Regulatory T cells promote corneal endothelial cell survival following transplantation via Interleukin-10

Abstract

The functional competence of corneal endothelial cells (CEnCs) is critical for survival of corneal allografts, but these cells are often targets of the immune response mediated by graft-attacking effector T cells. Although regulatory T cells (Tregs) have been studied for their role in regulating host’s alloimmune response towards the graft, the cytoprotective function of these cells on CEnCs has not been investigated. The aim of this study was to determine whether Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and to elucidate the mechanism by which this cytoprotection occurs. Using two well-established models of corneal transplantation (low-risk and high-risk models), we show that Tregs derived from low-risk graft recipients have a superior capacity in protecting CEnCs against effector T cell-mediated and IFN-γ and TNF-x-induced cell death compared to Tregs derived from high-risk hosts. We further demonstrate that the cytoprotective function of Tregs derived from low-risk hosts occurs independent of direct cell-cell contact and is mediated by the immunoregulatory cytokine IL-10. Our study is the first to report that Tregs provide cytoprotection for CEnCs through secretion of IL-10, indicating potentially novel therapeutic targets for enhancing CEnC survival following corneal transplantation.

1. Introduction

Corneal transplantation is the most common form of tissue grafting worldwide¹. When performed on an uninflamed and non-vascularized host bed, the graft survival rate exceeds 90%². However, inflammation and vascularization of the host bed disrupt the cornea’s relatively tolerant immune microenvironment, and graft survival rates fall to approximately 50% despite maximum immunosuppression in high-risk recipients^3–5. Immune rejection is the leading cause of corneal graft failure⁶. Corneal endothelial cells (CEnCs) are the target of the effector immune response mediated primarily by graft-targeting CD4⁺IFNγ⁺ T helper type 1 (Th1) cells^7,8. When the CEnC count decreases beyond a certain threshold, they are no longer capable of maintaining corneal graft transparency and the graft fails^9,10.

Substantial progress has been made in determining the mechanisms by which host factors, such as recipient bed vascularity and inflammation influence the immune response to corneal allografts^11–15. Forkhead box protein 3 (Foxp3)-positive regulatory T cells (Tregs) are a subset of CD4⁺ T cells that play a critical role in maintaining immune tolerance¹⁶. Studies performed by our laboratory^17–24 and others^25–27 indicate the critical contribution of Foxp3⁺ Tregs to immune tolerance in the setting of corneal transplantation. Furthermore, studies have demonstrated that these cells exhibit marked phenotypic plasticity and functional adaptability according to cues they receive from their microenvironment^17,28–30. In the setting of corneal transplantation, we have demonstrated that Tregs derived from mice with inflamed and vascularized host beds that are prone to quick rejection (i.e. high risk hosts) have decreased expression of Foxp3 (the transcription factor that is crucial for their development and function) relative to low risk hosts that survive long term^17,23,24. Furthermore, we have established that these Tregs derived from high-risk hosts have: (i) decreased ability to suppress graft-attacking CD4⁺CD25⁻ effector T cells, (ii) decreased expression of CTLA-4 co-inhibitory molecule, (iii) higher expression of IFN-γ (a cytokine involved in graft rejection), and (iv) significantly diminished production of immunoregulatory cytokines such as IL-10 and transforming growth factor (TGF)-β^17,23.

The mechanisms by which Tregs exert their immunosuppressive effects remain an area of intense investigation^31–38. In addition to modulating dendritic cell function, Tregs are known to suppress effector T cells through different mechanisms, including cytolysis, competition for metabolites, and release of soluble immunosuppressive cytokines such as IL-10^18,38. Of note, there is evidence that IL-10 itself provides cytoprotection for hepatocytes, neurons and brain microvascular endothelial cells^39–42. However, the cytoprotective effects of Tregs or IL-10 on CEnCs have not been previously studied.

This study aimed to investigate the capacity of Tregs to suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death. Furthermore, we evaluated whether the cytoprotective effects of Tregs are dependent upon the host immune microenvironment, i.e. whether the Treg cytoprotective effects are reduced in the high-risk setting, and determined whether direct cell-cell contact is necessary for such cytoprotection. Finally, we examined the contribution of the immunosuppressive cytokine IL-10 to Treg-mediated CEnC cytoprotection.

