Role of PD-L1 in Regulatory T Cell–Mediated Suppression of Corneal Neovascularization

Shima Dehghani; Manuel Chacon; Katayoon Forouzanfar; Seokjoo Lee; Akitomo Narimatsu; Rohan Bir Singh; Aytan Musayeva; Francesca Kahale; Sonia Anchouche; Neda Heydarian; Shilpy Bhullar; Thomas H Dohlman; Yihe Chen; Tomas Blanco; Reza Dana

doi:10.1016/j.ajpath.2025.08.008

. 2025 Sep 9;195(12):2463–2476. doi: 10.1016/j.ajpath.2025.08.008

Role of PD-L1 in Regulatory T Cell–Mediated Suppression of Corneal Neovascularization

Shima Dehghani ¹, Manuel Chacon ¹, Katayoon Forouzanfar ¹, Seokjoo Lee ¹, Akitomo Narimatsu ¹, Rohan Bir Singh ¹, Aytan Musayeva ¹, Francesca Kahale ¹, Sonia Anchouche ¹, Neda Heydarian ¹, Shilpy Bhullar ¹, Thomas H Dohlman ¹, Yihe Chen ¹, Tomas Blanco ¹, Reza Dana ^1,^∗

PMCID: PMC12799401 PMID: 40946796

Abstract

Corneal neovascularization (NV) leads to inflammation and fibrosis, thereby compromising visual acuity and corneal graft survival. Despite existing antiangiogenic therapies, clinical outcomes remain suboptimal. This study explores the antiangiogenic potential of regulatory T cells (Tregs) through programmed death-ligand 1 (PD-L1). In vitro tube formation assays were performed by co-culturing MS1 endothelial cells with Tregs. In addition, murine corneal suture–induced NV and high-risk corneal transplantation models were used to evaluate the effects of subconjunctival Treg injections in vivo. Wild-type (WT) Tregs inhibited endothelial tube formation, whereas PD-L1^−/− Tregs failed to exert this effect. Blocking B7-1 on MS1 cells attenuated the inhibitory effect of WT Tregs, indicating a PD-L1–B7-1 interaction as a key mechanism. Furthermore, vascular endothelial growth factor A expression in MS1 cells was significantly reduced on co-culturing with WT Tregs, a response that was absent with PD-L1^−/− Tregs. In vivo, subconjunctival Treg injections significantly reduced corneal NV in both corneal suture–induced NV and high-risk corneal transplantation models, and this effect was lost on blocking PD-L1. These results show that PD-L1 expressed by Tregs plays a pivotal role in suppressing corneal angiogenesis, acting through a contact-dependent mechanism through B7-1, leading to down-regulation of vascular endothelial growth factor A, highlighting the function of PD-L1 in Treg modulation of corneal angiogenesis.

Graphical Abstract

Corneal neovascularization (NV) is a pathologic process causing vision loss, and it is a common complication in ocular trauma and inflammatory, infectious, and degenerative disorders.¹ In the United States, an estimated 1.4 million individuals are affected by corneal NV annually.² The pathologic changes associated with cornea NV can result in scarring, edema, lipid deposition, and inflammation, which not only impair vision but also have a deleterious impact on the outcomes of corneal transplantation.³^,⁴ Although penetrating keratoplasty achieves a 90% survival rate in avascular, uninflamed, low-risk host eyes, the presence of corneal NV in the host bed at the time of transplantation (termed high-risk penetrating keratoplasty) reduces graft survival, with high rejection rates ranging from 30% to >50%.⁵

Despite the available interventions, such as anti–vascular endothelial growth factor (VEGF) therapies, matrix metalloproteinase inhibitors, multi-kinase inhibitors, and surgical ablation, no therapy has been fully effective in suppressing corneal NV.⁶ Corticosteroids, although widely used, are associated with adverse effects such as elevated intraocular pressure, cataract formation, and increased susceptibility to infections.³^,⁶ Similarly, anti-VEGF therapies have limited efficacy, with pilot studies showing that subconjunctival and topical administration does not significantly improve graft survival in high-risk penetrating keratoplasty.⁷

Given this therapeutic gap, novel strategies are a priority in managing corneal NV. Regulatory T cells (Tregs) have been shown to play a critical role in promoting immune tolerance in corneal transplantation, with multiple studies reporting their capacity to suppress the generation of alloreactive T cells and secrete anti-inflammatory cytokines such as IL-10 and transforming growth factor-β.8, 9, 10, 11 Previous work by this laboratory has specifically showed that adoptive transfer of Tregs significantly improves graft survival in low-risk corneal transplantation.⁸

However, whether Tregs possess additional functions such as modulating corneal angiogenesis has not been fully explored. The evidence in the literature indicates both antiangiogenic and pro-angiogenic effects of Tregs, depending on the tissue milieu.¹² Notably, the antiangiogenic effect of Tregs on retinal neovessels has been shown in a murine model of oxygen-induced retinopathy.¹³ These findings raise the possibility that Tregs could serve a dual role in corneal transplantation by both suppressing alloimmunity8, 9, 10, 11 and limiting NV, thus improving graft outcomes.

Building on the previous work, the current study explores a previously unrecognized function of Tregs in suppressing corneal NV. Among several Treg-associated molecules, programmed death-ligand 1 (PD-L1) was selected for mechanistic investigation due to its high expression in naive Tregs and its established role in immune regulation. Previous work by this laboratory has also suggested a direct antiangiogenic role for PD-L1,¹⁴ beyond its classical immune checkpoint function. Herein, we investigate the mechanisms by which Treg-expressed PD-L1 modulates angiogenesis in vitro and assess its therapeutic relevance in vivo by using murine models of suture-induced corneal NV and high-risk corneal transplantation.

