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

Ayano Imai, Sunao Sugita, Yuko Kawazoe, Shintaro Horie, Yukiko Yamada, Hiroshi Keino, Kazuichi Maruyama, Manabu Mochizuki

Research output: Contribution to journalArticlepeer-review

17 Citations (Scopus)

Abstract

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 TGFb2-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 TGFb2. By flow cytometry sorting, CD25{thorn}CD45RA- Tregs were separated. Expressions of CD25high, 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 CD25high, Foxp3, CD152, and TNFRSF 18. In addition, RPE-induced Tregs included significant numbers of CD4{thorn}CD25highFoxp3highCD45RA- active effector Tregs that significantly suppressed the activation of Th1/Th17 cell lines, indicating that they have immunosuppressive properties. Furthermore, CD4{thorn}CD25lowFoxp3lowCD45RA- 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.

Original languageEnglish
Pages (from-to)7299-7309
Number of pages11
JournalInvestigative Ophthalmology and Visual Science
Volume53
Issue number11
DOIs
Publication statusPublished - 2012 Oct

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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