2. Materials and Methods

2.1. Animals

Six- to eight-week-old BALB/c and C57BL/6 male mice were purchased from Charles River Laboratories (Wilmington, MA). The study was approved by the Institutional Animal Care and Use Committee, and all animals were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

2.2. Orthotopic Corneal Transplantation

Two well-established models of allogeneic corneal transplantation were used, as described previously^43–46. For the low-risk model, C57BL/6 donor corneas were grafted onto normal BALB/c host beds. For the high-risk model, three intrastromal 11–0 sutures were placed in the central cornea of BALB/c recipient mice 14 days prior to transplantation to induce an inflamed and vascularized host bed. Mice exhibiting substantial corneal neovascularization at the time of transplant surgery were selected as high-risk graft recipients. A standard protocol for murine orthotopic corneal transplantation was followed, as described previously^43–46. Briefly, a 2-mm central corneal button was excised from donor C57BL/6 mice, grafted onto a 1.5-mm BALB/c recipient bed and secured with eight interrupted 11–0 nylon sutures. All procedures were performed under anesthesia with intraperitoneal injection of Ketamine (120 mg/kg) and Xylazine (20 mg/kg). Post-operatively, buprenorphine (0.05–0.1 mg/kg) was injected subcutaneously to minimize pain. Sutures were removed 7 days after surgery. Grafts that were opaque at postoperative week 2 were excluded from further analysis, as were eyes that underwent complications during or after surgery such as cataract, infection, intraocular hemorrhage or synechiae.

2.3. Cornea-in-the-cup Assay

In order to evaluate the cytoprotective function of Tregs derived from low-risk or high-risk hosts in the context of effector T cell-mediated and IFN-γ and TNF-α-induced CEnC death, a cornea-in-the-cup assay was used⁴⁷. All cell types were harvested from draining cervical lymph nodes ipsilateral to the grafted eye 14 days after corneal transplantation, and purified using magnetic cell sorting (CD4⁺CD25⁺ Regulatory T cell Isolation kit, Miltenyi Biotec, Auburn, CA, USA) as per the manufacturer’s instructions. The purity of isolated cells was investigated by evaluating their Foxp3 expression using flow cytometry and CD4⁺CD25⁺Foxp3⁺ cell purity of >90% was confirmed in sorted cells. To examine the efficacy of Tregs from low-risk or high-risk hosts in suppressing effector T cell-mediated CEnC death, naïve C57BL/6 (graft donor) corneal cups were cultured in RPMI 1640 medium with 10% fetal bovine serum either (i) alone, (ii) with 100,000 effector T cells from low-risk BALB/c recipients or (iii) with 100,000 effector T cells from low-risk BALB/c recipients and 100,000 Tregs derived from either low-risk or high-risk graft recipients. To examine the protective function of Tregs derived from low-risk or high-risk hosts against inflammatory cytokine-induced CEnC death, naïve C57BL/6 corneal cups were cultured in RPMI 1640 medium with 10% fetal bovine serum either (i) alone, (ii) with a combination of recombinant mouse IFN-γ (100ng/mL; Biolegend) and TNF-α (100ng/mL; Biolegend, Recombinant mouse TNF-α, carrier-free) or (iii) with a combination of IFN-γ and TNF-α and Tregs derived from either low-risk or high-risk graft recipients.

To determine whether Tregs were able to protect CEnCs in a non-contact-dependent manner, the same experimental approach was performed using a 1-μm pore size Transwell cell culture insert (Sigma Aldrich, HST Transwell-96 Permeable support with 1.0-μm pore polyester membrane, TC-treated, sterile). Naïve C57BL/6 corneal cups in RPMI 1640 medium with 10% fetal bovine serum and IFN-γ (100ng/mL) and TNF-α (100ng/mL) were placed in the bottom chamber while Tregs (derived from low-risk or high-risk hosts) were placed in the upper chamber.