Materials and Methods

Cell Culture and Tube Formation Assay

The MS1 cell line (CRL-2279; ATCC, Manassas, VA) was cultured in Dulbecco’s modified Eagle’s medium (Corning Inc., Corning, NY) supplemented with 1.0 g/L d-glucose, 862 mg/L l-glutamine, 110 mg/L sodium pyruvate, and 10% heat-inactivated bovine serum at 37°C in 5% carbon dioxide–containing humidified air. MS1 mouse endothelial cells (CRL-2279; ATCC) were used for in vitro tube formation assays. This cell line was selected for its reproducible tube-forming ability and prior use in corneal NV studies.14, 15, 16

For the tube formation assay, MS1 cells were seeded onto Cultrex Reduced Growth Factor Basement Membrane Extract Type 2 (Bio-Techne Corporation, Minneapolis, MN) in 96-well plates.¹⁷ Each well was coated with 50 μL of Cultrex, followed by the addition of 15 × 10³ MS1 cells suspended in 50 μL Dulbecco’s modified Eagle’s medium, with or without 2 × 10³ Tregs. After 4 hours of incubation at 37°C, tube formation was assessed by using bright-field microscopy. Quantitative image analysis was conducted by using the Angiogenesis Analyzer¹⁸ in ImageJ software version 1.53t (NIH, Bethesda MD; https://imagej.net/ij) to measure tube length and junction number as indicators of angiogenesis.¹⁷ Treg-conditioned media (wild-type Tregs, PD-L1 knockout Tregs, or PD-L1–blocked Tregs) were generated by incubating Tregs in Dulbecco’s modified Eagle’s medium for 4 hours. Supernatants were collected and used for tube formation assays.

In separate experiments, recombinant mouse PD-L1 (R&D Systems, Minneapolis MN) was supplemented at 25 ng or 100 μg to the culture medium. To neutralize B7-1 (CD80) on MS1 cells, anti-mouse B7-1 monoclonal antibody (1G10; Bio X Cell, Lebanon, NH) was applied at 0.6 μg/mL for 30 minutes at 37°C, based on prior studies showing its specific interaction with B7-1.¹⁹^,²⁰ The addition of the latter antibody itself did not produce a significant effect on tube formation. In all Treg-related experiments, CD4⁺CD25^– conventional T cells were used as controls.

Animals and Anesthesia

Eight-to ten-week–old female Balb/c (H-2^d) and C57BL/6 (H-2^b) mice were obtained from Charles River Laboratories (Wilmington, MA). The animals were housed in a pathogen-free animal vivarium at the Schepens Eye Research Institute. All the procedures conducted on the animals adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the Public Health Policy on Humane Care and Use of Laboratory Animals. Anesthesia was induced with ketamine (120 mg/kg) and xylazine (20 mg/kg). For in vitro experiments, splenic Tregs were derived from male PD-L1 knockout (PD-L1^−/−) C57BL/6 mice (provided by Arlene Sharpe, MD), as described previously.²¹

T-Cell Isolation and PD-L1 Blockade

CD4⁺CD25⁺ Tregs and CD4⁺CD25^– T cells were isolated from naive Balb/c and PD-L1^−/− C57BL/6 mice using magnetic separation with Treg isolation kits (Miltenyi Biotec, Somerville, MA). These freshly isolated cells were used without prior activation, expansion, or exposure to inflammatory stimuli.

For PD-L1 blockade, 2 × 10⁵ freshly isolated Tregs were incubated in RPMI 1640 (Lonza Inc., Lexington, MA) with 10 μg/mL neutralizing anti-mouse PD-L1/B7-H1 antibody (R&D Systems) for 1 hour. The cell number was selected based on preliminary experiments showing preserved viability and FoxP3 expression postincubation. The PD-L1 blocking antibody concentration was selected based on preliminary experiments showing this concentration to yield similar effects on tube formation as PD-L1 knockout Tregs. PD-L1–blocked Tregs were washed with Dulbecco’s modified Eagle’s medium before co-culture in tube formation assays or with phosphate-buffered saline before subconjunctival injection.

Suture-Induced Inflammatory Corneal Angiogenesis

Corneal NV was induced by placing three interrupted 11-0 nylon sutures (Mani, Utsunomiya, Japan) intrastromally with two stromal incursions extending over 120 degrees of the corneal circumference in Balb/c (H-2^d) mice.²² In one experimental series, mice were administered a subconjunctival injection of either saline (50 μL) or 1 × 10⁵ freshly isolated CD4⁺CD25⁺ Tregs (in 50 μL saline) at the time of suture placement. In a separate series designed to assess the role of PD-L1, three treatment groups were used: i) control (saline), ii) Treg-treated (1 × 10⁵ Tregs), and iii) PD-L1–blocked Treg-treated (1 × 10⁵ Tregs pretreated with anti–PD-L1 antibody). All treatments were administered via subconjunctival injection (50 μL) at the time of suturing.

High-Risk Corneal Transplantation and Subconjunctival Treg Injection

To establish high-risk graft beds, corneal NV was induced 14 days before transplantation using the method described in Suture-Induced Inflammatory Corneal Angiogenesis. Allogeneic corneal transplantation was performed by using vascularized Balb/c recipient beds and age-matched C57BL/6 donor corneas. Donor corneas (2-mm diameter) were excised and sutured onto the recipient graft beds created by excising a 1.5-mm site in the central cornea of Balb/c mice.²²

