cytometry analyses revealed upregulation of the surface markers CD26, CD54, and CD273 and intracellular molecules IDO1 and PTGS2 in cMSC. In contrast, IFN-γ activation predominantly increased HLA-related markers while also enhancing the homogeneity of the populations. Together, these results underlined the treatment dependence of transcriptomic and phenotypic signatures. Secretome profiling identified 6 categories of modulated proteins, out of which 22 molecules potentially involved in PBMC conditioning and 40 implicated in cMSC-mediated immunomodulation. Functionally, cMSC induced modulation in PBMC subsets, raising the proportions of lymphocyte populations (CD4 Treg, CD8, B memory), underlining the multimodal effect of conditioning. Also, both a direct cell-cell contact and cMSC supernatants significantly suppressed activated T-cell proliferation in vitro. To confirm immunomodulation efficacy in vivo, cMSC were administrated to our humanized mouse model of Myasthenia Gravis and the treatment significantly halved disease severity from 2 weeks post-injection.Conclusions This integrative study establishes distinct conditioning signatures, suggests molecular mechanisms, and underscores the therapeutic potential of cMSC, offering a robust framework for advancing cell-based therapies in autoimmune diseases.