To control for the possibility of Tregs acting as a sink^{38, 48, 49} for inflammatory cytokines, Tregs derived from low-risk or high-risk graft recipients were stimulated in anti-CD3ε precoated plates (10 μg/mL; Biolegend, LEAF™ purified anti-mouse CD3ε, clone 145–2C11, for 3 hours at 37°C, then washed three times with PBS) with IL-2 (4ng/mL, Recombinant mouse IL-2 carrier-free, Biolegend) for 24 hours at 37°C. The supernatant of stimulated Tregs was collected and cultured with naïve C57BL/6 corneal cups in RPMI 1640 medium with 10% fetal bovine serum in the presence of IFN-γ (100ng/mL) and TNF-α (100ng/mL).

To determine whether IL-10 antagonism interferes with the suppressive effect of Tregs derived from low-risk hosts on inflammatory cytokine-induced CEnC death, an anti-IL-10 antibody was used in the co-culture (1μg/mL; Mouse IL-10 Antibody, Monoclonal Rat IgG₁ clone #JES052A5, R&D System). Naïve C57BL/6 corneal cups were cultured in RPMI 1640 medium with 10% fetal bovine serum either (i) alone, (ii) with a combination of IFN-γ and TNF-α, (iii) with IFN-γ, TNF-α and Tregs from low-risk hosts or (iv) with IFN-γ, TNF-α, Tregs from low-risk hosts and anti IL-10 antibodies (1μg/mL). To test the hypothesis that IL-10 can restore the impaired function of Tregs from high-risk hosts in suppressing inflammatory cytokine-induced-CEnC death, we added recombinant IL-10 (10 μg/mL, BioLegend) to cultures with naïve C57BL/6 corneal cups, IFN-γ and TNF-α and Tregs from high-risk hosts. Finally, to examine the direct role of IL-10 on inflammatory cytokine-induced CEnC death independent of Tregs, we added recombinant IL-10 (10 μg/mL) to the naïve C57BL/6 corneal cups cultured with IFN-γ and TNF-α. All cultures were incubated for 24 hours in 5% CO₂ at 37°. Experiments were performed three times with 5–6 corneas in each group.

2.4. Immunohistochemistry

To determine CEnC death, corneal cups were stained and analyzed by confocal microscopy. After incubation, corneal cups were washed in PBS and then fixed in absolute ethanol for 20 minutes in 96-well plates at room temperature. Fixed corneas were then washed in PBS (3x), incubated in 5% bovine serum albumin for 60 min at room temperature and stained with a FITC-conjugated zonula occludens-1 (ZO-1) monoclonal antibody (1:200; ThermoFisher Scientific) overnight at 4°C in the dark. After another washing, corneas were permeabilized with 0.1% Triton X in 0.1% sodium citrate for 10 min at room temperature. Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) was performed using the In Situ Cell Death Detection Kit, TMR red (Sigma Aldrich) for 60 minutes in humidified atmosphere at 37°C in the dark. To evaluate the expression of IL-10RA by naïve CEnCs and inflammatory cytokine-stimulated CEnCs, after fixation corneas were stained with a primary antibody for IL-10RA (Anti-IL10RA antibody, ab225820, Abcam) and a FITC-conjugated secondary antibody (Goat anti-Rabbit IgG [H+L] Cross-Adsorbed Secondary Antibody, FITC; Invitrogen, Thermofisher). The control corneas were stained with secondary antibody only. Corneas were mounted on glass slides with the endothelium facing up, cut on four sides to flatten them, and embedded with DAPI-containing mounting medium. Flat mount corneas were analyzed using a confocal microscope (x400 magnification; Leica TCS-SP2, Leica, Wetzlar, Germany) and two images were taken from the central area of each cornea.

2.5. Image analysis

Live cells were defined as ZO-1-positive TUNEL-negative cells. ZO-1 staining (rather than DAPI) was used to determine live CEnCs to avoid staining of other cell types such as immune cells and keratocytes, as described previously⁴⁷. Apoptotic cells were defined as ZO-1-positive and TUNEL-positive. In each experiment, the number of live cells in the naïve C57BL/6 corneal cups cultured in medium alone was set as the reference. The number of live cells in each sample was then divided by the reference number, and expressed as a percentage of live cells. Percentage of cell death was derived from the percentage of live cells as per the following equation: % cell death = (100-% alive cells). The percentage of suppressive function was calculated per the following equation: % suppression of CEnC death = (1-% cell death_i / % cell death_r) x100, where i= the group analyzed and r=reference group⁴⁷. A masked observer was responsible for capturing images by confocal microscopy and counting cells using the ImageJ software.