Tregs were isolated from the spleen of naive Balb/c mice (using the method described in T-Cell Isolation and PD-L1 Blockade), suspended in phosphate-buffered saline, and injected into the subconjunctival space of graft recipients using a 30-gauge metal needle after corneal transplantation, as described previously.⁸ Mice (n = 24) were randomized into three groups (n = 8 per group): i) control (subconjunctival saline, 50 μL), ii) Treg-treated (subconjunctival Treg, 10⁵ cells/50 μL), and iii) PD-L1–blocked Treg-treated (subconjunctival PD-L1-blocked Treg, 10⁵ cells/50 μL). NV was assessed at days 7 and 14 by using slit-lamp biomicroscopy. NV was quantified by using a standardized grading system (0 to 3 per quadrant; total range: 0 to 12), as previously described.²² Corneal sutures were removed 7 days after surgery, and grafts were evaluated for NV and opacity on days 3, 7, and 14 posttransplantation using slit-lamp biomicroscopy. A standardized scoring system was used to quantify NV (range, 0 to 8+) and opacity (range, 0 to 5+) after transplantation.²²^,²³

Corneal Whole-Mount and Immunofluorescent Staining

Freshly excised corneas (day 14 posttransplantation) were fixed in 99.5% ethanol; blocked with 5% bovine serum albumin, 5% goat serum, and 0.3% Triton X-100; and then incubated overnight with anti-mouse CD31 (PECAM-1; FITC, eBioscience, Thermo Fisher Scientific, San Diego CA) and anti-mouse LYVE1 (eFluor 660; eBioscience, Thermo Fisher Scientific) at 1:100 dilution. Corneas were mounted in Vectashield with DAPI (Vector Laboratories, Burlingame, CA) and imaged using a laser scanning confocal microscope (Heidelberg Retina Tomograph III with Rostock Corneal Module; Heidelberg Engineering GmbH, Heidelberg, Germany). ImageJ software 1.53t and the plugin of Rabiolo et al²⁴ were used to quantify vessel-covered area. Area of corneal NV was calculated as: (vessel area in the graft/corneal graft area) × 100.

Real-Time Quantitative PCR

Total RNA was extracted by using the RNeasy kit (Qiagen, Hilden, Germany), and cDNA was synthesized by using the QuantiTect Reverse Transcription Kit (Qiagen). Gene expression was quantified by real-time quantitative PCR using the StepOne Real-Time PCR System (Applied Biosystems Inc.) and Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). GAPDH was used as an internal control, and each sample was analyzed in triplicate for each experiment (n = 3). The primer sequences are listed in Table 1.

Table 1.

Primer Sequences of Genes Assessed Using Real-Time Quantitative PCR

Gene	Primer sequence
Mouse Vegfa	F: 5′-CAGGCTGCTGTAACGATGAA-3′ R: 5′-AATGCTTTCTCCGCTCTGAA-3′
Mouse Vegfr2	F: 5′-GCTTTCGGTAGTGGGATGAA-3′ R: 5′-TTGGTGAGGATGACCGTGTA-3′
Mouse Il10	F: 5′-CATGGGTCTTGGGAAGAGAA-3′ R: 5′-AACTGGCCACAGTTTTCAGG-3′
Mouse Ifng	F: 5′-GCGTCATTGAATCACACCTG-3′ R: 5′-TGAGCTCATTGAATGCTTGG-3′
Mouse Dll4	F: 5′-TGCCTCTCGAACTTGGACTT-3′ R: 5′-TGGAAATACAGATGCCCACA-3′
Mouse Tnfrsf1a	F: 5′-ACCAAGTGCCACAAAGGAAC-3′ R: 5′-ATTCTGGGAAGCCGTAAAGG-3′

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F, forward; R, reverse.

Statistical Analysis

Data were analyzed by using an unpaired two-tailed t-test and two-way analysis of variance with Tukey’s post hoc test. Statistical significance was set at P < 0.05. Analyses were performed by using GraphPad Prism version 10.5.8 for Mac (GraphPad Software, Boston, MA).

Results

Tregs Suppress Vessel Formation in Vitro and in Vivo

To assess the effect of Tregs on angiogenesis, a tube formation assay was performed on a gelled basement matrix. Under control conditions, MS1 endothelial cells formed an interconnected tubular network within 4 hours. However, the addition of Tregs to the co-culture system significantly reduced both the number of junctions (8.0 ± 4.5 vs 51.0 ± 4.0; P = 0.002) and total tube length (3968.0 ± 932.5 pixels vs 7949.0 ± 395.2 pixels; P = 0.01) (Figure 1).

Regulatory T cells (Tregs; CD4⁺CD25⁺) suppress angiogenesis *in vitro* and *in vivo*. A: Representative images of the tube formation assay performed using MS1 mouse endothelial cells under three experimental conditions: MS1 cells cultured alone, MS1 cells co-cultured with Tregs, and MS1 cells cultured in Treg-conditioned medium. Images were captured at 4 hours’ post-culture. B: Quantitative analysis of tube formation indices in MS1 cells co-cultured with Tregs. Image analysis revealed a significant reduction in both the number of junctions and total tube length upon co-culture with Tregs compared with control conditions. C: Quantitative analysis of tube formation indices in MS1 cells cultured in Treg-conditioned medium, displaying a similar inhibitory effect compared with direct co-culture, suggesting a contact-independent antiangiogenic mechanism. D: Representative images of murine corneas illustrating the inhibitory effect of subconjunctival Treg injection on corneal neovascularization (NV) in the suture-induced NV model. E: Quantitative analysis of corneal NV scores at day 7, showing a significant reduction in suture-induced NV in Treg-treated eyes compared with controls. Data represent a representative experiment from three independent replicates. Error bars indicate SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Original magnification: ×200 (A). Scale bars: 200 μm (A); 0.5 mm (D). CM, control culture medium; N-Treg, Tregs derived from normal mice; TM, Treg-conditioned medium.

