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.

Despite the successful outcome of corneal transplantation in nonvascularized, or so-called low-risk (LR), recipient beds, corneal transplantation performed in vascularized and inflamed high-risk (HR) host beds has shown little improvement in survival in the past several decades. Indeed, though systemic corticosteroids and immunosuppressive agents may be partially effective in preventing graft rejection, their use is limited because of a wide range of side effects, including infection, cataract, and glaucoma. Moreover, even with potent immunosuppression, rejection rates in HR corneal transplantation can be as high as 50% to 90%. ^{1 2} There is, therefore, a compelling need to improve the survival of HR grafts in a manner that minimizes the impact on host immune competence. 

The most common cause of corneal graft failure remains allograft rejection, during which host alloreactive T cells are activated and, once peripheralized to the graft, can result in graft destruction. ³ Allograft rejection is triggered by two distinct, but not mutually exclusive, pathways of allorecognition mediated by either donor-derived antigen-presenting cells (APCs) or recipient APCs—the direct and indirect pathways of allosensitization, respectively. In the direct pathway, donor APCs present intact major histocompatibility complex (MHC) class II molecules residing on their surfaces to T cells. In contrast, in the indirect pathway, recipient APCs present processed MHC or minor antigens to T cells. ^{4 5 6 7 8} The direct and indirect pathways play distinct roles in corneal alloimmunity. With the use of a murine orthotopic corneal transplantation model, Huq et al. ⁶ demonstrated that T cells activated by the direct pathway are detected, and donor-derived APCs are functionally capable of priming host T cells in HR graft recipients. They also found that transplantation with MHC class II knockout donor tissue leads to significantly improved survival of HR allografts. ⁶ Simon et al. ⁹ discovered that prolonged storage of donor corneas prevented allograft rejection, particularly in HR corneal transplantation, but offered no mechanistic information to explain this finding, although they postulated that the depletion of donor APCs through prolonged storage could reduce graft immunogenicity. 

CD45 is a transmembrane molecule found on the surfaces of all bone marrow–derived nucleated hematopoietic cells and their precursors; hence, it is a leukocyte common antigen. ¹⁰ It is well known that the cornea contains a heterogeneous population of bone marrow–derived cells that express the leukocyte common antigen CD45. ^{11 12 13 14} Given the capacity of CD45⁺ cells to potentially prime T cells, and thereby initiate alloimmune responses, it is of interest to investigate the effect of graft passenger leukocyte depletion on corneal transplant survival. To this end, complement-dependent cytotoxicity (CDC) is a mechanism of killing cells in which antibody binds to the receptor of a target cell, then fixes and activates the complement system. The result is the formation of a membrane attack complex that makes a hole within the cell membrane, causing cell lysis and death. CDC can potentially provide a powerful strategy for depleting graft passenger leukocytes before corneal transplantation and has been shown to be effective in renal transplantation. ¹⁵ The present study was conducted to compare the efficiency of ex vivo donor passenger leukocyte depletion using CDC and other strategies and to investigate the acceptance of the APC-depleted corneal grafts in HR corneal transplants. 

Methods

Mice

C57BL/6 and BALB/c mice (male, 6–14 weeks) were purchased from Taconic Farms (Germantown, NY). Corneal buttons from C57BL/6 mice were used as sources of donor tissue because they are MHC and multiple minor H disparate to BALB/c mice, which were used as recipients in the corneal transplantation experiments. For ex vivo experiments, mice were humanely killed by cervical dislocation, and the corneal buttons were harvested. All protocols were approved by the Schepens Eye Research Institute Animal Care and Use Committee, and mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Immunofluorescent Staining of CD45 on Corneal Buttons