2.6. Real Time PCR

Corneas were collected in Trizol (Ambion, Life Technologies, Grand Island, NY), and then homogenized on ice. Briefly, the RNA was extracted and purified by using RNeasy Micro kit (Qiagen, Valencia, CA). Total RNA was reverse transcripted using a Superscript III kit (Invitrogen, Carlsbad, CA). Real-time PCR was performed using TaqMan Universal PCR Mastermix (Applied Biosystems, Foster City, CA) and preformulated primers for IL-10RA (ThermoFisher Scientific, Mm00434147/ Mm00434151_m1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ThermoFisher Scientific, Mm99999915_g1).

2.7. Statistical analysis

The ANOVA test for multiple comparisons was used to determine differences among groups. Mann-Whitney U test was then used to compare both percentage of CEnC death and percentage of suppression of CEnC death between each pair of groups. Data are presented as the mean ± standard error of mean (SEM). P <0.05 was considered statistically significant.

3. Results

3.1. Regulatory T cells suppress effector T cell-mediated corneal endothelial cell death

Naïve C57BL/6 corneal cups were cultured in medium alone, with effector T cells from low-risk BALB/c recipients, or with effector T cells cells and Tregs derived from either low-risk or high-risk hosts. Effector T cells alone induced 45.1±4.3% CEnC death. The percentage of cell death was reduced when either Tregs from low-risk grafted hosts (16.5±1.5%; p<0.001) or from high-risk grafted hosts (32.2±2.8%; p=0.034) were added to the culture (Fig. 1A and 1B). However, Tregs derived from low-risk hosts exerted a more significant suppression in Teff-induced CEnC death compared to Tregs derived from high-risk hosts (63.3±3.3% vs. 28.5±6.2% suppression, p<0.001).

Figure 1. Regulatory T cells suppress effector T cell-mediated corneal endothelial cell death.

Naïve C57BL/6 corneal cups were cultured with 1×10⁵ effector T cells (Teff) from low-risk BALB/c recipients, and with 1×10⁵ regulatory T cells (Tregs) derived from either low-risk or high-risk transplant recipients. Teff and Tregs were isolated from the draining lymph nodes (dLNs) of low-risk or high-risk recipients at day 14 post-transplantation. (A) Corneas were fixed and stained with ZO-1 (Green) and TUNEL (Red), and were analyzed using confocal microscope (X400 magnification; Scale bar=50μm) (B) Bar graph showing percentage of corneal endothelial cell (CEnC) death in different groups. The number of live cells in the group in which naïve C57BL/6 corneal cups were cultured alone served as the reference. The number of live cells in each of the remaining groups was divided by the reference number, and expressed as a percentage of live cells. % CEnC death was derived from the percentage of live cells, as explained in the Methods section. Tregs from low-risk hosts and, to a lesser extent, Tregs from high-risk hosts were able to suppress Teff mediated-CEnC death (p<0.001 and p=0.03, respectively). Data are presented as mean ± SEM of three independent experiments (n= 4–6/group). Mann-Whitney Test. * p<0.05, *** p<0.001.

3.2. Regulatory T cells from low-risk graft recipients suppress IFN-γ and TNF-α-induced corneal endothelial cell death

To determine the capacity of Tregs derived from low-risk or high-risk hosts to suppress CEnC death induced by inflammatory cytokines, naïve C57BL/6 corneal cups were cultured in the presence of IFN-γ (100ng/mL) and TNF-α (100ng/mL) with or without Tregs derived from either low-risk or high-risk graft recipients. As shown in Fig. 2A and 2B, the combination of IFN-γ and TNF-α induced 50.6±1.8% CEnC death. Tregs derived from low-risk hosts significantly reduced CEnC death induced by IFN-γ and TNF-α by 41.8±5.2% (p<0.001). However, percentage of dead CEnCs in cultures containing Tregs derived from high-risk recipients did not differ significantly from the number of dead CEnCs induced by IFN-γ and TNF-α (Fig. 2B), demonstrating a significant difference in the capacity of Tregs to suppress CEnC death based on whether they were derived from low-risk or high-risk graft recipients.

