Conditioning mesenchymal stromal cells for immunomodulation in a model of autoimmune disease, Myasthenia gravis – CONDIMESO

Mesenchymal Stem/Stromal Cells (MSC) possess broad immunoregulatory capabilities, modulating both adaptive and innate immunity by direct cell-cell contact, secretion of potent molecules, and production of extracellular vesicles (EVs). Our previous work has indicated that MSC conditioned via co-culture with peripheral blood cells (PBMC) could present optimized immunomodulatory effects in the preclinical context of an auto-immune disease, Myasthenia gravis (MG). This neuromuscular disorder frequently involves thymus dysfunction and is due to the production of pathogenic autoantibodies directed against the neuromuscular junction. MG is characterized clinically by chronic abnormal and invalidating muscle fatigability which can be life-threatening. The immunosuppressive treatments have severe side-effects, therefore cellular therapy based on conditioned MSC (cMSC) or acellular therapy based on the EVs derived from cMSC, may represent innovative therapeutic tools.
Three objectives were defined to shift towards clinical perspectives. (A) We aim to understand the mechanisms of conditioning at molecular and cellular levels, and to provide useful phenotypic and functional readouts for the validation of cMSC and their EVs. (B) We aim to identify the soluble molecules responsible for conditioning to propose a combination of the most active ones as a substitute to the co-culture with PBMC. (C) We aim to evaluate and compare the immunomodulatory efficacy of cMSC and of their EVs in vitro and in our humanized animal model.
For this purpose, 5 Tasks were defined. (1) We will identify gene regulations upon conditioning, using the RNASeq analysis of MSC and PBMC interactions. This study will point the new immunomodulatory pathways linked to conditioning, and will suggest deregulated membrane-associated proteins as new candidate markers. A preliminary RNASeq comparison between resting and conditioned MSC showed differential expression of 244 genes. The validation of RNASeq study by RT-PCR, flow cytometry, and single-cell mass cytometry clustering (CyTOF) will provide mechanisms of action and useful readouts among the new candidate markers. A preliminary CyTOF study identified differences between cMSC and rMSC. (2) We will characterize the EVs produced by cMSC (size, quantity, protein contents, membrane markers, structure) using classical and new methodologies (Cytoflex S, MACSPlex). (3) We will assess the immunomodulation capacities of cMSC and EVs in vitro using PBMC inhibition assay, the analysis of changes in PBMC sub-populations by CyTOF, and the ELISA dosage of secreted immunomodulatory proteins. (4) Using a proteomic approach (Proseek Multiplex by Olink, Uppsala, Sweden), we identified proteins involved in conditioning and we will evaluate, compare, combine the efficacy of these proteins using medium-throughput screening up to define an optimal combination. (5) We will assess and compare the efficacies in vivo of MSC conditoned by either PBMC, or the optimal combination of molecules, and their EVs in our humanized mouse model, established by grafting fragments of MG thymus in immunodeficient mice. Biological and clinical parameters will be followed using classical tools (grip test, behavior, inverted grid) and newly added ones (electrmyography, voluntary exercise) and this will allow to compare, back to back, the potentials of these cellular and acellular approaches.
The project fosters original collaborations between academic research, clinicians and cell therapy laboratories. Our 4 teams share expertise in the production and characterization of cells and EVs, in screening, and in the pathophysiology of MG. The project will attempt to link regulated gene pathways, specific phenotypes, and immunomodulatory capacities. It may lead to new cell- or EV-based products poised to clinical applications, and the study of their therapeutic efficacy in MG may open perspectives towards treating other autoimmune pathologies.