Abstract
BackgroundDue to their “natural immune privilege” and immunoregulatory properties human fibroblast-like limbal stem cells (f-LSCs) have acquired great interest as a potential tool for achieving immunotolerance. Hashimoto’s thyroiditis (HT) is the most common thyroid autoimmune disease and cause of hypothyroidism. To date, conventional hormone replacement therapy and unspecific immunosuppressive regimens cannot provide a definitive cure for HT subjects. We explored the immunosuppressant potential of human f-LSCs on circulating lymphomonocytes (PBMCs) collected from healthy donors and female HT patients.MethodsWe assessed the immunophenotyping of f-LSCs, both untreated and after 48 h of proinflammatory cytokine exposure, by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and flow cytometry. The immunosuppressant effects of f-LSCs on healthy activated PBMCs were investigated in cell-cell contact and transwell settings through cell cycle assay, acridine orange staining, and caspase-3 detection. We also studied T-cell responses and possible Treg conversion by means of flow cytometry. Functional assays were conducted in activated HT lymphocytes cocultured with f-LSCs after carboxyfluorescein succinimidyl ester labeling and intracellular detection of pro- and anti-inflammatory cytokines.ResultsThe hypo-immunogenicity of the f-LSC population depended on both cell contact and soluble factors produced, 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) while maintaining their negative phenotype for major histocompatibility (MHC) class II and costimulatory molecules. During coculture, f-LSCs suppressed up to 40% of proliferation in healthy activated PBMCs, arrested them in the G0/G1 cell cycle phase without inducing apoptosis cascade, inverted the CD4/CD8 ratio, and promoted sustained expression of the immunomodulator marker CD69. Under coculture conditions the Th imbalance of autoreactive T cells from female HT patients was fully restored.ConclusionsOur 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.
Highlights
Due to their “natural immune privilege” and immunoregulatory properties human fibroblast-like limbal stem cells (f-LSCs) have acquired great interest as a potential tool for achieving immunotolerance
Further investigations are necessary to establish whether this stem cell-based therapy approach in Hashimoto’s thyroiditis (HT) could avoid lifetime hormone replacement therapy by inducing T-cell education
Immunophenotype of f-LSCs f-LSCs at a relatively early passage exhibited the expected fibroblast-like morphology and expressed the phenotypic markers shown in Fig. 1a and 1b. quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and flow cytometry analysis were performed to determine the presence of key factors involved in immunoregulation
Summary
Due to their “natural immune privilege” and immunoregulatory properties human fibroblast-like limbal stem cells (f-LSCs) have acquired great interest as a potential tool for achieving immunotolerance. It has been proposed that mesenchymal stem cells (MSCs) can contribute to the control of inflammatory diseases, as has been demonstrated by the MSCmediated attenuation of inflammation in myocarditis [1], rheumatoid arthritis [2], and experimental autoimmune diseases [3, 4]. Due to their regenerative and immunosuppressive properties, MSCs derived from different adult tissue have become a preferred cell type in the field of regenerative medicine and immunotherapy. Limitations for the use of CB-MSCs include insufficient quantity and quality of MSCs obtainable from a single unit of CB, the potential for transfer of genetically abnormal or premalignant cells in patients, and the lack of a National Cord Blood Policy able to manage daily operations of cord blood banking and allocation nationwide
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