B7–H1–Mediated Protection of Corneal Endothelial Cells From Killing by Allo–Reactive T Cells in vitro

Abstract

Purpose: : Programmed Death–1 (PD–1) / B7–H1 costimulatory pathway has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We have demonstrated that cornea of normal mouse eyes constitutively express B7–H1, and that PD–1/B7–H1 pathway has a role in corneal allograft survival by inducing apoptosis of infiltrating cells. To further substantiate the B7–H1–mediated protection of corneal allografts from effector T cells, we evaluated the corneal endothelial cell (CEC) destruction by allo–reactive T cells in vitro.

Methods: : Fresh normal cornea from C57BL/6 eyes was incubated with anti–B7–H1 mAb or control rat IgG for 2 h. Magnetic cell sorting and separation was used to purify T cells from the spleen of BALB/c mice that were pre–sensitized by subcutaneous immunization with C57BL/6 spleen cells or with third–party (C3H/He) spleen cells, or from the spleen of naive BALB/c, C57BL/6, or C3H/He mice. The cornea pre–treated with anti–B7–H1 monoclonal antibody (mAb) or control rat IgG was incubated with 2.5x10⁵ T cells. The unfixed corneal samples were incubated with 50 ug/ml propidium iodide (PI) to stain nuclei of dead CEC. PI–positive cells were counted at three randomly selected areas in the corneal endothelium of each corneal sample using confocal microscopy.

Results: : The killing of CEC by allo–reactive T cells in vitro was significantly enhanced in the corneas pre–treated with anti–B7–H1mAb as compared to those pre–treated with control IgG (p=0.0016). Interestingly, B7–H1–mediated protection was also observed after incubation with third–party–reactive T cells (p=0.0248). Moreover, the B7–H1–mediated protection was still observed even after incubation with naive allogeneic T cells (p=0.0051). In contrast, no significant difference was observed between the anti–B7–H1 mAb and control rat IgG–treated corneas when syngeneic C57BL/6 T cells were used as the effector.

Conclusions: : B7–H1 expressed on CEC plays a substantial role in the protection of CEC from destruction by allo–reactive effector T cells. B7–H1 protects cornea from bystander injury by activated T cells. Moreover, constitutive expression of B7–H1 in cornea may inhibit the peripheral sensitization, since naive T cells were sensitized by B7–H1–deficient allogeneic cornea to promote their injury.

Keywords: immune tolerance/privilege • transplantation • immunomodulation/immunoregulation