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Published in final edited form as: Exp Eye Res. 2024 Oct 11;249:110123. doi: 10.1016/j.exer.2024.110123

Foxp3+ regulatory T cells reside within the corneal epithelium and co-localize with limbal stem cells

Maryam Tahvildari 1,2,*, Rao Me 1, Mizumi Setia 1, Nan Gao 1, Pratima Suvas 1, Sharon A McClellan 1, Susmit Suvas 1,*
PMCID: PMC11622170  NIHMSID: NIHMS2031027  PMID: 39396695

Abstract

In this study we investigated the presence of resident Foxp3+ regulatory T cells (Tregs) within the cornea and assessed the role of resident Tregs in corneal epithelial wound healing. Using a mouse model, we showed that in the steady state Foxp3+Tregs are either in close proximity or co-localize with ABCG2+ limbal stem cells. We also showed that these Tregs reside within the epithelial layer and not the corneal stroma. In addition, using a mouse model of mechanical injury, we demonstrated that depletion of Tregs from the cornea prior to corneal mechanical injury, using subconjunctival injection of anti-CD25, was associated with delayed epithelial healing. These results suggest a role for cornea resident Tregs in corneal epithelial cell function and wound healing and opens doors for further exploration of the role of Tregs in limbal stem cell function and survival.

Keywords: Foxp3+ regulatory T cells, corneal epithelial wound healing, limbal stem cells

1. Introduction:

The corneal epithelial layer is the first line of defense against external pathogens, and healthy corneal epithelium is integral in preventing corneal infections and protecting the damage to the eye (Chi and Trinkaus-Randall, 2013). Various conditions such as mechanical or chemical injuries, infections, or inflammatory conditions (e.g. ocular graft-versus-host disease, Stevens John Syndrome, peripheral ulcerative keratitis) can lead to an epithelial defect. The wound healing process can be divided into three distinct, consecutive, but overlapping phases: a pro-inflammatory initiation phase, a tissue formation phase, and a resolution and tissue re-organization phase (Zaiss et al., 2019). However, despite the resolution of the inflammation (initiation phase), a successful healing process is not always achieved, leading to a persistent epithelial defect (PED). Dry eyes, diabetes, limbal stem cell deficiency, exposure keratopathy, and conditions that lead to neurotrophic corneal diseases, can all lead to PED, which in turn predispose the eye to infections, corneal scarring, corneal melt and even corneal perforation (Chen et al., 2022; Maqsood et al., 2021).

Foxp3-expressing regulatory T (Tregs) cells play an indispensable role in establishing and maintaining immune homeostasis in the body (Zaiss et al., 2019). It is shown that the decreased function of Tregs in the draining lymph nodes (as shown in the level of the expression of Tregs’ key transcription factor, Foxp3) is associated with an increase in corneal allograft rejection (Chauhan et al., 2009). We also know that systemic expansion of Tregs (through low-dose IL-2 treatment) (Tahvildari et al., 2016) or local increase in its population (through subconjunctival injection of Tregs) (Shao et al., 2019) promotes corneal allograft survival. The IL-2/anti-IL-2 antibody complex-mediated systemic expansion of Foxp3+Treg has also been shown to prevent the development of herpes simplex virus- (HSV-1)-induced stromal keratitis (Gaddipati et al., 2015). However, the role of Foxp3+Tregs in epithelial wound healing is not fully understood. Multiple studies suggest that Tregs play a key role in wound healing by suppressing local inflammation during the transition from the initial pro-inflammatory phase into the tissue formation phase. It is shown that Tregs rapidly migrate to and accumulate at sites of inflammation, e.g., in the injured muscle or the inflamed nerve tissues or HSV-1 infected corneal tissue, and are pivotal in the resolution of inflammation during wound healing (Burzyn et al., 2013; Ito et al., 2019; Suvas et al., 2004). The cornea is home to resident leukocytes and the presence of antigen presenting cells (such as dendritic cells) and plasmacytoid dendritic cells have been well established (Hamrah et al., 2003). Presence of Tregs in the cornea and their potential role for regulation of ocular surface homeostasis is an area of ongoing investigation. In a recent study numerous CD45+ cells including Foxp3+Tregs were found in the “outer” limbal area of the mouse cornea; authors also show that mice with severe combined immunodeficiency (SCID) show delayed corneal epithelial wound healing (Altshuler et al., 2021). In this study, we provide evidence of Tregs residing mainly in the corneal epithelial layer and within the limbal stem cell niche. We also demonstrate that depletion of Tregs from the ocular surface, using subconjunctival injection of anti-CD25, delays corneal epithelial healing after mechanical injury.

