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 that 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 medium at different temperatures for prolonged periods. A third strategy used complement-dependent cytotoxicity (CDC) by treating the buttons with anti-CD45 mAb plus complement. Wholemount 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 hours. 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.

Figure 1

Enumeration of CD45⁺ cells in corneal buttons stored in Optisol-GS® medium. Representative Confocal micrograph of whole-mount corneal buttons, fresh (A) or stored for 14 days (B) in Optisol-GS^® at 4°C. The unit bar in the pictures represents 100 μm. (C) Corneal CD45⁺ cells in the buttons decreased over time when the buttons were stored in medium at either 4°C (n = 5, 3, 8, 9, 5, 4, 5 corneal buttons per time group, respectively. P < 0.005 at day 7 and afterwards) or 37°C (n = 9, 7, 7, 7, 10, 9 corneal buttons per time group, respectively. P < 0.0005 in all storage time groups). Trendlines were fitted to the data and the quality of the fit was calculated as R².

Figure 2

Detection of apoptotic (TUNEL⁺) CD45 cells in corneal buttons. (n = 5, 6, 4, 4, 4 corneal buttons per time group, respectively). (A) Confocal micrographs of whole-mount corneal buttons stored in Optisol-GS^® medium at 4°C for 2 weeks compared with the fresh cornea (green = CD45-Alexa 488, red = TUNEL ). (B) Proportion of apoptotic cells (bars) as percentage of total CD45⁺ cells (line) in the buttons stored in medium at 4°C over time. The percentage shown on the bars indicates the proportion of apoptotic cells. Apoptotic CD45⁺ cell densities in the 2-, 3-, and 4-week groups were significantly higher than those of the fresh corneas (P < 0.05 in the 14-day group, and P < 0.01 in the 21-day, and 28-day groups).

Figure 3

Detection of CD45⁺ cells in Optisol-GS^® medium at 37°C. (A) By 3 days of storage a morphologically heterogeneous population of cells could be detected in the medium. (B) Total CD45⁺ cells in the storage medium detected with laser scanning cytometry and calculated per corneal button.

Figure 4

Evaluation of ex vivo treatment of complement-dependant cytotoxicity (CDC) on corneal buttons. (A) Percent reduction of CD45⁺ cells in the corneal buttons with CDC treatment when compared to the untreated control group (n =7 [control], and 5, 10, 5, 3 corneal buttons per complement concentration groups respectively, *P < 0.00005). (B) Two-color flow cytometric analysis for CD45 expression (FITC) and cell death (LIVE/DEAD^®-Red) on digested corneal buttons treated with CDC. CD45⁺ gates are indicated in cell density plots (arrow). Frequencies of cell death in CD45⁺ gated populations were analyzed in the adjacent histogram. (C) Confocal micrographs of the endothelial layer of the CDC-treated corneas stained for ZO-1 in grayscale. Normal endothelial mosaic and cell density are shown. (D) Cell viability vs. CD45 expression of BM-derived dendritic cells stored in Optisol-GS for up to 7 days, showing cell viability was lost before complete loss of surface CD45 expression.

Figure 5

In vivo studies of CDC-treated donor buttons on HR corneal transplantation. (A) Kaplan-Meier survival curves of CDC-treated corneal grafts and the control groups in HR corneal transplantation; no significant difference was found (P > 0.05). (B) Flow cytometric analysis of infiltrating total CD45⁺ cells and CD3⁺ T cells in transplanted corneas at week 8 post-transplantation.