To evaluate the extent of the antiangiogenic effect of Tregs through contact-independent mechanisms, the tube formation assay was also performed using conditioned media from Treg cultures. A significant reduction in the number of junctions (10.6 ± 2.3 vs 51.0 ± 4.0; P = 0.001) and tube length (4627.0 ± 78.2 pixels vs 7949.0 ± 395.2 pixels; P < 0.001) was observed in MS1 cells exposed to Treg-conditioned medium compared with control medium (Figure 1). These results indicate that Tregs suppress angiogenesis via both contact-dependent and contact-independent mechanisms.

The antiangiogenic properties of Tregs were further validated in vivo by using the corneal suture–induced NV model. Subconjunctival injection of Tregs at the time of suture placement significantly reduced NV at both day 7 (NV score: 0.5 ± 0.2 vs 10.6 ± 2.3; P = 0.001) and day 14 postsuture placement (NV score: 3.0 ± 0.3 vs 2.2 ± 0.6; P = 0.005) compared with controls treated with phosphate-buffered saline (Figure 1).

Mechanistic Factors Underlying Treg-Mediated Antiangiogenic Effects

To elucidate the molecular basis of Treg-mediated antiangiogenic effect, the expression profile of antiangiogenic factors in Treg was analyzed by using real-time quantitative PCR. High levels of PD-L1 gene expression in Tregs was observed, which was corroborated by flow cytometry showing high PD-L1 surface expression (Figure 2).

Expression profile of antiangiogenic proteins in regulatory T cells (Tregs). Tregs were isolated through MACS sorting from naive Balb/c mice. A: Real-time quantitative PCR analysis exhibited high [programmed death-ligand 1 (PD-L1)] mRNA expression in wild-type Tregs. B: Representative flow cytometry plots showing the frequency of PD-L1–positive Tregs, gated on CD4⁺CD25⁺FOXP3⁺ cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PEDF, pigment epithelium-derived factor; PLG, plasminogen; sFLT-1, soluble fms-like tyrosine kinase-1; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; TSP1, thrombospondin.

To further investigate the role of PD-L1 in Treg-mediated suppression of angiogenesis, the effect of PD-L1^−/− and wild-type Tregs on tube formation was compared. The antiangiogenic effect observed with wild-type Tregs was markedly diminished when PD-L1^−/− Tregs were used, both in direct co-culture and in conditioned medium assays (Figure 3).

Programmed death-ligand 1 (PD-L1) is essential for regulatory T cell (Treg; CD4⁺CD25⁺)-mediated antiangiogenic effects. A: Representative images of the tube formation assay at 4 hours’ post–co-culture under five experimental conditions: MS1 mouse endothelial cells alone, MS1 cells co-cultured with wild-type Tregs, MS1 cells co-cultured with PD-L1 knockout (KO) Tregs, MS1 cells in Treg-conditioned medium, and MS1 cells in PD-L1^−/− Treg-conditioned medium. B: Image analysis showing a significant reduction in the number of junctions and tube length in MS1 cells co-cultured with wild-type Tregs, an effect that was abrogated when PD-L1^−/− Tregs were used. C: Similarly, the inhibitory effect of Treg-conditioned medium was reversed when using the medium derived PD-L1^−/− Tregs. D and E: Representative images and corresponding quantitative analysis of corneal neovascularization (NV) scores at days 7 and 14, showing that subconjunctival injection of wild-type Tregs significantly reduces corneal NV in the suture-induced NV model, whereas PD-L1–blocked Tregs fail to exert this effect. Data represent a representative experiment from three independent replicates. Error bars indicate SEM. ∗P < 0.05, ∗∗P < 0.01. Original magnification: ×200. Scale bars: 200 μm (A); 0.4 mm (D). CM, control culture medium (Dulbecco’s modified Eagle’s medium DMEM); PD-L1^−/− Treg, PD-L1-knockout Treg; TM, Treg-conditioned medium; PD-L1.KO.TM, programmed death-ligand 1–knockout regulatory T cell–conditioned medium; PD-L1.B.Treg, programmed death-ligand 1–blocked regulatory T cells.

The role of PD-L1 in mediating Treg-dependent angiogenesis suppression was further confirmed in vivo by using the corneal suture model. Subconjunctival injection of PD-L1–blocked Tregs failed to suppress NV to the same extent as wild-type Tregs. The result was significantly higher NV scores at both day 7 (3.7 ± 0.2 vs 0.5 ± 0.2; P = 0.001) and day 14 (7.2 ± 1.0 vs 3.0 ± 0.3; P = 0.005) after suture placement (Figure 3).

PD-L1:B7-1 Interaction Mediates the Antiangiogenic Function of Treg

To investigate the mechanism underlying PD-L1–mediated inhibition of angiogenesis, recombinant PD-L1 protein was added to MS1 cell cultures. PD-L1 significantly suppressed tube formation, as evidenced by a reduction in both the number of junctions and total tube length (Figure 4).

rogrammed death-ligand 1 (PD-L1) suppresses tube formation via interaction with B7-1 on endothelial cells. The effect of PD-L1 protein on angiogenesis was assessed via tube formation assay. A: Recombinant PD-L1 protein (25 ng/mL and 1 μg/mL) was added to cultured MS1 mouse endothelial cells, significantly reducing tube formation indices, including the number of junctions and total tube length. B: The inhibitory effect of PD-L1 was reversed when B7-1 on MS1 cells was blocked with a monoclonal antibody (0.6 μg/mL) before PD-L1 administration, confirming that PD-L1–mediated angiogenesis suppression is B7-1 dependent. C: The B7-1 blocking antibody alone did not affect tube formation, showing that the observed inhibition was specifically mediated through PD-L1–B7-1 interactions. Data represent a representative experiment from three independent replicates. Error bars indicate SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001.