2. Methods:

2.1. Animals

Ten to sixteen-week-old female C57BL/6J and B6.Cg-Foxp3tm2Tch/J (also referred to as GFP-Foxp3 in the rest of the text) were used in our experiments and were purchased from the Jackson Laboratory (Bar Harbor, ME). In the GFP-Foxp3 mice, GFP (Green Fluorescent Protein) expression was evident in Foxp3+ Treg cells, which was confirmed through flow cytometry of the lymphoid tissues (data not shown). All mice were housed in the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited pathogen-free animal facility at Wayne State University School of Medicine (WSUSOM). Animals were gender- and age-matched for all experiments. All experimental procedures were in complete agreement with the Association for Research in Vision and Ophthalmology resolution on the use of animals in research. All experimental procedures undertaken were in accordance with the Institutional Animal Care and Use Committee of Wayne State University.

2.2. Corneal immunofluorescence staining

Mice were euthanized, and the entire cornea including the limbus with a scleral rim was excised under the operating microscope. Excised corneas were fixed in 4% paraformaldehyde and stored at 4°C until further processing. Before staining, radial incisions were made to produce six pie-shaped wedges. Corneas were washed in PBS and incubated with a 0.2% solution of Triton X-100 in PBS plus 1% bovine serum albumin (BSA) with Fc block for 20 minutes at room temperature. After blocking, the corneas were incubated overnight at 4°C with 100 μL of ABCG2 antibody (Novus Biologicals, Clone:3G8). The tissues were then washed x6 times in PBS (10min for each wash). Stained tissue whole mounts were placed in mounting medium (Vector Laboratories, Burlingame, CA, USA) onto glass slides and covered with a coverslip. Corneal whole mounts were examined using confocal microscopy (TCSSP8; Leica, Wetzlar, Germany). Whole corneal images were obtained using recorded images of regions of interest and automatically assembled in the TCSSP8 microscope. LAS X Life Science Microscope Software was used to acquire images and create maximum projection Z-stacks.

2.3. Separation of corneal epithelial layer from stroma

The corneal epithelial sheet was isolated from the underlying stroma as previously described (Setia et al., 2023). Briefly, eyeballs were incubated with 15 mg/mL of Dispase® II (MilliporeSigma) for 16 hours at 4°C. Subsequently, the intact epithelial sheet was carefully peeled off and transferred into 500 μL of 0.25% trypsin EDTA solution (Thermo Fisher Scientific). The epithelial sheet was then incubated for 6 minutes at 37°C to facilitate dissociation into single cells. Following the trypsinization step, the single cell suspension was prepared by gently pipetting up and down to ensure thorough dissociation. The trypsin activity was then inhibited by adding 500 μL of 2 mg/mL of soybean trypsin inhibitor (MilliporeSigma). Thereafter, the cell suspension was washed twice with RPMI medium supplemented with 10% fetal bovine serum, vortexed, and counted using a hemocytometer.

2.4. Flow cytometry

All cell suspensions were incubated with an Fc receptor blocking antibody (R&D Systems, Minneapolis, MN). For cell surface staining, the cells were washed with FACS buffer followed by blocking Fc receptors and incubation with fluorochrome-conjugated antibodies. The following fluorochrome-conjugated antibodies were used for cell surface staining: APC-Cy7 Rat anti-Mouse CD45 (clone: 30-F-11), PE Rat anti-Mouse CD25 (clone:7D4), BV605 Rat anti-Mouse CD4 (clone:RM4–5) from BD Biosciences (San Diego, CA). LIVE/DEAD Fixable Aqua Dead Cell Stain (Invitrogen) was used to differentiate live from dead cells. At the end of cell surface staining, the cells were fixed overnight in 2% paraformaldehyde (PFA). For intracellular intranuclear staining of Foxp3 when wild type (non-GFP-Foxp3) C57BL/6 mice were used, cells were fixed and permeabilized with appropriate buffers (eBioscience). Alexa Fluor 647 Rat anti-Mouse Foxp3 (clone: MF14) from BioLegend was used to stain intranuclear Foxp3. Isotype controls were used for all antibodies. Samples were acquired using LSR-Fortessa (BD Biosciences) flow cytometer, and the data were analyzed using FlowJo version 10.7.1 software (Ashland, OR, USA).

2.5. Subconjunctival injection of Anti-CD25

Mice were given subconjunctival injections with a volume of 5 μL per injection containing anti-CD25 (5mg/0.7ml) (BioXcell, Clone: PC-61.5.3) or sterile saline (PBS) as a control. Injections were performed 1 hour prior to wound creation and under anesthesia via isoflurane inhalation.

