The inflamed corneal bed microenvironment abrogates the graft-protective function of regulatory T cells

Abstract

Purpose : We have previously shown a correlation between Treg dysfunction and enhanced APC maturation in the graft site of recipients at high-risk (HR) of rejection, at early stages after corneal transplantation. Herein, we investigated graft site environment-induced function changes of Treg in HR recipients.

Methods : High risk (HR) and low risk (LR) allogeneic corneal transplantations were performed using C57BL/6 mice as donors and BALB/c as hosts. On days 3 and 7, post-transplantation CD4⁺CD25⁺FoxP3⁺ Tregs and prototypical phenotypic maturation markers on CD11b⁺MHC-II^high APCs were assessed. Bone marrow GM-CSF-generated CD11b⁺ APCs were preconditioned with Tregs FACS-sorted from the graft-site of HR or LR in the presence of LPS (100ng/ml) and IL-2 (10ng/ml) and sonicated allogenic splenocytes (1:20:21) for 48 hours. Then, Tregs were removed, and CD4⁺CD25^- naïve T cells were added for 5 days. Next, MACS-sorted naïve Tregs from naïve mice were cultured with IL-6, with or without anti-IL-6R antibody for 12 hours, and co-cultured as above. Tregs from LR recipients were injected in newly HR host, and vice versa. Readouts included flow cytometry, ELISA, RT-PCR, and clinical follow-up.

Results : Treg frequencies and their expression levels of FoxP3 were significantly lower in HR compared to LR (p<0.01). The frequency of FoxP3⁺ Treg expressing alpha E integrin (CD103) was significantly lower in HR vs. LR (p<0.001) as well as the expression of IL-6R (p<0.001). Frequencies of MHC-II^high, CD80, CD86, and CCR7 in CD11b⁺APC, as well as IFN-g production by CD4+ T cells,⁺ T cells were found significantly higher when APCs were preconditioned with Treg from HR recipients vs. LR (p<0.001). This was recapitulated when APCs were preconditioned with naïve Treg pretreated with IL-6. Finally, adoptive transfer of Treg from LR to HR recipient mice suppressed the expression of maturation markers in the grafted cornea (p<0.001) and led to 55% allograft survival (p<0.0001); while adoptive transfer of HR Treg to LR host increased the expression of those markers (p<0.001) and led to 100% allograft survival (p<0.0001).

Conclusions : These results demonstrate that the transplant environment alters the regulatory function of the graft site’s early infiltrated Treg, and suggest that highly functional Treg could be a potential therapeutic approach to improve survival in HR corneal transplants.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.