IL-6/IL-6R Axis-mediated Treg Loss of Function Enhances Dendric Cell Immunostimulatory Function and Corneal Graft Failure

Abstract

Purpose : IL-6 is a pleiotropic cytokine involved in the physiology of every system; and many studies have shown IL-6 cause Treg dysfunction. Following corneal transplantation, IL-6 is upregulated in the graft microenvironment and systemic blockade of IL-6 enhances Treg suppressive function and graft survival. However, the mechanisms by which IL-6 promotes Treg loss of function and failure remain yet to be elucidated. Herein, we investigated the IL-6/IL-6R axis mediated Treg loss of function

Methods : High-risk (HR) or low-risk (LR) corneal transplantation was performed in BALB/c mice using C57BL/6 as donors. On days 3 and 7 post-transplantation, grafted corneas were excised. Treg profiled as CD4⁺CD25⁺FoxP3⁺ as well as prototypical phenotypic maturation markers on CD11b⁺MHC-II^high dendritic cells (DC) were assessed by flow cytometry. Additionally, Treg and CD11b⁺F480⁺macrophages were separately FACS-sorted from the graft site of HR or LR hosts and assessed by qRT-PCR and Western Blot. Bone marrow-derived cells (BMDC) were stimulated with LPS (100ng/ml) and sonicated allogeneic splenocytes and cocultured with Treg from the graft site of HR or LR recipients (20:21:1). Then, Tregs were removed, and CD4⁺CD25^- naïve T cells were added for 5 days. MACS-sorted Tregs from naïve mice were incubated with or without IL-6 for 12 hours, and subconjunctivally injected after corneal transplantation in LR recipient mice

Results : FoxP3 expression by Treg at the graft site was significantly lower in HR compared to LR recipients (p<0.01), while IL-6R expression by the Treg was significantly upregulated (p<0.001). The expression intensity of maturation markers, MHC-II, CCR7, CD80/CD86 in graft site DC as well as the expression of IL-6 by macrophages was higher in the HR as compared to LR recipients (p<0.001). The production of IFN-y by CD4⁺ T cells was significantly higher when BMDC were preconditioned with either Treg from HR recipients (p<0.01) or with naïve Treg pretreated with IL-6 (p<0.001) compared to preconditioned with naïve Treg alone. Finally, graft survival was significantly reduced in recipients that received IL-6 pretreated Treg (20%) compared to untreated Treg (75%) (p<0.0001)

Conclusions : These results confirm that the IL-6/IL-6R axis mediates the loss of function of early infiltrating Treg at the graft site, which facilitates APC immunostimulatory function and subsequent graft failure

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.