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Khrishen Cunnusamy, Peter W. Chen, Jerry Y. Niederkorn; Cytokine Requirement, Molecular Mechanisms, and Kinetics of CD4+CD25+ T Regulatory Cells in the Establishment of Corneal Immune Privilege. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1146.
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© ARVO (1962-2015); The Authors (2016-present)
This study examined the mechanisms CD4+CD25+ T regulatory cells (Tregs) utilize in promoting corneal allograft survival.
BALB/c mice bearing C57BL/6 corneas were injected with anti-IL-17A, anti-CD25 or a rat IgG isotype control antibody. CD4+CD25+ Tregs were isolated from graft acceptors and rejectors 3 weeks post transplantation. A CFSE-based suppression assay was used to assess the IL-17A requirement for Treg function by incubating Tregs with naïve CFSE-labeled CD4+ effector T cells in vitro with anti-CD3 antibody. Cytokine production from acceptor CD4+ T cell subsets was quantified by ELISA. Transwell assays were performed to test contact-dependent suppression. For blocking assays, antibodies to cytotoxic T-lymphocyte antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor family-related gene ligand (GITRL) and transforming growth factor beta (TGFβ) 1, 2, 3 were used.
BALB/c mice rejected 50% of the C57BL/6 corneal allografts. Treatment with either anti-IL-17A or anti-CD25 antibody led to 90% and 100% rejection, respectively. In vivo and in vitro neutralization of IL-17A decreased the suppressive activity of Tregs by ~50-70%. Acceptor CD4+CD25- T cells selectively produced IL-17A. In transwell assays, contact-inhibition between the IL-17R expressing acceptor Tregs and CD4+ effector T cell responders led to ~80% loss in suppression. Acceptor Tregs treated in vitro with anti-IL-17A displayed a ≥2 fold decrease in CTLA-4, GITR and TGF-β1 expression by qPCR analysis. Simultaneous blockade of all three molecules in vitro impaired acceptor Treg-mediated suppression by ~50%. In vivo Treg activity was necessary only during the early phases following corneal transplantation as treatment with either anti-CD25 or anti-IL-17A did not affect the survival of corneal allografts if initiated 30 days following corneal transplantation.
Maintenance of ocular immune privilege is mediated transiently by IL-17A and Tregs during initial establishment of the corneal allograft. Depletion of IL-17A impairs CD4+CD25+ Treg function by diminishing the expression of CTLA-4, GITR and membrane-bound TGF-β1.
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