Abstract
Purpose :
Following corneal transplantation, IL-6 is upregulated in the graft microenvironment and negatively affects Treg suppressive function. The mechanisms by which IL-6 induces Treg dysfunction are yet to be elucidated. We investigated the role of IL-6/IL-6R axis in Treg loss of function.
Methods :
High-risk (HR,vascular host bed) or low-risk (LR, avascular host bed) corneal transplantation was performed in BALB/c mice using C57BL/6 mice as donors. 7 days post-transplantation, ipsilateral lymph nodes were harvested. Treg (CD4+CD25+) or migratory APC (CD11b+MHC-IIhighB220+) were isolated with FACS. APC expression levels of IL-1, IL-6, IL-12 and Treg expression levels of IL-1R, IL-6R and IL-12R were evaluated using RT-PCR, ELISA and Western Blot. APCs were transfected with IL-6 siRNA and cocultured with CD4+CD25- Treg (MACS-sorted from naïve mice) in 1:5 ratio. Naïve APCs were co-cultured with Treg pretreated with anti-IL-6 receptor-blocking antibody (1µg/ml) for 24 hours.Treg suppressive function was assessed by flow cytometry and Treg capacity for inhibiting CFSE-labeled CD4+ proliferating T cells was measured. Finally, the HR corneal transplantation mice were treated with anti-IL-6 antibody and graft survival was assessed for 8 weeks.
Results :
APCs expressed higher levels of IL-1 (p=0.012), IL-6 (p<0.0001), and IL-12 (p=0.043) in HR compared to LR recipients.Tregs from HR recipients had higher IL-6R expression (p=0.008), but IL-1R and IL-12R levels were comparable.Treg cocultured with IL-6 silenced APCs showed significantly higher expression levels of Foxp3 (79% vs. 43%, P <0.001), IL-10 (69% vs. 40% P <0.001), and TGF-β1 (74% vs. 37%, P <0.001) compared to Treg cocultured with IL-6 expressing APCs. Blocking IL-6R prevented the reduction of FoxP3 (63%, P < 0.001), IL-10 (61%, p<0.001) and TGF-β1 (63%, p<0.001) upon co-culture with APCs.Tregs cocultured with IL-6-silenced APCs demonstrated an enhanced suppressive capacity on effector T cell proliferation compared to those cocultured with IL-6 expressing APCs (16% vs 22% proliferation, p=0.01).Finally, IL-6 blockade in HR recipients increased graft survival rates (70% vs. 30%, p=<0.05).
Conclusions :
These results confirm for the first time that migratory APC induce IL-6 mediated Treg dysfunction. Targeting the IL-6/IL-6R axis may enhance Treg-mediated immunoregulation and improve graft survival in high-risk corneal transplantation.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.