Regulatory T cells Derived from Hosts at High Risk of Corneal Graft Rejection Have Impaired Protective Effect on Corneal Endothelial Cells

Abstract

Purpose : The functional competence of corneal endothelial cells (CEnC) is critical for graft survival. We have previously demonstrated that regulatory T cells (Tregs) derived from hosts at high risk (HR) of corneal graft rejection have dysfunctional immune-regulatory function. Herein, we investigated whether Tregs protect CEnC from effector T cell and inflammatory cytokine-induced loss, and whether such protective functions are maintained in HR Tregs

Methods : Eight-week-old BALB/c (as recipient) and C57BL/6 (as donor) mice were used in two well-established models of corneal transplantation: a low-risk (LR) model in which the host bed is uninflamed, and a high-risk (HR) model in which stromal sutures are placed 14 days prior to transplantation to induce inflammation. Draining lymph nodes were harvested at day 14 and CD4⁺CD25^hi Tregs were purified using magnetic sorting. Corneal cups derived from naïve C57BL/6 mice were cultured with Tregs derived from either LR or HR hosts in direct or indirect (transwell) contact. Either CD4⁺CD25^- effector T cells or inflammatory cytokines (IFN-γ 100ng/mL and TNF-α 100ng/mL) were added to the culture to induce CEnC death. The role of IL-10 was investigated by antagonizing and enhancing its function in the co-culture system. After 24 hours of culture, immunostaining for ZO-1 and TUNEL was performed, and CEnC death was analyzed via confocal microscopy

Results : Co-culture of corneal cups with effector T cells alone resulted in 45.1% CEnC loss. Addition of HR Tregs to the culture decreased CEnC loss to 32.2%(p=0.034), while addition of LR Tregs suppressed CEnC loss more significantly to 16.5%(p<0.001). Inflammatory cytokines led to 50.6% CEnC loss, which was reduced to 29.5% after culture with LR Tregs(p<0.001). Addition of HR Tregs, however, did not exert a significant protective effect against CEnC death(p=0.61). Notably, LR Tregs reduced CEnC death to a similar degree in the presence of the transwell system (p=0.002). Finally, addition of IL-10 neutralizing antibody abrogated the protective effect of LR Tregs on CEnC(p<0.001). IL-10 alone was also found to be effective in reducing inflammatory cytokine-induced CEnC death(p<0.001)

Conclusions : Our data demonstrate that Tregs from LR, but not HR, transplant recipients protect CEnC from effector T cell-mediated and inflammatory cytokine-induced cell death through expression of IL-10

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.