The impact of migratory graft-derived APC on host Treg function, host allosensitization, and graft survival in mice receiving diabetic corneal allografts

Abstract

Purpose : We have shown that transplanting corneas from diabetic donor mice enhances host immunity to graft. Here, we specifically investigated the contribution of diabetic graft-borne APC to host immunity and graft survival.

Methods : Diabetic Cx3cr1^YFPCreER^tdTomato, CD11c^GFPDTR (C57BL/6 background) transgenic and wild-type mice were used as donors, and non-diabetic BALB/c as recipients. Diabetes was induced by injecting 50mg/kg streptozocin (STZ) for 5 days. Twelve hours before transplantation, corneas from CD11c^GFPDTR mice were excised, and APC were ex vivo depleted by using diphtheria toxin (0.3 ng/ml) and clodronate-containing liposomes (2.5 mg/ml). Corneal transplantation was then performed. Three days post-transplant, grafted corneas and draining lymph nodes (DLN) were excised. Corneal explants were imaged by confocal microscopy. The kinetics of graft-derived migratory APC was evaluated by flow cytometry. IFNγ production by alloreactive CD4+ Th1 cells was quantified by flow cytometry and ELISPOT. The functionality of FoxP3+ Tregs was evaluated by flow cytometry and their capacity to suppress proliferation of CD4+ T cells was assessed in coculture experiments.

Results : The presence of tdTomato+ APC was significantly reduced in grafts from diabetic donors. The frequency of tdTomato+ graft-derived APC was significantly higher in the DLN of hosts receiving diabetic donors (p<0.001). Depletion of APC led to a significant reduction of host’ IFNγ+ CD4+ T cells and that was reflected in the abolition of the direct pathway of sensitization (p<0.001). Conversely, APC depletion led to the reduction of DLN Treg as well as diminished functionality profiled by expression of FoxP3+ (p<0.01), IL-10 (p<0.01), and TGFB1 (p<0.01), and their suppressive capacity (p<0.001). APC-depleted diabetic allografts had a higher survival rate (40%) compared to non-depleted diabetic allografts (0%) (p<0.001).

Conclusions : Graft-derived APC from diabetic donors governate host alloimmunity and determines graft survival. However, their depletion impairs the development of host tolerance to the graft. These results emphasize the dual functions of corneal APC, the balance of which depends in large part on the microenvironment in which they operate.

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