Abstract
Purpose :
Regulatory T cells (Tregs) play a critical role in suppressing corneal allograft rejection. c-Rel, a member of the NF-kB family, is required for the proper function of Tregs. However, the role of c-Rel in Tregs under inflammatory microenvironment is still elusive. We hypothesize that c-Rel impairs the function of Tregs from corneal graft rejected hosts and targeting c-Rel in those Tregs can improve the efficacy of Treg-based therapy of corneal transplant rejection.
Methods :
C57BL/6 Foxp3YFP mice and BalB/c mice were used as corneal recipients and donors, respectively. The frequency of Tregs were assessed by flow cytometry. Expression level of c-Rel and Foxp3 in Tregs was determined by intracellular staining and western-blot. The production of cytokines by Tregs was determined by ELISA. The immunosuppressive function of Tregs was assessed using proliferation assay.
Retrovirally delivered anti-c-Rel shRNA (shRel) was used to knock down the expression of c-Rel in Tregs. The function of shRel-treated Tregs was evaluated using proliferation assay, cornea-in-the-cup assay, and adoptive transfer experiments. Long-term allograft survival was evaluated using Kaplan-Meier survival analysis.
Results :
The number of Treg cells is increased in the mouse heterologous corneal keratoplasty model (P<0.01), the immunosuppressive function of Tregs was comprised, accompanied by the increased expression of c-Rel in Tregs (P<0.01). Tregs derived from rejected hosts produced elevated inflammatory cytokines IL-17A (P<0.001) and IFN-g (P<0.001), which are direct targets of c-Rel.
The immunosuppressive function of shRel-treated Tregs was significantly improved along with the decreased production of IL-17A (P<0.001) and IFN-g (P<0.05). Those Tregs exhibited improved capacity in protecting corneal endothelial cells against inflammatory cytokines-induced cell death in vitro. Tregs isolated from rejected hosts showed improved ability to reduce the risk of corneal transplant rejection when c-Rel expression was suppressed (Median Survival Time (MST): control group (11.2±2.6 days) vs shRel group (17.0±4.8 days)).
Conclusions :
Our current study revealed that c-Rel plays a negative role in regulating Treg function under inflammatory microenvironment. Targeting c-Rel in Treg cells may be a promising therapeutic strategy to treat corneal transplant rejection.
This is a 2020 ARVO Annual Meeting abstract.