Zhang Xian, Shen Linling, Jin Yiping, Saban Daniel R, Chauhan Sunil K, Dana Reza
Schepens Eye Research Institute, Boston, Massachusetts 02114, USA.
Invest Ophthalmol Vis Sci. 2009 Jul;50(7):3137-44. doi: 10.1167/iovs.08-1899. Epub 2009 Jan 10.
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.
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.
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.
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.
开发并比较有效清除移植物来源的过客白细胞（包括角膜植片中的抗原呈递细胞）的策略，并使用角膜移植的高风险（HR）模型评估该策略在促进移植物存活方面的有效性。
从C57BL/6小鼠获取的角膜植片用于三种体外清除过客白细胞的策略。两种策略涉及在不同温度的培养基中长时间保存。第三种策略是通过用抗CD45单克隆抗体加补体处理植片，利用补体依赖的细胞毒性（CDC）。分别使用共聚焦显微镜或流式细胞术，对全层角膜植片或酶消化角膜的细胞进行分析，以检测全白细胞表面标志物CD45。创建HR宿主床，用于评估清除过客白细胞对移植存活的效果。
所有三种处理均使植片中的过客白细胞数量显著减少。CDC是清除过客白细胞最有效的策略，可使CD45(+)细胞减少39%（P < 0.00005），且对内皮细胞层的损伤可忽略不计，在24小时内即可实现。然而，清除过客白细胞未能提高HR移植物的寿命。
抗CD45抗体加补体介导的供体组织靶向是清除角膜过客白细胞最有效的方法，可大幅缩短细胞清除所需时间。然而，即使在HR环境下，清除移植物中的过客白细胞对促进移植物存活也没有显著影响。
