Locally Administered Regulatory T Cells Modulate Corneal Neovascularization in High-Risk Corneal Transplantation

Abstract

Purpose : While in solid tumors and leukemia Treg are correlated with a heightened vascular response, in ischemia and ocular tissues they have been reported to inhibit angiogenesis. Here, we examined the role of Treg in modulating corneal neovascularization (CNV) following high-risk corneal transplantation.

Methods : Treg were MACS-sorted from naïve BALB/c mice, and the expression of PD-L1 was assessed by RT-PCR and flow cytometry. Treg were preincubated with or without anti-PD-L1 antibody (10 µg/ml) and cocultured with endothelial cells (MS-1) on Matrigel for 4 hours. Imaging was performed by bright field microscope and tube formation indices were quantified using ImageJ. High-risk corneal transplantation was performed using C57BL/6 as donors and BALB/c mice as recipients. After transplantation, recipient mice received subconjunctival injection of (1) saline, (2) Treg (10⁵ cells), or (3) PD-L1-blocked Treg (10⁵ cells). Mice were followed for 2 weeks and CNV was scored. Corneas were then harvested and stained with CD31 (blood vessels) and LYVE-1 (lymphatic vessels), imaged with confocal microscopy, and assessed for CNV area using VesselJ.

Results : PD-L1 was highly expressed in naive Treg. The mean number of vascular junctions and mean tube length were significantly lower when MS-1 was cocultured with Treg (10±2 and 3594±906) compared to MS-1 alone (75±6 and 8995±630) (p<0.001 and p<0.001, respectively). This inhibitory effect was abrogated by blocking PD-L1 on Treg (27±3 and 6317±362) (p=0.001 and p=0.009, respectively). In vivo assessment showed decreased CNV in graft recipients treated with Treg (6.8±0.4) compared to saline (7.9 ± 0.0) (p=0.01) and this inhibitory effect was not evident when PD-L1-blocked Treg were injected. Similarly, grafted-corneal explants from recipients treated with Treg showed a significantly smaller CNV area (35.7±1.5%) compared to those treated with saline (46.2±2.3%) (p=0.01) and this inhibitory effect was not observed in recipients treated with PD-L1-blocked Treg. Lymphangiogenesis was not affected by Treg (p=0.9 for all comparisons).

Conclusions : Locally administered Treg modulates CNV following high-risk corneal transplantation. Although PD-L1 expressed in Treg seems to be critical, other relevant antiangiogenic factors need to be additionally investigated.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.