To count the number of CD45⁺ cells in the cornea, the corneal buttons were stained immunohistochemically with anti-CD45. All staining procedures were conducted in 96-well plates at room temperature (RT). Antibodies were purchased from BD PharMingen (San Diego, CA) unless otherwise specified. All antibodies were diluted 1:100 in 2% bovine serum albumin in phosphate-buffered saline (PBS-BSA). Wholemount corneal buttons (2-mm diameter) from C57BL/6 mice, either stored in medium (Optisol-GS; Bausch & Lomb Surgical, Irvine, CA) or freshly excised from the mice, were fixed with acetone for 15 minutes, then blocked with anti-Fc receptor (anti-FcR) monoclonal antibody (mAb) CD16/CD32 for 30 minutes to prevent nonspecific staining. The corneas were then stained with rat anti-mouse CD45-FITC (clone30-F11) and the isotype-matched control antibody rat IgG2b-FITC or with Alexa Fluor 488 anti-mouse CD45 and the isotype control antibody Alexa Fluor 488 rat IgG2b; both Alexa Fluor 488 antibodies were obtained from BioLegend (San Diego, CA). After CD45 staining, the corneal buttons were labeled with a nuclear stain (To-Pro-3, 1 μM; Invitrogen-Molecular Probes, Carlsbad, CA) for 30 minutes at RT. After three washings with PBS for 5 minutes each, corneas were mounted with a mounting medium (Vectashield; Vector Laboratories, Burlingame, CA) and analyzed by confocal microscopy. 

Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling Assay

TUNEL assay was used along with CD45 staining to detect the apoptotic CD45⁺ cells in the wholemount corneal buttons stored for prolonged periods. First, wholemount corneal buttons were fixed with acetone for 15 minutes at RT. TUNEL staining was then carried out according to the instructions provided by the manufacturer (Roche, Basel, Switzerland). Afterward, the procedures described for CD45 and nuclear staining were performed. CD45⁺ cell apoptosis was assessed by confocal microscopy. 

Cell Counting and Imaging with Laser Scanning Confocal Microscopy

Confocal laser scanning microscopy was carried out with a spectral confocal laser scanning microscope equipped with an air-cooled, argon-krypton ion-laser system (TCS SP; Leica Microsystems Heidelberg, Heidelberg, Germany). Magnification was achieved using a Nikon oil-immersion lens at 60×. Corneal cells were counted in a masked manner. The central area of each 2-mm button was defined as the central 1-mm section; the remainder was defined as the paracentral cornea (the full diameter of a murine cornea is 3 mm). To count the cells, one to three and six to eight consistent and equally spaced fields were assessed from the central and the paracentral areas, respectively. Images were recorded by continuously focusing the optical dissection through the full-thickness of the cornea within the fixed area of a field. Any cells straddling the upper and left adjacent boundaries of each field were excluded. Because the cells were not uniformly distributed in the cornea, the cell counts of the unit area from different locations were averaged and multiplied by the corresponding amount of the central and paracentral areas to obtain the total number of cells in each corneal button, then expressed and plotted as average cell densities by dividing the total number of cells by the total area of the corneal button. 

Detection of CD45⁺ Cells in Medium with Laser Scanning Cytometry

Corneal buttons were stored in 12-well culture dishes at 37°C for different periods of time. To determine whether CD45⁺ cells emigrated from the corneas into the medium, adherent cells and nonadherent cells were collected from the medium of culture dishes. Isolated cells were washed once with PBS and blocked by incubation with anti-FcR mAb (CD16/CD32) for 20 minutes. The cells were then stained with anti–CD45-FITC. To label the nuclei, cells were fixed with 0.5% paraformaldehyde at 4°C overnight, washed twice with PBS, and stained (Hoechst 33258; Invitrogen) according to the manufacturer’s instructions. After washing with PBS, 10 μL resuspended pellet resuspension was placed onto a spot in the center of a glass slide. The slides were coverslipped and sealed for laser scanning cytometry (LSC; Compucyte Inc., Cambridge, MA) measurements. LSC results were expressed as percentages of positive cells over and above the negative isotype control–stained cells. 