Figure 2. Regulatory T cells derived from low-risk hosts suppress inflammatory cytokine-induced corneal endothelial cell death.

Naïve C57BL/6 corneal cups were cultured with IFN-γ (100ng/mL) and TNF-α (100ng/mL), as well as 1×10⁵ Tregs from either low-risk (LR) or high-risk (HR) hosts. (A) Corneas were fixed and stained with ZO-1 (Green) and TUNEL (Red), and were analyzed using confocal microscope (X400 magnification; Scale bar=50μm) (B) Bar graph showing the percentage of corneal endothelial cell (CEnC) death in different groups. The number of live cells in naïve C57BL/6 corneal cups cultured alone was set as the reference. The number of live cells in each of the other samples was divided by the reference number and expressed as a percentage of live cells. % CEnC death was derived from the percentage of live cells, as explained in the Methods section. Tregs derived from low-risk hosts suppressed inflammatory cytokine-induced CEnC death (p<0.001) in contrast to Tregs from high-risk hosts (p=0.61). Data are presented as mean ± SEM of three independent experiments (n= 4–6/group). Mann-Whitney Test. * p<0.05, ***p<0.001, ns: not significant.

3.3. Regulatory T cells are able to protect corneal endothelial cells in a non-contact dependent manner

Next to determine whether Tregs suppress inflammatory cytokine-induced CEnC death in a cell-cell contact dependent manner or through secretory factors, cornea cups were separated from Tregs in the culture using Transwell cell culture inserts. Our results demonstrated that Tregs derived from low-risk hosts retained their protective effect on CEnC when separated from the cornea cups by a Transwell insert and reduced CEnC death by 31.4±7.4% (p=0.002). However, Tregs derived from high-risk hosts failed to exert a significant suppressive effect on CEnC death induced by IFN-γ and TNF-α (Fig. 3A). Of note, the efficacy of Tregs derived from low-risk hosts in reducing IFN-γ and TNF-α-induced CEnC death did not differ significantly in the Transwell and non-Transwell culture systems (% suppression of CEnC death of 41.8±5.2% and 31.4±7.4%, respectively; p=0.221; Fig. 3B), indicating that Tregs derived from low-risk graft recipients exert their cytoprotective function through soluble factors and independent of cell-cell contact.

Figure 3. The protective effect of regulatory T-cells on corneal endothelial cells does not depend on direct cell-to-cell contact.

Naïve C57BL/6 corneal cups treated with IFN-γ (100ng/mL) and TNF-α (100ng/mL) were placed in the lower chamber of a Transwell insert (1-μm pore size) and cultured with 1×10⁵ Tregs derived from either low-risk (LR) or high-risk (HR) hosts that were placed in the upper chamber. (A) Bar graph showing the percentage of corneal endothelial cell (CEnC) death in different groups. The number of live cells in the naïve C57BL/6 corneal cups cultured alone was set as the reference. The number of live cells in each of the other samples was divided by the reference number, and expressed as the percentage of live cells. % CEnC death was derived from the percentage of live cells, as explained in the Methods section. Tregs from low-risk hosts maintained their ability to suppress CEnC death when separated from corneas by Transwell inserts (p=0.002). (B) Bar graph showing comparable capacity of Tregs in suppressing inflammatory cytokine-induced CEnC death in direct co-culture and Transwell culture systems (p=0.22). (C) Representative confocal images of corneas cultured with IFN-γ and TNF-α with or without Tregs in the Transwell culture system (ZO-1 [Green] and TUNEL [Red]; X400 magnification; Scale bar=50μm). (D) 1×10⁵ Tregs were stimulated in anti-CD3ε precoated plates with IL-2 (4ng/mL) for 24 hours at 37°C, and the supernatant of stimulated Tregs was then collected and used instead of Tregs in the culture system. Bar graph demonstrates the percentage of CEnC death in different groups. The supernatant of Tregs from low-risk hosts was significantly more effective in suppressing inflammatory cytokine-induced CEnC death compared to the supernatant from their high-risk counterparts (p<0.001). Data are presented as mean ± SEM of three independent experiments (n= 4–6/group). Mann-Whitney Test. * p<0.05, ** p<0.01, *** p<0.001, ns: not significant.

