IL-6 amplifies corneal transplant alloimmunity by inducing dysfunctional IFN-γ- secreting Treg through the VEGFA-VEGFR1 axis.

Background: Corneal allografts generally exhibit high acceptance due to the immune-privileged ocular microenvironment. Regulatory T cells (Treg) promote graft tolerance; however, in a vascularized milieu, they may acquire a pro-inflammatory phenotype. We investigated how dysfunctional Treg contribute to graft rejection through the VEGFA-VEGFR1 axis.

Methods: Treg were treated with IL-6 and VEGFA, and their phenotype-including FoxP3, IL-10, and IFN-γ-was evaluated with or without a VEGFR1 antagonist (αVEGFR1) using PCR, immunoblotting, ELISA, and flow cytometry. Suppressive function was assessed by T-cell proliferation assays, corneal cell death by Annexin V staining, and graft survival by adoptive transfer of dysfunctional Treg into a transplantation model.

Results: IL-6 reduced FoxP3 (p < 0.0001) and upregulated VEGFR1 (p < 0.001). IL-6 plus VEGFA further decreased FoxP3 (p < 0.0001) and IL-10 (p < 0.0001) while increasing IFN-γ (p < 0.0001). αVEGFR1 reversed these effects, restoring FoxP3 (p < 0.0001) and IL-10 (p < 0.05) while reducing IFN-γ (p < 0.001). Functionally, IL-6/VEGFA-pretreated Treg showed impaired suppression of effector T-cell proliferation (p < 0.0001), an effect partially rescued by αVEGFR1 (p < 0.001). In ex vivo assays, fresh Treg prevented, whereas dysfunctional IFN-γ-secreting Treg induced, corneal cell death (p < 0.0001). In vivo, grafts injected with dysfunctional Treg were rejected within 3-7 weeks, while those with αVEGFR1-treated Treg showed improved survival and reduced cell death (p < 0.0001).

Conclusion: IL-6 impairs Treg via VEGFR1 upregulation, driving IFN-γ secretion through the VEGFA-VEGFR1 axis and promoting corneal cell death and graft rejection.