Corneal CD45⁺ Cell Depletion by Complement-Dependent Cytotoxicity

To efficiently deplete CD45⁺ cells from the corneal buttons rather than passively extending the storage time, CDC was carried out using rat anti-mouse CD45 (clone 30-F11) and rabbit complement (Low-Tox-M, CL3051; Cedarlane; Burlington, ON, Canada) according to the manufacturer’s protocol, with minor modifications. The control mAb was the isotype-matched rat IgG2b. Corneal buttons were incubated at 4°C overnight with anti-CD45 or isotype control mAb in storage medium (Optisol-GS; Bausch & Lomb Surgical) at a dilution of 1:100. After three gentle washings with HBSS⁺ for 2 minutes each on ice, the corneal buttons were incubated at 37°C for 1 to 3 hours with different concentrations of complement. The corneal buttons were then gently washed again on ice with HBSS⁺ three times and underwent either confocal microscopic enumeration of CD45⁺ cells after staining of wholemount corneal buttons or flow cytometric analysis on digested corneal buttons. 

To confirm the depletion of CD45⁺ cells from the tissue (as determined by the confocal microscopic enumeration), digestion of corneal buttons was carried out for flow cytometric analysis. After they were washed with PBS on ice, the CDC-treated corneal buttons were incubated for 30 minutes at 37°C in HBSS⁺ medium supplemented with 2 mg/mL collagenase D and 0.5 mg/mL DNase (both from Roche). Corneal buttons were then pressed gently and passed through a nylon mesh screen to remove debris and to generate single-cell suspensions. Cells were washed in PBS twice, blocked by anti-FcR for 20 minutes on ice, and incubated with anti–CD45-FITC alone or in combination with a dead cell marker (Live/Dead Fixable Red Dead Cell Stain Kit; Live/Dead-Red; Invitrogen-Molecular Probes) according to the manufacturer’s instructions. Cell suspensions were washed in PBS-BSA and then resuspended in PBS for flow cytometric analysis. Flow cytometry was performed with a flow cytometer (Epics-XL; Beckman Coulter, Inc., Fullerton, CA), and software (Summit Software version 4.3; Dako, Carpinteria, CA) was used for data analysis. 

Corneal Endothelium Morphologic Study with Zonula Occludens-1 Staining

To assess the status of the endothelial layer as a result of the depletion strategies, zonula occludens (ZO)-1 staining was performed to delineate the tight junctions between endothelial cells, ^{16 17} thereby allowing their enumeration and morphologic characterization. Rabbit anti–ZO-1 mAb (40–2300; Zymed Laboratories, Inc., South San Francisco, CA) was used as the primary antibody, and was diluted at a concentration of 1 μg/mL in PBS-BSA. Normal rabbit IgG was used for the isotype control. The duration of staining of the primary mAb was 2 hours, followed by staining of the secondary antibody, goat anti-rabbit IgG-rhodamine (Santa Cruz Biotechnology, Santa Cruz, CA), for 1 hour, both at RT. After ZO-1 staining, the corneal buttons were labeled with nuclear stain (To-Pro-3; Invitrogen-Molecular Probes) for 30 minutes at RT. After three washings with PBS for 5 minutes each, corneas were mounted with mounting medium (Vectashield; Vector Laboratories) and coverslipped for confocal microscopic analysis. 