To control for the possibility of cells binding to the inflammatory cytokines in the culture and thereby suppressing their bioavailability (e.g. a “sink” mechanism), we substituted Tregs with the supernatant of anti-CD3ε-stimulated Tregs. Our data demonstrated that the supernatants of both Tregs derived from low-risk and high-risk hosts were effective in suppressing IFN-γ and TNF-α-induced CEnC death. While we observed 34.2±2.8% CEnC death in corneas cultured in the presence of IFN-γ and TNF-α, addition of supernatants of stimulated Tregs derived from low-risk and high-risk hosts reduced CEnC death to 11.0±1.6% (p=0.001) and 23.0±2.8 (p=0.020), respectively (Fig. 3D). Similar to our previous observations, Tregs derived from low-risk graft recipients demonstrated a significantly greater capacity in suppressing CEnC death (67.7±4.8%) as compared to Tregs derived from high-risk hosts (32.7±8.3%; p=0.004).

3.4. IL-10 is a key factor in suppressing IFN-γ and TNF-α-induced corneal endothelial cell death

IL-10 is a key regulatory factor secreted by Tregs, which mediates the alloantigen-specific function of Tregs. Previous work from our laboratory has demonstrated that Tregs derived from high-risk graft recipients secrete significantly lower levels of IL-10 compared to their counterparts derived from low-risk hosts²³. We thus next investigated the role of IL-10 in mediating the cytoprotective function of Tregs. We first confirmed the expression of the IL-10 receptor by murine CEnCs using qPCR (data not shown) and confocal microscopy (Fig. 4A). Next, to determine the role of IL-10 in Treg-mediated suppression of CEnC death, IL-10 neutralizing antibody was added to the cornea cups cultured with Tregs derived from low-risk hosts in the presence of IFN-γ and TNF-α. Our results demonstrated that IL-10 neutralization completely abrogated the efficacy of Tregs from low-risk hosts in suppressing IFN-γ and TNF-α-induced CEnC death (61% vs. 2% [with IL-10 blockade] suppression in CEnC death; p<0.001; Fig. 4B). Since we consistently observed a lower cytoprotective function by Tregs derived from high-risk hosts, we next set to determine whether addition of IL-10 could restore the suppressive function of these Tregs against IFN-γ and TNF-α-induced CEnC death. To this aim, recombinant IL-10 was added to the culture of cornea cups with Tregs derived from high-risk hosts in the presence of IFN-γ and TNF-α. While Tregs derived from high-risk hosts did not exert a protective effect against CEnC death (p=0.953), as we observed earlier, supplementation of the culture with IL-10 significantly improved their capacity to suppress CEnC death resulting from exposure to IFN-γ and TNF-α (p<0.001; Fig. 4C).

Figure 4. IL-10 is a key factor in suppressing inflammatory cytokine-induced corneal endothelial cell death.

(A) Confocal images showing IL-10 receptor (IL-10R) expression in [A] isotype control, [B] naïve C57BL/6 corneal cups and [C] naïve C57BL/6 corneal cups cultured with IFNγ (100ng/mL)+TNFα (100ng/mL) for 24 hours at 37°C and 5%CO₂. Corneal endothelial cells (CEnCs) are shown to express IL-10R (green), expression of which is increased after exposure to inflammatory stimuli. (B) Naïve C57BL/6 corneal cups were cultured with Tregs from low-risk hosts in the presence of IFN-γ (100ng/mL) and TNF-α (100ng/mL) with or without anti-IL-10 (1μg/mL) antibody. Bar graph showing percentage of CEnC death in different groups. The number of live cells in the naïve C57BL/6 corneal cups cultured alone was set as the reference. The number of live cells in each of the other samples was divided by the reference number and expressed as a percentage of live cells. %CEnC death was derived from the percentage of live cells, as explained in the Methods section. The capacity of Tregs from low-risk hosts to suppress CEnC death was abrogated by neutralization of IL-10 in the culture (p<0.001). (C) Naïve C57BL/6 corneal cups were cultured with Tregs from high-risk hosts in the presence of IFN-γ (100ng/mL) and TNF-α (100ng/mL) with or without recombinant IL-10 (10μg/mL). Bar graph showing the percentage of CEnC death in different groups. Addition of IL-10 potentiated the cytoprotective function of dysfunctional Tregs derived from high-risk hosts on CEnCs (p<0.001). (D) Naïve C57BL/6 corneal cups were directly cultured with or without IL-10 (10μg/mL) in the presence of IFN-γ (100ng/mL) and TNF-α (100ng/mL). Bar graph demonstrates the capacity of IL-10 alone in suppressing inflammatory cytokine-induced CEnC death (p<0.001). Data are presented as mean ± SEM of three independent experiments (n= 4–6/group). Mann-Whitney Test. *p<0.05, **p<0.01, ***p<0.001, ns: not significant.

