Abstract
Our previous studies documented that human fibroblast-limbal stem cells (f-LSCs) possess immunosuppressive capabilities, playing a role in regulating T-cell activity. This study highlights the molecular activities by which human f-LSCs can attenuate the inflammatory responses of self-reactive peripheral blood mononuclear cells (PBMCs) collected from patients with autoimmune endocrine diseases (AEDs). Anti-CD3 activated PBMCs from twenty healthy donors and fifty-two patients with AEDs were cocultured on f-LSC monolayer. 2D-DIGE proteomic experiments, mass spectrometry sequencing and functional in vitro assays were assessed in cocultured PBMCs. We identified the downmodulation of several human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) isoforms in healthy and AED activated PBMCs upon f-LSC interaction. The reduction of hnRNPA2/B1 protein expression largely affected the cycling ki67+, CD25+, PD-1+ reactive cells and the double marked CD8+/hnRNPA2B1+ T cell subset. Anti-PD1 blocking experiments evoked hnRNPA2/B1 overexpression, attributing putative activation function to the protein. hnRNPA2/B2 transient silencing inverted immunopolarization of the self-reactive PBMCs from AEDs toward a M2/Th2-type background. Pharmacological inhibition and co-immunoprecipitation experiments demonstrated the involvement of NF-ĸB in hnRNPA2/B activity and turnover. Our data indicate cardinal involvement of hnRNP A2/B1 protein in peripheral mechanisms of tolerance restoration and attenuation of inflammation, identifying a novel immunoplayer potentially targetable in all AEDs.