The evidence in the literature suggests that programmed death receptor protein-1 (PD-1), a known receptor for PD-L1,²⁵ is not expressed on MS1 cells.¹⁴ However, recent studies indicate that PD-L1 can also bind with B7-1,²⁶^,²⁷ a receptor expressed by MS1 cells.¹⁴ It was observed that blocking B7-1 on MS1 cells before PD-L1 treatment significantly diminished its inhibitory effect on tube formation, confirming a PD-L1–B7-1 interaction as the key antiangiogenic mechanism (Figure 4).

Co-culture experiments further validated this mechanism. When MS1 cells were pretreated with a B7-1–blocking antibody before Treg co-culture, the antiangiogenic effect of Treg was significantly attenuated. Real-time quantitative PCR analysis of MS1 cells after Treg co-culture revealed a marked reduction in VEGF-A expression (P < 0.0001), an effect that was not observed in PD-L1^−/− Treg co-cultures. These findings highlight the role of Treg-expressed PD-L1 in suppressing endothelial cell VEGF-A expression via B7-1 interaction (Figure 5). A similar reversal of the inhibitory effect was observed when Tregs were treated with a PD-L1/B7-H1 neutralizing antibody before co-culture (Supplemental Figure S1).

Mechanistic insights into the programmed death-ligand 1 (PD-L1)–mediated antiangiogenic function of regulatory T cells (Tregs; CD4⁺CD25⁺). A: Tube formation assay showing that the antiangiogenic effect of Tregs was significantly reversed when B7-1 was blocked on MS1 mouse endothelial cells, confirming that Treg-mediated inhibition of angiogenesis occurs via PD-L1–B7-1 interaction. B: Real-time quantitative PCR (qPCR) analysis of MS1 cells co-cultured with wild-type Tregs showed a significant reduction in vascular endothelial growth factor A (VEGF-A) expression, an effect that was abolished in PD-L1^−/− Treg co-cultures. Data represent a representative experiment from three independent replicates. Error bars indicate SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. PD-L1^−/− Treg, programmed death-ligand 1–knockout regulatory T cell; B7-1.B.MS1, B7-1-blocked MS1 cells.

Treg-Mediated Suppression of Corneal NV in High-Risk Corneal Transplantation and the Role of PD-L1

Given the efficacy of Tregs in suppressing NV in the suture model, their effect was next evaluated in a high-risk corneal transplant setting characterized by severe corneal NV (Supplemental Figure S2).

Corneal transplantations were performed in high-risk recipients, and graft vascularization was monitored in three treatment groups: i) allogeneic hosts receiving subconjunctival saline, ii) allogeneic hosts receiving subconjunctival Tregs, and iii) allogeneic hosts receiving subconjunctival PD-L1–blocked Tregs (see High-Risk Corneal Transplantation and Subconjunctival Treg Injection). NV scores were recorded by using slit-lamp biomicroscopy over a 2-week period posttransplantation.

Treg-treated mice exhibited significantly lower NV scores compared with the saline-treated and PD-L1–blocked Treg groups. The NV scores were significantly reduced in Treg-treated mice compared with saline-treated and PD-L1–blocked Treg-treated mice at day 3 (NV score: 4.0 ± 0.1 vs 5.0 ± 0.9 and 4.2 ± 0.1; P < 0.0001) and day 7 (NV score: 6.8 ± 0.4 vs 7.8 ± 0.0 and 7.4 ± 0.2; P = 0.01) posttransplantation (Figure 6). However, in contrast to the prolonged antiangiogenic effect observed in the suture model, the Treg-mediated suppression of NV in high-risk transplantation was transient, with no significant difference observed at day 14. This suggests that the short-term efficacy of subconjunctival Treg injection may be insufficient to provide long-term suppression of NV in the high-risk transplant setting.

Subconjunctival regulatory T cell (Treg; CD4⁺CD25⁺) injection reduces corneal neovascularization (NV) in high-risk corneal transplantation, while programmed death-ligand 1 (PD-L1) blockade dampens this effect. A: Representative slit-lamp images of murine corneas at day 7 post-transplantation in three treatment groups: saline injection, Treg injection, and PD-L1–blocked Treg injection. B: Immunofluorescence staining of corneal whole mounts at day 14, labeled with anti-CD31 (green) for vascular endothelial cells, captured via laser scanning *in vivo* confocal microscopy. C: Schematic diagram illustrating the experimental design for *in vivo* studies. D: Quantification of corneal NV scores at days 3 and 7, showing significantly lower NV scores in the Treg-treated group compared with saline and PD-L1–blocked Treg groups. E and F: Image analysis of CD31-immunostained corneas at day 14, revealing significantly reduced NV area (%) in both the whole-mount corneas (E) and graft sites (F) of the Treg-treated group compared with controls. Data represent n = 8 per group. Error bars indicate SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001. Original magnification: ×10 (B). Scale bars = 0.5 mm (A and B). HR-PK, high-risk penetrating keratoplasty; PD-L1.B.Treg, programmed death-ligand 1–blocked regulatory T cells; SC, subcutaneous.

Discussion

The current study identifies, for the first time, a novel function of PD-L1 expressed by Tregs in modulating angiogenesis (Figure 7). Although PD-L1 is well established for its immunoregulatory functions, particularly in maintaining immune tolerance and suppressing alloreactive responses in corneal transplantation,28, 29, 30 these findings reveal a novel role of PD-L1 in inhibiting angiogenesis, specifically mediated by Tregs.

