Corneal Myofibroblasts Preserve Regulatory T Cell Functional Phenotype

Abstract

Purpose : Fibrosis is a physiological response to tissue injury in which myofibroblasts produce extracellular matrix materials to repair damaged tissue. While myofibroblasts have been well-characterized with respect to wound healing, their ability to interact with immune cells and modulate inflammation is relatively unknown. Given that fibrosis is a physiological response aimed at resolving injury and regulatory T cells (Tregs) are a critical immunosuppressive cell population, here we evaluate the interactions of myofibroblasts and Tregs and hypothesize that corneal myofibroblasts support Tregs by preserving Treg expression of FoxP3, IL-10, TGFβ-1, and CTLA-4.

Methods : In vitro, MK/T-1 corneal fibroblasts were treated with transforming growth factor-beta-1 (TGF-β1; 100 ng/ml) for 48 hours to induce differentiation into myofibroblasts and then washed. CD4+CD25+ Regulatory T-cells (Tregs) were isolated from the spleens of naïve 8-10-week-old male BALB/c mice by MACS sorting. 5x10⁵ Tregs were cultured in the presence of IL-2 (50 ng/ml) alone or with corneal myofibroblasts at 70% confluence. After 12 hours, Tregs were collected, and FoxP3 expression was assessed by flow cytometry. mRNA expression of IL-10, TGF-β1, and CTLA-4 were assessed by real-time PCR.

Results : When cultured alone, the frequency of FoxP3+CD4+CD25+ Tregs was 70.25 ± 0.78%. When cultured with myofibroblasts, the frequency of FoxP3+ Tregs was 88.8 ± 0.14% (p<0.0001). FoxP3 MFI among CD4+CD25+ Tregs was 230 ± 2.8 in the Treg-only group and 277.5 ± 9.2 in the Treg plus myofibroblast group (p= 0.005). Treg mRNA levels of TGF-β1 were 1.9-fold higher in the Treg plus myofibroblast group compared to the Treg-only group (p= 0.0220). mRNA levels of IL-10 and CTLA-4 were higher in the Treg plus myofibroblast group compared to the Treg-only group with a trend towards significance (IL-10: 3.9-fold higher (p= 0.2343), CTLA-4: 1.5-fold higher (p=0.1251)).

Conclusions : In a co-culture system, corneal myofibroblasts preserve Treg expression of the signature transcription factor FoxP3. Tregs co-cultured with corneal myofibroblasts also demonstrate significantly higher TGFβ1 mRNA levels than Tregs cultured alone. These findings suggest that corneal myofibroblasts may contribute to Treg-mediated immunoregulation by preserving the functional phenotype of Tregs.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.