GITR-Ligand-Dependent Local Expansion of Regulatory T Cells as a Mechanism of Immune Privilege for Corneal Allografts

Abstract

Purpose: : The pathway between glucocorticoid-induced TNF receptor family-related protein (GITR) and GITR ligand (GITRL) has been shown to control the function of regulatory T cells (Treg). We have demonstrated Foxp3⁺CD25⁺CD4⁺ T cells infiltrating into corneal allografts play a role in graft acceptance, and that GITRL expressed on corneal endothelium plays a role in Treg-mediated immune suppression within the cornea. To further substantiate the GITRL-mediated protection of corneal allografts from effector T cells, we evaluated the killing of corneal endothelial cells (CECs) by CD4⁺ T cells and induction of Tregs by GITRL expressed in cornea, in vitro.

Methods: : Fresh normal corneas from C57BL/6 eyes were incubated with anti-GITRL mAb or control rat IgG for 2 h. Magnetic cell sorting and separation was used to purify CD4⁺ T cells from the spleen of BALB/c mice that were pre-sensitized by subcutaneous immunization with C57BL/6 splenocytes or with third-party (C3H/He) splenocytes, or from the spleen of naive BALB/c, C57BL/6 or C3H/He mice. Cornea pre-treated with anti-GITRL mAb or control rat IgG was incubated with 2.5 ×10⁵ T cells. Unfixed corneal samples were incubated with propidium iodide (PI) to stain nuclei of dead CECs. PI-positive cells were counted at 3 randomly selected areas in the corneal endothelium of each corneal sample using confocal microscopy. The population of Foxp3⁺CD25⁺CD4⁺ T cells after incubation with cornea was assessed by flow cytometry.

Results: : The killing of CEC by allo-reactive T cells in vitro was significantly enhanced in corneas pre-treated with anti-GITRL mAb as compared to those pre-treated with control IgG. Interestingly, GITRL-mediated protection was also observed after incubation with T cells, irrespective of prior sensitization. The population of Foxp3⁺CD25⁺CD4⁺ T cells was increased after incubation with GITRL-expressing cornea, but not with GITRL-blocked cornea.

Conclusions: : GITR-L expressed on CECs plays a substantial role in the protection of CECs from destruction by CD4⁺ T cells. GITRL-dependent expansion of induced-regulatory T cells within the cornea is one of the mechanisms underlying immune privilege in corneal allografts.

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