Regulatory T cell (Treg; CD4⁺CD25⁺)-mediated inhibition of angiogenesis requires programmed death-ligand 1 (PD-L1) signaling. **A, Aʹ:** Baseline angiogenesis conditions, showing MS1 endothelial cell proliferation, expression of B7-1 and vascular endothelial growth factor A (VEGF-A), and the expected neovascularization (NV) response following corneal suturing. **B, Bʹ:** Treg-mediated vascular suppression, where programmed death-ligand 1 (PD-L1) on Tregs engages B7-1 on MS1 cells, leading to VEGF-A down-regulation and reduced angiogenesis *in vitro* and reduced corneal (NV *in vivo*. **C, Cʹ:** Impact of PD-L1 deficiency on Treg-mediated angiogenesis suppression. PD-L1 KO Tregs fail to inhibit NV, both *in vitro* and *in vivo*. PD-L1^−/− Treg, PD-L1-knockout Treg.

The role of Tregs in promoting immune tolerance and suppressing alloimmune responses, particularly in corneal transplantation, is well established.8, 9, 10, 11 Tregs promote immune tolerance by suppressing the generation of alloreactive T cells from naive T cells, through direct contact and by secreting anti-inflammatory cytokines such as IL-10 and transforming growth factor-β; this creates a localized immunosuppressive environment at the graft site and in draining lymph nodes.³¹^,³²

Although the immunomodulatory functions of Tregs are well known, their potential role in regulating corneal angiogenesis has not been fully explored.8, 9, 10, 11 Corneal NV is a major risk factor for graft failure, as it disrupts the immune privilege of the avascular cornea.33, 34, 35 Recent studies suggest that Tregs can influence angiogenesis in a tissue- and context-dependent manner.¹² In solid tumors and hematologic malignancies, Tregs promote angiogenesis.36, 37, 38, 39 Conversely, in pathologic conditions such as limbal ischemia and oxygen-induced retinopathy, Tregs exhibit antiangiogenic functions.¹³^,⁴⁰

The findings of the current study add a new potential dimension to the role of Tregs in corneal transplantation by showing that, beyond their immunoregulatory effects,8, 9, 10, 11 they can suppress angiogenesis through a PD-L1–dependent mechanism. This suggests that Tregs may help regulate both alloimmunity8, 9, 10, 11 and vascular growth,¹^,³^,³⁴^,³⁵ two processes that significantly affect graft survival in high-risk settings.

In the current study, freshly isolated CD4⁺CD25⁺ Tregs suppressed endothelial tube formation and reduced VEGF-A expression in vitro, consistent with previous findings in nontumor models. Bansal et al⁴¹ reported that Tregs inhibited tube formation in cardiac endothelial cells. Similarly, Huang et al⁴² found a similar effect in pulmonary endothelium through Delta-like ligand 4–Notch signaling. However, contrasting results have been reported in certain tumor models and inflammatory or metabolic conditions. Li et al⁴³ showed that Helios^high Tregs from umbilical cord blood promoted tube formation and increased VEGF signaling in a leukemia setting. Similarly, Leung et al⁴⁴ found that Tregs enhanced angiogenesis in hyperglycemic conditions, in part through IL-10, and amphiregulin. These differences likely reflect differences in the Treg phenotype, tissue microenvironment, and the presence of inflammatory or metabolic stimuli. In this study, nonactivated, freshly isolated CD4⁺CD25⁺Tregs were used, which may explain the consistent antiangiogenic effect observed.

Similar findings were seen in vivo using two established models of corneal NV: suture-induced inflammation and high-risk corneal transplantation. In both settings, local delivery of freshly isolated CD4⁺CD25⁺ Tregs significantly reduced corneal NV. These results are consistent with prior studies showing that adoptive transfer of Tregs can suppress pathologic NV, including in the retina during oxygen-induced retinopathy¹³ and the lung in asthma.⁴² Together, these in vitro and in vivo findings suggest that Tregs may play a role in suppressing corneal angiogenesis beyond their established alloimmune regulation. These dual roles make them uniquely positioned as potential modulators of high-risk graft outcomes. Although the current study focused on Tregs, comparing their effects with those of standard anti-VEGF therapy in future experiments could provide helpful context for evaluating therapeutic potential. Interestingly, these effects were abrogated when PD-L1 was genetically deleted or pharmacologically blocked, indicating that PD-L1 played a critical role in the observed antiangiogenic effect.

Although several immunomodulatory molecules have been associated with Treg function, the current study prioritized PD-L1 for mechanistic investigation based on preliminary studies and its unique capacity to modulate both immune and vascular responses. The real-time quantitative PCR and flow cytometry analyses showed that PD-L1 was the most highly expressed candidate among candidate antiangiogenic proteins. In addition, both the in vitro tube formation assays and in vivo corneal NV model supported a functional role for PD-L1 in mediating the antiangiogenic effects of Tregs. Furthermore, previous work from this laboratory has shown that PD-L1 is involved in the regulation of corneal NV,¹⁴ providing additional support for its relevance for the corneal model.

Other Treg-associated mediators such as IL-10,⁴⁵ cytotoxic T-lymphocyte-associated protein 4,⁴⁶ and Delta-like ligand 4⁴² were considered but not selected as the primary focus due to their limited mechanistic relevance to angiogenesis in the corneal setting. IL-10, although immunosuppressive, has been shown to promote angiogenesis and in certain settings, including through modulation of macrophage responses to hypoxia⁴⁵ or in ischemic tissues of diabetic mice.⁴⁴ Delta-like ligand 4–Notch signaling has been implicated in angiogenesis but was primarily studied in inflammation-experienced Tregs, such as in asthma models,⁴² limiting its applicability to the use of unprimed Tregs in the current study. Cytotoxic T-lymphocyte-associated protein 4, although a well-known checkpoint molecule,⁴⁶ lacks direct evidence of involvement in endothelial signaling or angiogenic regulation. These contextual and mechanistic differences further supported the prioritization of PD-L1 for the current study.

