Immunosuppressive properties of regulatory T cells generated by incubation of peripheral blood mononuclear cells with supernatants of human RPE cells.

PURPOSE To determine whether supernatants of human retinal pigment epithelium (RPE) cells can convert CD4⁺ T cells into regulatory T cells (Tregs) under Treg-induction conditions in vitro and in vivo. METHODS Peripheral blood mononuclear cells were cocultured with supernatants from TGFβ2-pretreated human RPE lines on anti-CD3-coated plates. Cells were then separated with a CD4⁺CD25⁺ Treg isolation kit and cultured with supernatants from RPE, anti-CD3/CD28 antibodies, high-dose IL-2, and TGFβ2. By flow cytometry sorting, CD25⁺CD45RA⁻ Tregs were separated. Expressions of CD25(high), Foxp3, CD152, and TNFRSF 18 on Tregs were analyzed by flow cytometry. Cytokine production by Tregs was measured by ELISA. Proliferation of target T cells was assessed by [³H]thymidine incorporation or CFSE incorporation. In addition, mouse RPE-induced Tregs were used for the in vitro assay and in vivo experimental autoimmune uveitis (EAU) models. RESULTS Human RPE-induced Tregs expressed higher levels of the Treg markers CD25(high), Foxp3, CD152, and TNFRSF 18. In addition, RPE-induced Tregs included significant numbers of CD4⁺CD25(high)Foxp3(high)CD45RA⁻ active effector Tregs that significantly suppressed the activation of Th1/Th17 cell lines, indicating that they have immunosuppressive properties. Furthermore, CD4⁺CD25(low)Foxp3(low)CD45RA⁻ nonsuppressing cytokine-secreting T cells were removed from the in vitro-manipulated Treg population. Administration of mouse RPE-induced Tregs significantly suppressed ocular inflammation in mice with EAU. In addition, the Tregs suppressed retinal antigen-specific T cells in vitro. CONCLUSIONS It is hoped that through the data provided in this study that Tregs might become useful as individualized therapeutic agents for ocular autoimmune diseases.