"Unveiling novel mechanisms of immune tolerance loss through small RNA-seq of CD4+ T cell-derived extracellular vesicles in multiple sclerosis" in ISEV2022 Abstract Book

Introduction: An impairment of immune tolerance is a determining factor in relapsing-remitting multiple sclerosis (RR-MS) and defects in CD4+ regulatory T (Treg) cell function are believed to be a major pathogenic factor. While CD4+ conventional T (Tconv) cells are known to substantially contribute to the autoimmune and inflammatory processes occurring in MS, the biological effect of extracellular vesicles released by Tconv cells (Tconv-EVs) on the differentiation and function of Treg cells remains uncharacterized. This study aimed at filling this gap in knowledge. Methods: Human Tconv cells isolated from blood of naïve to treatment RR-MS patients and healthy controls were in vitro stimulated and Tconv-EVs isolated by size exclusion chromatography and characterized by electron microscopy, flow cytometry and nanoview’s technology. Small RNA-seq was used to profile Tconv-EV RNA cargo. Then, the transcriptome of human Treg cells in vitro treated with Tconv-EVs was quantified by RNA-seq. Relevant experimental data have been submitted to the EVTRACK knowledgebase (EV-TRACK ID: EV210300). Results: Tconv-EVs from RR-MS patients demonstrated a significantly different molecular content in terms of small RNA moieties, compared with healthy controls. Through a bioinformatic approach, some of these molecules, in particular belonging to the microRNA family, could be linked to the differential effect of these two types of Tconv-EVs on Treg cell transcriptome, in particular on the transcripts involved in Treg cell growth and regulatory function. Summary/Conclusion: Our results have unveiled novel molecular mechanisms which may causally link the Tconv cell dysregulation in MS with a potential suppressive effect of their released EVs on Treg cell-mediatedmaintenance of self-tolerance in these patients. Funding: This work is supported by Fondazione Italiana Sclerosi Multipla (FISM, grant 2018/R/4) to P.d.C. and V.D.R.