Although the immune regulatory functions of PD-L1 are well established,³⁷ its role in angiogenesis has only recently gained attention.¹⁴^,47, 48, 49 Jin et al¹⁴ showed that PD-L1 expressed by corneal epithelial cells decreased expression of VEGF receptor 2 in MS1 cells and thus reduced their proliferation. They also observed that PD-L1 knockout mice exhibited heightened angiogenesis in inflamed corneas compared with wild-type mice. A key finding of this study is the functional significance of Treg-derived PD-L1 in regulation of angiogenesis. Blocking PD-L1 on Tregs reversed their antiangiogenic effects in both in vitro and in vivo models, underscoring its essential role in vascular suppression. Furthermore, these results align with the observations by Jin et al,¹⁴ showing that PD-L1 deficiency enhances corneal NV.

Recent studies suggest that PD-L1 directly interacts with B7-1 (CD80), influencing T-cell function through a unique ligand-ligand interaction.¹⁹^,²⁰^,²⁷ Structural analyses have shown that PD-L1 and B7-1 share significant sequence and structural homology, with an overlapping binding interface that modulates immune signaling.⁵⁰ This interaction leads to inhibition of T-cell activation and reduced cytokine production.¹⁹^,²⁷ In addition, B7-1:PD-L1 binding is specific and can be blocked by application of antibodies targeting either B7-1 or PD-L1.²⁰ These findings extend this concept to angiogenesis regulation, showing for the first time that PD-L1 on Tregs inhibits NV through interaction with B7-1 expressed on endothelial cells.

VEGF-A is a critical proangiogenic factor that mediates NV in ocular tissues.51, 52, 53, 54 These findings show that co-culturing MS1 endothelial cells with Tregs results in a significant reduction in VEGF-A expression, an effect that is reversed upon PD-L1 knockout in Tregs. This observation suggests that the PD-L1–B7-1 interaction plays a central role in modulating VEGF-A expression and angiogenesis. Consistent with this theory, an inverse relationship between B7-1 (CD80) and VEGF expression has been previously reported in esophageal cancer cells.⁵⁵ The restoration of angiogenic capacity in MS1 cells after PD-L1 knockout or blockade suggests that PD-L1–B7-1 signaling directly regulates VEGF-A expression, aligning with prior studies linking Tregs to VEGF-A levels in the microenvironment.¹²^,⁴⁴^,⁵⁶ Although this study did not directly assess the intracellular signaling pathways downstream of the PD-L1–B7-1 interaction, further investigations are warranted to elucidate the molecular mechanisms governing VEGF-A suppression in endothelial cells. The MS1 endothelial cell line was selected due to its prior use in studies of corneal angiogenesis14, 15, 16 and expression of B7-1,¹⁴ making it particularly relevant for investigating PD-L1–mediated mechanisms. Although MS1 cells are not derived from ocular tissue¹⁴ and may not fully replicate the corneal vascular environment, they provided a practical and mechanistically relevant model. To support the physiological relevance, the findings were validated in two in vivo models of corneal NV. Future studies using primary ocular endothelial cells may provide additional tissue-specific insights.

Similarly, prior studies have reported positive correlations between PD-L1 expression on tumor cells and elevated VEGF levels or increased mean vascular density,57, 58, 59, 60 but these findings are not directly comparable to the current study. Unlike tumor-derived PD-L1, which may facilitate immune evasion and vascular remodeling, this study focused on PD-L1 expressed on Tregs and its interaction with endothelial cells. Importantly, these findings suggest that naive Tregs exhibit intrinsic antiangiogenic properties, which are altered upon PD-L1 knockout or blockade.

Given the robust antiangiogenic effects of Tregs observed in vivo, the evaluation was extended to a clinically relevant model of high-risk corneal transplantation, in which corneal NV is a major contributor to graft failure.⁵ These results show that subconjunctival injection of Tregs significantly reduces corneal NV in transplant recipients. However, the therapeutic effect was transient, suggesting that a single injection may not be sufficient to provide long-term vascular suppression. This finding aligns with previous studies, which showed that subconjunctival Treg levels peak around day 7 but decline thereafter, indicating that their effects are localized and time dependent in the ocular environment.⁸ Although this study focused on high-risk models, corneal NV also poses a risk to graft survival in low-risk transplants.⁶¹ Even limited vascular ingrowth can disrupt immune privilege and increase the chance of rejection.³⁴^,⁶² Prior work has shown that PD-L1 contributes to graft tolerance in low-risk settings.⁶³ These findings suggest that the PD-L1–mediated mechanisms observed may also play a role in maintaining avascularity in less inflamed transplant environments. Future work may explore how Treg-derived PD-L1 supports long-term graft survival in minimally inflamed or avascular environments.

A limitation of this study is the use of PD-L1–blocked Tregs rather than PD-L1–knockout Tregs for the in vivo experiments. This use was dictated by the availability of experimental models in Balb/c mice, which allow for accurate visualization and scoring of corneal NV. Although PD-L1–knockout Tregs were available in a C57BL/6 background, injecting these cells into Balb/c mice would have triggered alloreactive immune responses due to major histocompatibility complex mismatch, leading to T cell–mediated cytotoxicity and inflammation. To circumvent this issue, parallel in vitro and in vivo experiments were conducted by using PD-L1–blocked Tregs; these experiments validated that the chosen PD-L1 blocking concentration (10 μg/mL) produced effects comparable to PD-L1–knockout Tregs (Supplemental Figure S1). The downstream signaling pathways modulated by PD-L1 in Tregs were not assessed in this study. Future studies could investigate how PD-L1 blockade or knockout affects Treg differentiation and signaling cascades. Moreover, longitudinal studies assessing repeated Treg administration would be valuable to determine whether sustained therapy enhances the durability of the antiangiogenic effect and improves long-term graft survival. In addition, this study primarily focused on contact-dependent mechanisms for Treg PD-L1–mediated angiogenesis suppression, but the role of Treg-derived paracrine factors remains an important area for further investigation. Lastly, further evaluation of VEGF expression⁵¹ and inflammatory cell involvement in vivo,¹ potentially through the use of RAG1^−/− or RAG2^−/− mice, could provide additional insights into the complex interplay between immune regulation and NV in the context of corneal transplantation.

