Depletion of Passenger Leukocytes from Corneal Grafts: An Effective Means of Promoting Transplant Survival?

Abstract

Purpose To develop and compare effective strategies for depleting graft-derived passenger leukocytes which include antigen-presenting cells from corneal buttons, and to assess the effectiveness of this strategy in promoting graft survival using a high-risk (HR) model of corneal transplantation. Methods Corneal buttons harvested from C57BL/6 mice were used in three ex vivo strategies of passenger leukocyte depletion. Two strategies involved storage in Optisol-GS medium at different temperatures for prolonged periods. A third strategy utilized complement-dependent cytotoxicity (CDC) by treating the buttons with anti-CD45 mAb plus complement. Whole-mount corneal buttons or cells from enzyme digested corneas were analyzed using confocal microscopy or flow cytometry, respectively, for the pan-leukocyte surface marker CD45. HR host beds were created and used to evaluate the efficacy of passenger leukocyte depletion on transplant survival. Results Passenger leukocyte numbers in the buttons were significantly reduced by all three treatments. CDC was the most efficient strategy for passenger leukocyte depletion with 39% reduction (P < 0.00005) of CD45+ cells, and negligible damage to the endothelial layer, achievable within 24 h. However, passenger leukocyte depletion failed to improve HR graft longevity. Conclusions Anti-CD45 antibody plus complement-mediated targeting of donor tissue is the most efficient way to deplete corneal passenger leukocytes and can considerably reduce the time required for cell depletion. However, depletion of graft passenger leukocytes does not have a significant effect on promoting graft survival even in the HR setting.