Human limbal fibroblast-like stem cells induce immune-tolerance in autoreactive T lymphocytes from female patients with Hashimoto’s thyroiditis

AbstractBackground: Due to their “natural immune privilege” and immunoregulatory properties human fibroblast-likelimbal stem cells (f-LSCs) have acquired great interest as a potential tool for achieving immunotolerance. Hashimoto’sthyroiditis (HT) is the most common thyroid autoimmune disease and cause of hypothyroidism. To date, conventionalhormone replacement therapy and unspecific immunosuppressive regimens cannot provide a definitive cure for HTsubjects. We explored the immunosuppressant potential of human f-LSCs on circulating lymphomonocytes (PBMCs)collected from healthy donors and female HT patients.Methods: We assessed the immunophenotyping of f-LSCs, both untreated and after 48 h of proinflammatory cytokineexposure, by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and flow cytometry. Theimmunosuppressant effects of f-LSCs on healthy activated PBMCs were investigated in cell-cell contact and transwellsettings through cell cycle assay, acridine orange staining, and caspase-3 detection. We also studied T-cell responses andpossible Treg conversion by means of flow cytometry. Functional assays were conducted in activated HT lymphocytescocultured with f-LSCs after carboxyfluorescein succinimidyl ester labeling and intracellular detection of pro- and antiinflammatorycytokines.Results: The hypo-immunogenicity of the f-LSC population depended on both cell contact and soluble factorsproduced, as well as the undetectable expression of all those molecules required to fully activate T lymphocytes.Following exposure to Th1 cytokines, f-LSCs augmented expression of programmed death-ligand 1 and 2 (PDL-1 and-2), indoleamine-pyrrole-2,3-dioxygenase (IDO), interleukin (IL)-6, and monocyte chemotactic protein 1 (MCP-1) whilemaintaining their negative phenotype for major histocompatibility (MHC) class II and costimulatory molecules. Duringcoculture, f-LSCs suppressed up to 40% of proliferation in healthy activated PBMCs, arrested them in the G0/G1 cellcycle phase without inducing apoptosis cascade, inverted the CD4/CD8 ratio, and promoted sustained expression ofthe immunomodulator marker CD69. Under coculture conditions the Th imbalance of autoreactive T cells from femaleHT patients was fully restored. Conclusions: Our study describes an in vitro coculture system able to prevent inappropriate activation of autoreactive T lymphocytes of female HT patients and to generate a tolerogenic environment even in an inflammatory background. Further investigations are necessary to establish whether this stem cell-based therapy approach in HT could avoid lifetime hormone replacement therapy by inducing T-cell education.