The current study advances the understanding of the role of Tregs and PD-L1 in angiogenesis suppression, identifying the PD-L1–B7-1 axis as a novel mechanistic pathway in corneal NV regulation. These findings have significant therapeutic implications for managing corneal NV and high-risk transplantation. By leveraging the dual immunoregulatory and antiangiogenic properties of Tregs, future research could pave the way for innovative cell-based therapies to mitigate NV and enhance graft survival.

Disclosure Statement

None declared.

Acknowledgments

We extend sincere gratitude to Arlene Sharpe, M.D., for providing PD-L1^−/− mice for these studies, and Hamid Alemi, M.D., M.P.H., for his help in setting up the in vitro assays and capturing images.

Footnotes

Supported by NIH National Eye Institute grant R01 EY12963 (R.D.) and Core Grant for Vision Research 5P30EY003790.

M.C. and K.F. contributed equally to this work.

Supplemental material for this article can be found at https://doi.org/10.1016/j.ajpath.2025.08.008.

Supplemental Data

Supplemental Figure S1

Surface programmed death-ligand 1 (PD-L1) blockade on regulatory T cells (Tregs; CD4⁺CD25⁺) reverses their antiangiogenic effect, similar to PD-L1 knockout (KO) Tregs. A: Representative images of tube formation assay, 4 hours after coculture, in four experimental conditions: MS1 mouse endothelial cells cultured in isolation, MS1 cells co-cultured with Tregs, MS1 cells co-cultured with PD-L1–blocked Tregs, and MS1 cells cultured with conventional Tregs. B–D: Image analysis comparing number of junctions and total tube length between groups. B: Tube formation indices significantly decreased upon Treg co-culture with MS1 cells, but this inhibitory effect was lost when PD-L1 KO Tregs or PD-L1–blocked Tregs (treated with PD-L1/B7-H1 antibody, 10 μg/mL) were used. C: MS1 and Treg-conditioned media. Similarly, the inhibitory effect of Treg-conditioned media was reversed when using conditioned media from PD-L1 KO Tregs or PD-L1–blocked Tregs. D: MS1, Tregs, and B-7 blockade. Blocking B7-1 on MS1 cells impeded the antiangiogenic effect of Tregs on tube formation. E: Vascular endothelial growth factor A (VEGF-A) expression in MS1 cells decreased upon co-culture with Tregs; however, this effect was absent when PD-L1 KO Tregs or PD-L1–blocked Tregs were used. Data are presented as a representative experiment of three independent experiments conducted. Error bars indicate SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Original magnification: ×100 (A). Scale bars = 200 μm (A). Con. T Cell, conventional T cells (CD4⁺CD25^– T cells); PD-L1.B Tregs, programmed death-ligand 1–blocked regulatory T cells.

figs1.jpg^{(1MB, jpg)}

Supplemental Figure S2

Corneal transplantation on a vascularized corneal bed. A: Representative slit lamp images. a: Images of the vascularized corneal bed in Balb/c mice at day 14 post-suture placement, prior to transplantation. b: Images immediately after high-risk corneal transplantation, demonstrating a clear graft on a vascularized corneal bed. B: Baseline posttransplant neovascularization (NV) score (day 0), indicating no significant differences at baseline between groups. Group 1: Allogeneic hosts treated with subconjunctival saline. Group 2: Allogeneic hosts treated with subconjunctival regulatory T cells (Tregs; CD4⁺CD25⁺). Group 3: Allogeneic hosts treated with subconjunctival PD-L1 blocked Treg. Scale bars = 0.5 mm (A). N = 8 per group. Error bars indicate SEM. ns, nonsignificant; PD-L1.B Treg, programmed death-ligand 1–blocked regulatory T cells.

figs2.jpg^{(924.2KB, jpg)}

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Associated Data

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Supplementary Materials

Supplemental Figure S1

figs1.jpg^{(1MB, jpg)}

Supplemental Figure S2

figs2.jpg^{(924.2KB, jpg)}

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PERMALINK

Role of PD-L1 in Regulatory T Cell–Mediated Suppression of Corneal Neovascularization

Shima Dehghani

Manuel Chacon

Katayoon Forouzanfar

Seokjoo Lee

Akitomo Narimatsu

Rohan Bir Singh

Aytan Musayeva

Francesca Kahale

Sonia Anchouche

Neda Heydarian

Shilpy Bhullar

Thomas H Dohlman

Yihe Chen

Tomas Blanco

Reza Dana

Abstract

Graphical Abstract

Materials and Methods

Cell Culture and Tube Formation Assay

Animals and Anesthesia

T-Cell Isolation and PD-L1 Blockade

Suture-Induced Inflammatory Corneal Angiogenesis

High-Risk Corneal Transplantation and Subconjunctival Treg Injection

Corneal Whole-Mount and Immunofluorescent Staining

Real-Time Quantitative PCR

Table 1.

Statistical Analysis

Results

Tregs Suppress Vessel Formation in Vitro and in Vivo

Figure 1.

Mechanistic Factors Underlying Treg-Mediated Antiangiogenic Effects

Figure 2.

Figure 3.

PD-L1:B7-1 Interaction Mediates the Antiangiogenic Function of Treg

Figure 4.

Figure 5.

Treg-Mediated Suppression of Corneal NV in High-Risk Corneal Transplantation and the Role of PD-L1

Figure 6.

Discussion

Figure 7.

Disclosure Statement

Acknowledgments

Footnotes

Supplemental Data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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