High-Risk Corneal Transplantation

The well-established procedures for corneal HR transplantation have been described previously. ^{6 18} Briefly, three interrupted 8-shaped sutures (11-0 nylon, 50-μm diameter needle; Sharpoint, Vanguard, TX) were placed in the central cornea of BALB/c mice to induce inflammation and neovascularization (HR graft beds) 14 days before orthotopic corneal transplantation. Corneal grafts of 2-mm diameter were excised from C57BL/6 mice with a trephine (Storz Instrument Co., St. Louis, MO) and microscissors. Three groups of donor buttons were treated with anti-CD45 plus complement, isotype control mAb plus complement, or storage medium (Optisol-GS; Bausch & Lomb Surgical) just before the transplantation. The recipient bed was prepared by excising a 1.5-mm tissue in the central cornea of BALB/c host mice. Prepared donor corneal grafts were then transplanted onto the host bed with eight interrupted 11-0 nylon sutures. The sutures were removed 7 days after transplantation. Corneal grafts were assessed for graft survival by slit lamp biomicroscopy twice a week for 8 weeks according to a standardized corneal opacity scoring system, ¹⁹ with score 0 representing a clear graft and score 5 representing maximum stromal opacity. Grafts with consistent opacity scores of 3 or higher at 2 weeks or of 2 or higher at or after 3 weeks were defined as rejected. At week 8 after transplantation, the grafted corneas of different groups were harvested and analyzed for infiltrating immune cells. Corneal digestion for preparing single-cell suspension and flow cytometry (using anti–CD45-FITC and anti–CD3-PE mAbs; eBioscience Inc., San Diego, CA) was performed as described. 

Statistical Analyses

Results were expressed as mean ± SEM. When the data were fitted with a regression line, R ² was calculated as a parameter for the quality of the fit (Excel; Microsoft, Redmond, CA). Statistical software (GraphPad Prism, version 3.0; GraphPad, San Diego, CA) was used for all the statistical analyses. When comparing more than two groups for cell density in the corneal buttons, one-way analysis of variance was performed, followed by the Dunnett Multiple Comparison Test in which each experimental group was compared with the control group. Statistical comparisons of CD45⁺ cell counts between the complement plus anti-CD45 mAb and the complement plus isotype control mAb groups were performed with the unpaired Student’s t-test. P < 0.05 was considered statistically significant. Graft survival results were plotted as Kaplan-Meier curves. 

Results

Decrease of CD45⁺ Cell Numbers over Time with Prolonged Storage

Bone marrow–derived cells were successfully deleted from the cornea when the corneas were stored in the medium at both 4°C and 37°C over time. The FITC-stained cells in the corneal buttons were CD45⁺ (Figs. 1A 1B) . Staining was verified by the inclusion of a negatively stained isotype control antibody in each batch of slides. Representative images in Figure 1show that the number of CD45 ⁺ cells decreased significantly when the corneal buttons were stored in medium (Optisol-GS; Bausch & Lomb Surgical) at 4°C for 14 days (Fig. 1B)compared with baseline numbers in fresh corneas (Fig. 1A) . Cell enumeration data are plotted in Figure 1Cand fitted with regression lines. R ² values indicated that approximately 99% of the data could be fitted with a 2° polynomial function for the 4°C curve, and approximately 90% of the data could be fitted with an exponential function for the 37°C curve. The number of CD45⁺ cells decreased faster at 37°C than at 4°C. 

Increase of Apoptotic CD45⁺ Cells with Storage Time

To determine whether the CD45⁺ cells underwent apoptotic death in corneas stored for prolonged periods, groups of corneal buttons stored in the medium for up to 28 days were stained for the TUNEL assay along with Alexa Fluor 488 anti-CD45 staining. CD45⁺ TUNEL⁺ cells were detected in corneas that were stored longer than 2 weeks (Fig. 2A) . Figure 2Bdisplays the densities of CD45⁺ cells in the corneal buttons, showing a significant decrease over time (line; P < 0.05) and an increasing proportion of apoptotic CD45⁺ cells (bars and the percentages). Apoptotic CD45⁺ cell densities in the 2-, 3-, and 4-week groups were significantly higher than those in the fresh corneas (P < 0.05 in the 2-week group and P < 0.01 in the 3- and 4-week groups). 

