Regulatory T cells (Tregs) Suppress Corneal Neovascularization in High-Risk Corneal Transplantation via PD-L1

Abstract

Purpose : To examine the role of programmed death-ligand 1 (PD-L1) expressed by Tregs in modulating neovascularization in the transplanted grafts in a murine model of high-risk corneal transplantation.

Methods : Tregs were magnetically sorted from naïve or PD-L1^-/- C57BL/6 mice and cocultured with a mouse vascular endothelial cell line (MS1 VEC) for 4 hours on Matrigel. Subsequently, imaging was performed using bright field microscopy and tube length and junctions were analyzed by image J. We performed high-risk corneal transplantation where corneal tissue derived from C57BL/6 mice was transplanted in BALB/c mice. Post-transplantation, recipient mice were treated with (1) saline, (2) naïve Treg (1x10⁵ cells), or (3) PD-L1^-/- Tregs (1x10⁵ cells), subconjunctivally. The recipient mice were assessed for corneal neovascularization (CNV) for 2 weeks. At the end of the follow-up period the corneas were harvested and assessed for blood (stained with anti-CD 31 antibody) using confocal microscopy and CNV area was assessed by VesselJ.

Results : We observed significantly lower junction numbers and tube length on co-culturing VEC with Tregs compared to VEC alone (Junction numbers: p<0.0001; Tube length: p<0.0001). In contrast, co-culturing PD-L1^-/- Tregs with VEC cells resulted in significantly higher junction numbers (p=0.0013) and tube length (p=0.009) compared to co-culture with naïve Tregs. The recipients treated with naïve Tregs showed significantly lower CNV scores compared to saline (p=0.01) treated controls. However, this anti-angiogenic effect of Tregs was not observed on injecting animals with PD-L1^-/- Tregs (p=0.81). Moreover, the CNV area in recipients treated with saline was significantly higher than Treg-treated recipients (p=0.01) and comparable to animals injected with Tregs isolated from PD-L1^-/- mice (p=0.83).

Conclusions : PD-L1 expressed by Tregs plays an essential role in regulating CNV formation following high-risk penetrating keratoplasty.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.