Finally, to evaluate the direct cytoprotective effects of IL-10 on CEnCs in the absence of Tregs, IL-10 was added to naïve corneal cups cultured with IFN-γ and TNF-α, and CEnC death was determined. Our data demonstrated that IL-10 alone was effective in reducing IFN-γ and TNF-α-induced CEnC death (22.1±2.5% CEnC death with IL-10 added compared to 44.9±3.4% without IL-10; p<0.001; Fig. 4D), indicating the central role of IL-10 in protecting CEnCs from death induced by inflammatory cytokines.

4. Discussion

Tregs have a critical function in suppressing alloimmune responses in the setting of tissue transplantation^21,49,50. However, the functional status of Tregs demonstrates significant plasticity, and is determined in part by cues from their microenvironment^29,51. In this study, we investigated the mechanisms by which Tregs suppress effector T cell-mediated and inflammatory cytokine-induced CEnC death, and evaluated whether these effects were abrogated in Tregs derived from high-risk graft recipients. Our data show that Tregs from low-risk hosts are able to protect CEnCs against both effector T cell-mediated and IFN-γ and TNF-α-induced cell death, and that this function is significantly compromised in Tregs derived from high-risk hosts. Furthermore, we demonstrate that the cytoprotective function of Tregs occurs irrespective of direct cell-cell contact and is mediated by the immunomodulatory cytokine IL-10.

Host alloimmunity is the principal cause of donor CEnC loss after corneal transplantation⁵². Although multiple alloimmune mechanisms, including humoral antibody-mediated CEnC loss⁵³ and CD8⁺ T cell cytotoxicity⁵⁴ have been implicated in CEnC death following transplantation, the principal effector cells mediating CEnC loss have been shown to be CD4⁺ T helper 1 (Th1) cells^7,8,52. Th1 cells are notable for their secretion of signature cytokine IFN-γ, as well as other inflammatory cytokines such as TNF-α and IL-1⁵⁵. These pro-inflammatory cytokines have also been shown to cause CEnC death independent of Th1 cells⁵⁶. Our data demonstrate that Tregs are capable of rescuing CEnC from effector T cell-mediated death in vitro. Indeed, we show that both Tregs from low-risk and high-risk hosts are effective in protecting CEnCs, with a greater cytoprotective capacity demonstrated by Tregs derived from low-risk hosts. Previous work from our group has shown that Tregs derived from both low-risk and high-risk corneal allograft recipients effectively suppress the proliferation of effector CD4⁺CD25⁻ T cells, although Tregs derived from low-risk hosts exhibit 20% more suppressive capacity relative to Tregs from high-risk hosts²³. It is therefore conceivable that in these experiments both Treg populations are limiting CEnC loss through modulating effector T cell responses and not necessarily by exerting a direct effect on endothelial cells. In order to delineate whether Tregs have a direct cytoprotective effect on CEnCs or whether increased CEnC survival is due to modulation of the effector T cell response, we developed an in vitro system with only two cell types – CEnCs and Tregs. Previous work from our laboratory and others has shown that CEnC death can be induced by effector T cell-associated inflammatory cytokines, such as IFN-γ and TNF-α^52,56. Thus, we added IFN-γ and TNF-α to our in vitro culture system to induce CEnC death. Our data show that Tregs from low-risk graft recipients, but not from high-risk hosts, suppress inflammatory cytokine-induced CEnC death. It has been shown that Tregs exert their immunoregulatory function via both contact-dependent and non-contact dependent mechanisms³⁸. Thus, we employed a Transwell system to determine whether the cytoprotective effects of Tregs on CEnCs occurs in a contact-dependent manner or is mediated through soluble factors. We show that Tregs from low-risk hosts continue to exert their cytoprotective effects on CEnCs in the presence of Transwell inserts, indicating that the observed phenomenon is mediated through soluble factors rather than direct cell-cell contact.

