Th1-mediated Alloimmunity in a New Mouse Model of Endothelial Keratoplasty

Abstract

Purpose : There is a considerable shift in clinical practice from penetrating keratoplasty (PK) to endothelial keratoplasty (EK). However, the lack of animal models to evaluate mechanisms associated with host alloimmunity and graft rejection in EK is impeding generation of new strategies to suppress graft failure in EK. Here, we developed a new mouse model of EK, enabling us to evaluate the host alloimmune response.

Methods : EK was performed using C57BL/6 as donors and BALB/c mice as recipients. The central endothelium and Descemet membrane was removed from the recipient cornea and replaced by a 1.5-mm donor graft of the posterior stroma and Descemet's membrane. Clinical evaluation of opacity (scored 0-5) and AS-OCT were performed over the 4 weeks of follow-up. Subjects with detached grafts were excluded. Grafted hosts were divided into two groups, low-grade (<2) and high-grade (≥2) opacity. Corneas were collected from both groups and stained with ZO-1 to evaluate endothelial cell density. Lymph nodes were also collected and CD4⁺ T cells were MACS-sorted and cocultured with syngeneic or allogeneic antigen-presenting cells (APC) to evaluate the expression of IFN-γ by alloreactive Th1 cells (ELISPOT) in response to the direct (donor) or indirect (host) pathways of sensitization.

Results : Low-grade opacity was observed in 55% of mice (average opacity score = 1.00±0.00) while high-grade opacity was observed in 45% of mice (average opacity score= 2.75±0.95). The thickness of the graft was significantly higher in corneas showing high-grade compared to low-grade opacity (p<0.05). The number of ZO-1 positive cells was significantly higher in the corneas showing low-grade compared to high-grade opacity (p<0.05). While IFN-γ⁺ positivity was not detected in any mouse with low-grade opacity, the number of IFN-γ⁺ spots significantly increased in the high-grade opacity-scored mice (p<0.05). Remarkably, in the high-grade opacity group, there was a significantly higher frequency of IFN-γ⁺ spots than observed for the low-grade opacity group (p<0.05).

Conclusions : This work is the first study showing generation of Th1 alloimmunity in EK, which is associated with higher graft opacity.

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