UNRAVELING THE POTENTIAL ROLE OF A SMALLEXTRACELLULAR VESICLE LYOPHILIZED ENRICHEDIN IMMUNE AND INFLAMMATORY REGULATOR FACTORS AS IMMUNORESPONSE MODULATOR INPATIENTS SUFFERING FROM AUTOIMMUNE THYROID DISEASES

In Autoimmune Thyroid Diseases (AITDs), a loss of toler-ance to thyroid antigens and lymphocyte infiltration into the thyroid gland occurs. An imbalance in T helper type (Th)-17 and regulatory T lymphocytes (Tregs) contributes to perpetuate the inflammatory state leading to thyroid dysfunction and disruption. Although the replacement of hormone production represents the main aim in therapeu-tic treatment, a deeper understanding of the mechanism regulating the immune response improving inflammation could represent an important outcome in AITDs. In our previous study, we described an in vitro coculture model elucidating the ability of Th-1 cytokines licensed fibrob-last-like Limbal mesenchymal Stem Cells (f-LSCs) to reg-ulate T-cell activity in patients suffering from AITDs by an altered cytokine profile via downregulation of human het-erogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) isoforms, which were found overexpressed in peripheral blood mononuclear cells (PBMCs) collected from patient suffering from AITDs1-2. Unfortunately, the clinical application of human mesenchymal stem cells (MSCs) is hampered by legal and ethical issues. Noteworthy, MSC immunomodulator capability depends also by soluble products, which could be conveyed by Small Extra Vesicles (sEVs), nanoscale cell-derived struc-ture of 200 nm less in size. Data here reported demon-strate for the first time that a reconstituted lyophilized of sEVs (sEVLyo) derived TH-1 cytokines licensed f-LSCs retains the anti-inflammatory and immunomodulation capabilities in activated PBMCs from AITD patients, simi-lar to f-LSCs. As the lack of comparative studies assessing the efficacy of different EV isolation techniques hinders clinical use of sEVs, we firstly compared two different sEV isolation methods tangential flow filtration (TFF) and pre-cipitation. Once obtained, sEVs were lyophilized and char-acterized for size distribution, protein content, and purity by detection of CD63, CD9, CD81, specific sEVs surface markers. To explore the sEV anti-inflammatory and immune modulation properties, activated PBMCs from AITD patients were exposed to different concentrations of sEVLyo and several functional biological assays were per-formed. We demonstrate that sEVs improve the autoreac-tive response in PBMC from AITD patients, inhibiting CD8+Tcell proliferation, CD69+ and CD25+ expansion with-in CD4+Tcells. Among several anti-inflammatory and immunosuppressive markers modulated, i.e. IDO, PDL-1, MCP-1 and IL-4, hnRNP A2/B1 were found downregulated in activated PBMC under sEVLyo exposure. Finally, we pro-pose that TH-1 licensing enriched sEV in specific benefi-cial factors, including PD-L1, COX-2, TXN-1. Indeed, we confidently promote the lyophilization as a valid storage method to satisfyingly preserve functional sEVs, encour-age their emerging role as an alternative approach to stem cell therapy highlighting the possibility to modulate their protein content, once again suggesting hnRNP A2/B1 as potential target in AITDs.