Cytokine Requirement, Molecular Mechanisms, and Kinetics of CD4+CD25+ T Regulatory Cells in the Establishment of Corneal Immune Privilege

Abstract

Purpose: : This study examined the mechanisms CD4⁺CD25⁺ T regulatory cells (T_regs) utilize in promoting corneal allograft survival.

Methods: : BALB/c mice bearing C57BL/6 corneas were injected with anti-IL-17A, anti-CD25 or a rat IgG isotype control antibody. CD4⁺CD25⁺ T_regs 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 T_reg function by incubating T_regs 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.

Results: : 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 T_regs by ~50-70%. Acceptor CD4⁺CD25^- T cells selectively produced IL-17A. In transwell assays, contact-inhibition between the IL-17R expressing acceptor T_regs and CD4⁺ effector T cell responders led to ~80% loss in suppression. Acceptor T_regs 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 T_reg-mediated suppression by ~50%. In vivo T_reg 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.

Conclusions: : Maintenance of ocular immune privilege is mediated transiently by IL-17A and T_regs during initial establishment of the corneal allograft. Depletion of IL-17A impairs CD4⁺CD25⁺ T_reg function by diminishing the expression of CTLA-4, GITR and membrane-bound TGF-β1.

Keywords: cornea: basic science • transplantation • immune tolerance/privilege