Corneal Grafts Proned to Rejection Exhibit Impaired Function of Peripherally Induced Regulatory T Cells

Abstract

Purpose: To determine whether allograft rejection onto inflamed (high-risk) graft beds exhibit dysfunctional regulatory T cells (Tregs)

Methods: To induce inflamed (high-risk) graft beds, intrastromal corneal sutures were placed two weeks prior to transplantation in mice. Ipsilateral draining lymph nodes (DLNs) and corneas were harvested at day 0, 7, and 14 post transplantation. Draining lymph nodes were analyzed for the frequencies of IFN-g⁺ CD4⁺ T effector cells, Nrp-1⁺ natural Tregs, Nrp-1^- induced Tregs, and Treg expression of coinhibitory molecules (CTLA-4 and SLAM). Foxp3 mRNA expression in the cornea was analyzed by real-time PCR, and secretory cytokines (IL-10, TGF-b, and IFN-g) were measured using ELISA. The suppressive function of Tregs on T effector cells was evaluated using an in vitro proliferation assay, and the proliferation of T effector cells was determined via BrdU incorporation at day 0, 7, and 14 post transplantation.

Results: The frequencies of Tregs were significantly reduced in high-risk (HR) compared to low-risk (LR) graft recipients 7 and 14 days post transplantation (day7; p<0.01, day14; p<0.05), whereas the frequencies of IFN-g⁺ CD4⁺ T cells were significantly increased in HR compared to LR recipients (day7; p <0.05, day14; p<0.05). Foxp3 mRNA expression in the grafted corneas were significantly lower in HR grafts than in LR grafts at day 14 (p<0.05). Frequencies of Nrp-1^- induced Tregs were significantly reduced in HR recipients compared to LR recipients (day7; 11.2%, p<0.05, day14; 11.8%, p<0.01). CTLA-4 and SLAM expression was significantly decreased in Nrp-1^-Tregs of HR recipients compared to LR recipients (CTLA-4; p<0.05, SLAM; p<0.05), and Nrp-1^- induced Tregs from HR grafts expressed less IL-10 than Nrp-1^- Tregs from LR grafts (p<0.001). Latent TGF-b and IFN-g among Nrp-1^-Tregs were significantly higher in HR compared to LR recipients (p<0.01). Total regulatory T cells and induced Nrp-1^- Tregs from HR recipients show reduced suppressive function than total Tregs (day7; p <0.01, day14; p <0.01) and induced Nrp-1^-Tregs (day14; p <0.05) from LR recipients.

Conclusions: Corneal allografts performed onto inflamed graft beds lead to Treg dysfunction, which renders these transplants prone to rejection.