Abstract
Purpose::
Prolonged storage of donor cornea has been shown to prevent allograft rejection in both human and mouse studies, particularly in high risk corneal transplantation for reasons that remain obscure. We hypothesize that the mechanism is associated with corneal antigen-presenting cell (APC) depletion from the donor tissue, thereby diminishing direct-type allo-sensitization by graft-derived APCs. Accordingly, the purpose of the current study was to find efficient procedures to deplete APCs from the corneal buttons.
Methods::
Corneal buttons harvested from C57BL/6 mice were divided into different groups. The first two groups were stored in Optisol-GS® medium at either 4 ºC or 37 ºC. The third group was treated with anti-CD45 mAb plus complement in Optisol-GS® medium. In addition, non-treated fresh corneas were included as a control. To determine the number of APCs in the corneas, all groups were immunohistochemically stained with the pan-leukocyte surface marker CD45 present on all the APCs of the cornea. CD45+ cells were then enumerated on whole-mount corneas using confocal microscopy. The percent of CD45+ cells was also analyzed for cells that emigrated from the donor button into the medium using laser scanning cytometry (LSC).
Results::
CD45+ cell numbers were dramatically decreased over time in corneas in all three experimental groups compared with the control group. There was a concomitant increase in the number of CD45+ cells in the medium at 37 ºC, suggesting that the emigration of CD45+ cells contributed to these cells’ depletion. Both 4 ºC and 37 ºC groups showed progressively increased numbers of apoptotic cells over time. The corneas that were stored at 37 ºC had a faster APC depletion than those stored at 4 ºC. Complement-dependent cytotoxicity (CDC) was the most efficient strategy for APC depletion in that most CD45+ cells in the cornea could be depleted within a few hours.
Conclusions::
Prolonged storage of corneal tissue, especially at higher temperatures (37 ºC vs. 4 ºC) reduces the number of APCs in the cornea. Anti-CD45 antibody plus complement-mediated targeting of donor buttons is the most efficient way to deplete APCs in the cornea, and could reduce the length of time required for prolonged storage. This may prove to be a valuable tool for promoting corneal graft survival especially in high-risk keratoplasty.
Keywords: cornea: basic science • transplantation • immunomodulation/immunoregulation