Local Delivery of Regulatory T Cells Promotes Corneal Allograft Survival

Abstract

Purpose : Regulatory T cell (Tregs)-based immunotherapies have been used as a novel strategy to promote graft survival in several organs. However, the therapeutic efficacy of local delivery of Tregs has not been thoroughly studied in corneal transplantation. We herein investigated the kinetics of migration of subconjunctivally-injected Tregs and their role in promoting corneal allograft survival.

Methods : To determine the kinetics of distribution of injected Tregs, GFP⁺CD4⁺CD25⁺ Tregs were isolated from draining lymph nodes (DLNs) of GFP transgenic mice, and were subconjunctivally injected to graft recipients immediately after allogeneic corneal transplantation. Next, either CD4⁺CD25⁺ Tregs or conventional T cells (Tconv, as the control) was locally injected to graft recipients. The frequencies of MHC-II⁺CD11b⁺ antigen presenting cells (APCs) in the corneas and DLNs, IFNγ⁺CD4⁺ Th1 cells in the DLNs, and CD45⁺ cells in the graft were evaluated on day 14 after transplantation using flow cytometry. Additionally, the mRNA and protein expression of IFNγ, IL-10 and TGF-ß in the grafts were assessed using RT-PCR and ELISA, respectively. Finally, corneal allograft survival was determined by weekly evaluation of graft opacity scores for 10 weeks.

Results : Subconjunctivally injected Tregs were detected in the ipsilateral cornea and DLNs of graft recipients as early as 6 hours post-injection. Treatment with Tregs significantly decreased the frequencies of mature APCs in the cornea (p=0.032) and DLNs (p=0.002), suppressed Th1 cell frequencies in the DLNs (p=0.017), and inhibited CD45⁺ cell infiltration to the grafts (p=0.016) compared to Tconv-treated controls. Additionally, Treg-treated recipients demonstrated significantly reduced expression of pro-inflammatory cytokine IFNγ (p=0.029), and enhanced expression of immunosuppressive cytokines IL-10 and TGF-ß in the cornea compared to the control group (p=0.029 and p=0.057, respectively). Finally, Treg-treated recipients demonstrated significantly higher graft survival rates compared to Tconv-treated controls (p=0.03).

Conclusions : Our data demonstrate that subconjunctivally delivered Tregs migrate to the cornea and draining lymph nodes of graft recipients where they suppress antigen presenting cell maturation and T cell allosensitization, and promote allograft survival. Our study suggests that local delivery of Tregs could be a promising strategy in preventing corneal allograft rejection.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.