2.6. Corneal Epithelial Debridement

Mice were anesthetized with intraperitoneal injections of 120mg/kg ketamine and 20mg/kg xylazine prior to the mechanical injury procedures. A 2 mm circular wound was first demarcated with a trephine in the cornea, followed by the removal of corneal epithelial cells (CECs) within the circle using an Algerbrush II burr (Precision Vision, IL, USA) under a Zeiss (Oberkochen, Germany) dissecting microscope. The progress of wound healing was monitored by fluorescence staining and photographed with a slit lamp microscope (Pan and Chan, 2021).

2.7. Statistical analysis

The statistical analyses were performed with GraphPad Prism 6 software. Data are presented as means ± SDs. Experiments with two treatments and/or conditions were analyzed for statistical significance using a two-tailed Student’s t-test. A Bonferroni posttest was performed to determine statistically significant differences. Significance was accepted at p < 0.05. Experiments were repeated at least twice to ensure reproducibility.

3. Results and Discussion:

Flow cytometric analysis of naive mouse corneas (Fig.1Aa) and conjunctivae (Fig.1Ab) showed a discrete population of CD25+Foxp3+ Tregs among CD4+ T cells. Using GFP-Foxp3 mice, we were also able to show that Tregs were only in the corneal epithelium and not the stroma (Fig. 1B). To further identify and localize Tregs in the cornea, confocal microscopy was used, which showed that the majority of the Foxp3+Treg cells localized at the limbal area, some near ABCG2+ limbal stem cells (LSCs) and some co-localizing with them (Fig. 1C). Next, we looked at the kinetics of epithelial wound closure after mechanical injury when Tregs were depleted using subconjunctival injection of anti-CD25. Sterile phosphate buffer saline (PBS) was used as a control. Figure 2A demonstrates a successful decrease in the population of CD4+CD25+ T cells in the cornea after anti-CD25 injection. We observed a significant delay in corneal epithelial wound healing in the group that received anti-CD25 compared to the saline group (Fig. 2B).

Figure 1.

Figure 1.

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A. Flow cytometric analysis of naïve C57BL/6 mice, demonstrating a distinct population of Foxp3+ Treg cells among CD4+CD25+ T cells, both in the cornea (a) and conjunctiva (b). B. Flow cytometric analysis of naïve B6.Cg-Foxp3tm2Tch/J (GFP-Foxp3) mice, demonstrating presence of Foxp3+ Treg cells among CD4+ T cells, in the corneal epithelium (a) but not the corneal stroma (b). Flow cytometry experiments were performed 3 times and 6 corneas were pooled for each experiment. C. Immunochemistry of a whole mount cornea (10x magnification) (a) showing presence of Foxp3 (GFP) signaling in the naïve cornea of a GFP-Foxp3 mouse. Enlarged area of one limbal stem cell (LSC) clock hour (b) shows that Foxp3+ cells (yellow arrow) are mostly in proximity to the ABCG2+ LSCs (white arrow) or co-localizing with LSCs, appearing yellow (blue arrow) (b). 20x magnification of another limbal area showing ABCG2 red signal (c) and Foxp3 green signal (d) and the merged maximum projection Z-stack image (e) showing ABCG2 and Foxp3 co-localizing as the yellow signal (blue arrow) (e). Immunofluorescence experiments were performed 3 times and 4 corneas were evaluated in each group for each experiment.

Figure 2.

Figure 2.

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A. Flow cytometric analysis of the C56BL/6 mouse corneas showing decreased population of CD25+ cells 24 hour after subconjunctival injection of anti-CD25 (upper row) compared to PBS injected group (middle row) and no injection group (bottom row). B. (a) Slit lamp photos of the corneas after mechanical injury (2 mm epithelial debridement) and application of fluorescein stain to demonstrate the wound area after 5h, 21h, 26h and 48h in the anti-CD25 treated group (upper panel) and sterile PBS injected control group (lower panel). (b) Graph showing healed (epithelialized) area percentages in the treatment group vs. controls. Experiments were performed 3 times and 5 eyes (of 5 mice) were used per group in each experiment.

In this study, we investigated the presence of Foxp3+ Tregs in the cornea and studied their potential role in corneal epithelial healing. We show that in the steady state, Foxp3+ Tregs reside within the corneal epithelial layer, either near the limbal stem cells or co-localizing with them. Using a murine model of mechanical injury, we further show that depletion of Tregs from the cornea is associated with delayed epithelial wound healing.

