Early infiltrating regulatory T cells (Tregs) impact graft-site antigen presenting cell (APCs) phenotype and survival

Abstract

Purpose : To evaluate the effect of infiltrating Tregs on the phenotype of graft site APCs following in high-risk versus low-risk corneal transplantation

Methods : High risk(HR) or low risk(LR) corneal transplantations were performed in BALB/c recipient mice & C57BL6 mice were used as donors. At 3- and 7-days post-procedure, Treg were FACS-sorted from the graft site (grafted cornea & conjunctiva) and analyzed. Tregs sorted at day 7 post-procedure from the graft site of HR recipients(5x10³ cells) were subconjunctivally injected into newly transplanted LR RAG1^-/-or wild type(WT) recipient mice, and vice versa (n=14/group). Splenic Tregs were pre-incubated with IL-6(100ng/ml) and injected into LR RAG-1^-/- and WT recipient mice. At 2 weeks post-transplantation(n=6/group) DLN and corneas were harvested and Tregs, Th1 cells & APCs were analyzed using flow cytometry. Mice(n=8/group) were followed up for 8 weeks to evaluate graft survival.

Results : We observed significantly lower Treg frequencies(p=0.012), FoxP3 expression(p=0.0031) and increased IL-6R expression(p=0.024) in HR recipients compared to LR recipients. Adoptive transfer of HR Tregs into LR RAG^-/- mice showed significantly higher frequencies(p=0.0039) of APCs maturation compared to PBS treated controls. Conversely, LR-Treg transferred into RAG^-/- mice showed lower APC frequencies (p=0.0019) compared to controls and this effect was abolished when Treg were incubated with IL-6 prior injection. Adoptive transfer of HR Treg to LR mice showed increased corneal APC frequencies(p=0.023) and enhanced Th1 infiltration(p=0.042) compared to controls. Moreover, we observed reduced Treg(from DLNs) function characterized by lower FoxP3(p<0.0001), IL-10(p=0.0082) and TGF-b1 expression(p=0.0034) and enhanced graft immune rejection compared to controls(p<0.0001). When LR Treg were injected in HR recipients, we observed significantly lower graft site APC migration(p=0.029), and Th1 infiltration(p=0.0019) in the corneal tissue. Additionally, we observed higher expression levels of FoxP3(p=0.0015), IL-10(p=0.038) and TGF-b1 expression(p=0.045) in Tregs isolated from DLNs, and enhanced graft survival(p<0.0001).

Conclusions : The infiltrating Tregs impact graft site APC maturation depending on the graft site milieu, underlining the critical impact of graft site inflammation on frequencies and function of Tregs.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.