We considered the possibility that Tregs were binding inflammatory cytokines in the culture (i.e. acting as a cytokine sink). Accordingly, we conducted experiments in which Tregs were removed from the culture and replaced by the supernatant of stimulated Tregs. Our results show that even in the absence of Tregs, the supernatant of stimulated Tregs exerts a protective function on CEnCs, further confirming that Treg promotion of CEnC survival is mediated through soluble factors. Interestingly, we observed that supernatant derived from both LR and HR Tregs were able to suppress CEnC death more than Tregs themselves. We believe that this finding is related to Tregs acting as a cytokine sink, reducing the amount of IL-10 or other immunoregulatory cytokines in the culture supernatant. However, by removing actual Tregs from the culture, and only using their supernatant, we circumvent the cells acting as cytokine sinks and focus on the function of their expressed cytokines.

In a previous study, our group reported that Tregs derived from low-risk allograft recipients secrete comparably higher levels of IL-10 compared to Tregs derived from high-risk hosts²³. IL-10 has been shown to protect various cell types from biological activities induced by other cytokines through the activation of transcription factors⁵⁷. This cytoprotective effect has been documented in a variety of conditions including diabetes⁵⁸ and microvascular endothelial cell function in hypertension⁵⁹, neuronal cells after brain infarction⁴⁰ and hepatic cells after hepatic cold ischemia/reperfusion injury³⁹. The observation of higher levels of IL-10 secreted by Tregs derived from low-risk hosts coupled with studies that have demonstrated the protective role of IL-10 prompted us to hypothesize that the promotion of CEnC survival by Tregs occurs through their secretion of IL-10. Using PCR and immunohistochemistry staining, we confirmed the expression of IL-10 receptor by murine CEnCs. Furthermore, we observed an upregulated expression of IL-10 receptor by CEnCs following exposure to inflammatory stimuli. Our data indicate that IL-10 plays a critical role in the cytoprotective function of Tregs, as neutralization of IL-10 completely abrogates the protective effects of Tregs from low-risk hosts on CEnC, while addition of IL-10 to cultures with less cytoprotective Tregs from high-risk hosts enhances their suppressive effect against CEnC death.

We note that IL-10 has been reported to expand Foxp3⁺ Tregs, to promote CTLA-4 expression by Tregs and increase their suppressive function⁶⁰. In order to determine whether IL-10 was having a direct effect on CEnC survival, or whether IL-10 was in fact upregulating non-IL-10-mediated Treg cytoprotective mechanisms, we excluded Tregs from the co-culture system and cultured CEnCs directly with inflammatory cytokines and IL-10. Our data clearly indicate that IL-10 alone is effective in protecting CEnCs from inflammatory cytokine-induced cell death. Interestingly, previous studies have reported that CEnCs promote the generation of Tregs through their expression of CTLA-2α and TGF-β^61,62. Our study provides evidence that these cellular interactions may in fact be bidirectional, with Tregs promoting CEnC survival by IL-10-mediated cytoprotection against inflammatory cytokine-induced death. However, these data shed further light on the diverse mechanisms by which regulatory mechanisms conspire to promote the observed high survival of corneal grafts, even without use of immunosuppressive medications.

Abbreviations:

CEnCs

corneal endothelial cells

Foxp3

Forkhead box protein 3

IFN-γ

interferon-gamma

IL-10

Interleukin-10

Teff

effector T cell

Th1

T helper 1

TNF-α

Tumor necrosis factor-alpha

Treg

regulatory T cell