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

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: Corneal epithelial wound healing; Foxp3(+) regulatory T cells; Limbal stem cells.

Figure 1.

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.

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.