CD4+CD25+Foxp3+ Tregs are widely recognized for their pivotal role in immune homeostasis (Sakaguchi et al., 2008). However, the mechanisms that govern the function and distribution of tissue-resident (peripheral or non-lymphoid) Tregs, in comparison to central (lymphoid) Tregs, remain a mystery (Hewavisenti et al., 2021). These tissue-resident immune cells play a crucial role in responding swiftly to disturbances of the tissue’s local homeostasis, such as infections, non-infectious inflammatory responses (e.g. in autoimmunity, transplant rejection and mechanical injuries) as well as in cancers. They also possess tissue-specific physiological roles in various tissues (Altmann, 2018; Niedzielska et al., 2018). Tissue-resident Tregs are shown to have distinct TCR function and repertoire, compared to central Tregs (Hewavisenti et al., 2021; Mannie et al., 2020). For instance, Tregs localized in lung and gut mucosa display several non-coding RNAs and express distinct TCR-associated surface markers compared to central Tregs (Sullivan et al., 2019).

The immunological characteristics of somatic stem cell niche is largely unknown; in the bone marrow, it is shown that there is abundance of Foxp3+Tregs within the hematopoietic stem cell niche, suggesting that Tregs maintain immune-privileged environment for the stem cells (Fujisaki et al., 2011). In the eye, resident Tregs are known to be present within the conjunctival associated lymphoid tissues (CALT) (Chen et al., 2022) and responsible for the induction of mucosal tolerance and protection of the ocular surface from infectious and non-infectious insults (Siebelmann et al., 2013). Our current study not only confirms the presence of tissue-resident Foxp3+Tregs in the cornea, but also reveals their distribution within the corneal epithelium and the limbal stem cell niche (Fig 1). We utilized a novel technique (Setia et al.), to separate the corneal epithelium from stroma and specifically analyze cell population present in each layer. We know that there may be some cells lost during the processing of tissues when performing flow cytometry, however, the presence of a distinct population of Tregs in the epithelium (and the absence of it in the stroma) further suggest importance of Tregs in epithelial cell function. A recent study has shown presence of Foxp3+ Tregs in the epithelial and anterior stroma (Altshuler et al., 2021); they also showed that depletion of Tregs using subconjunctival injection of anti-CD25 affects LSC function as shown by decreased expression of markers associated with LSC quiescence and increased cell proliferation. These findings pave the way for further studies to unravel the function of tissue-resident Tregs in the cornea and the eye, potentially revolutionizing our understanding of corneal and ocular homeostasis and wound healing.

Emerging evidence suggests that tissue-resident Tregs promote immune-mediated tissue homeostasis, but also have non-immune mediated roles in maintaining and re-establishing homeostasis during wound healing (Zaiss et al., 2019). For instance, peripheral Tregs are shown to have plasticity in their function when studied in various phases of regeneration and fibrosis in a kidney injury model (do Valle Duraes et al., 2020). One such mechanism is an expression of epidermal growth factor (EGF) receptor (EGFR), a transmembrane tyrosine kinase receptor in Tregs upon wounding. The EGFR pathway is also known to stimulate epidermal and dermal regeneration in the skin (Nosbaum et al., 2016), and inhibition of this pathway delays corneal epithelial wound healing (Nakamura et al., 2001). In addition, recent studies have shown that tissue-resident Tregs express EGFR ligand, amphiregulin (AREG), under inflammatory conditions, which enhance muscle and lung tissue repair after injury (Nosbaum et al., 2016). Expression of Amphiregulin during wound repair induces the release of TGF-beta in the tissues, a cytokine that has both immune-regulatory and tissue regenerating functions (Zaiss et al., 2019). In a recent study, subconjunctival administration of Tregs has been shown to facilitate corneal wound healing and show a higher expression level of AREG in Tregs upon chemical injury (Yan et al., 2020). In the current study, we have shown delayed epithelial wound healing after depletion of these Tregs (Fig 2), suggesting a role for these Tregs to modulate corneal (limbal) epithelial cell function and epithelial healing. These data suggested that Tregs may directly contribute to corneal epithelial wound healing, independent of their immune-regulatory function (Arpaia et al., 2015). Further studies are required to characterize cornea resident Foxp3+Tregs and elucidate their function in maintaining and re-establishing tissue homeostasis in the eye.

Funding sources:

The current study was supported by Startup funds, Wayne State University School of Medicine to Dr. Maryam Tahvildari and funding from the National Eye Institute (NEI) grant R01EY030129-04 awarded to Dr. Susmit Suvas.

Footnotes

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