Concomitant Increase of CD45⁺ Cell Numbers in the Medium at 37°C

Cells with variable morphology were readily observed in the medium when buttons were stored at 37°C. However, no evidence of cells and minimal sloughing of the epithelium were found in the medium at 4°C during the storage period. Because tissue APCs are capable of emigrating out of tissues, ^{20 21} the cells in the medium were assayed for CD45 expression. LSC (a slide-based cytometry system) was chosen over flow cytometry to define the cells because LSC is more suitable for detecting relatively small populations of cells. Concurrent with a decrease in the number of CD45⁺ cells in the corneal buttons (Fig. 1B , solid curve), there was an increase of cells in the medium during the first week (Fig. 3A) . LSC analysis on the cells in the medium indicated that the percentage of CD45⁺ cells started to increase on day 2, peaked around day 7, and started to decrease afterward (experiments were repeated several times; representative data are plotted in Fig. 3B ; n = 40 corneal buttons at each time point). 

Efficient Depletion of CD45⁺ Cells Achieved with Complement-Dependent Cytotoxicity

The primary protocol of the CDC method was developed and tested using bone marrow–derived dendritic cells as prototypic CD45⁺ cells. Cells were treated with anti-CD45 antibody and complement, and the degree of cell killing was assessed (data not shown). Optimized conditions for complement concentration and incubation time that could result in maximal depletion of resident CD45⁺ cells, with minimal toxicity to the endothelial cells, were tested and applied to the donor buttons to deplete resident CD45⁺ leukocytes. Different combinations of complement concentrations (5%–60%) and incubation time (0.5–3 hours) were further tested on donor buttons, including evaluation of endothelial cell health; representative data on CDC-treated corneal buttons are plotted in Figure 4A . Statistically, when compared with the untreated control groups, the anti-CD45 mAb plus complement-treated corneal buttons had significantly fewer CD45⁺ cells in the 20% and 60% (Low-Tox-M, CL3051; Cedarlane) groups, with the respective percentage reductions of CD45⁺ cells 39% (n = 5) and 46% (n = 3) (P < 0.00005; Fig. 4A ). The efficacy of this treatment was further supported by flow cytometric analysis on digested corneal buttons (Fig. 4B) . The two-color flow cytometric analysis for CD45 expression (FITC) and cell death (Live/Dead-Red; Invitrogen-Molecular Probes) revealed that nearly half (48.8%) of the CD45⁺ cells were dying/dead after CDC treatment, not including those that were already dead (Fig. 4B) . 

The corneal endothelium serves a critical role in maintaining corneal clarity. ^{22 23} To ensure the safety of CDC treatment to the endothelium, corneal buttons were stained for ZO-1 so as to delineate their numbers and morphology; indeed, CDC treatment using (Low-Tox-M, CL3051; Cedarlane) complement showed minimal damage to the endothelial cells (Fig. 4C) . Given that treatment for 1-hour incubation at 37°C with 20% complement after anti-CD45 mAb incubation overnight at 4°C caused negligible endothelial changes but substantial degrees of CD45⁺ cell depletion, these parameters were determined as optimal for in vivo transplantation. 

An additional experiment was performed to gain insight into the lifespan and degree of CD45 expression of bone marrow–derived dendritic cells stored in the storage medium for up to 10 days. Our flow cytometric analysis of cell viability and CD45 expression revealed that approximately 70% of the cells were dead/dying on day 2; significant diminishment in CD45 expression was not reached until 2 days later (Fig. 4D) , suggesting that cell viability was lost before complete loss of surface CD45 expression. 

Fate of CDC-Treated Donor Grafts on High-Risk Corneal Transplantation

Donor buttons were treated with anti-CD45 plus complement, treated with isotype antibody plus complement, or kept in the storage medium alone, before transplantation in high-risk host beds. All three groups experienced swift rejection starting on day 17 after transplantation. Survival rates were 22%, 33%, and 30% for the anti-CD45 plus complement, isotype antibody plus complement, and storage medium–only groups, respectively (Fig. 5A) . The anti-CD45 plus complement group did not differ significantly from its two control groups, isotype antibody plus complement and storage medium alone (Fig. 5A) . In addition, the transplanted corneas of different groups were harvested at week 8 after transplantation to quantify and analyze the phenotypes of infiltrating immunocytes (Fig. 5B) . The anti-CD45 plus complement-treated group showed slightly higher infiltration of CD45⁺ cells compared with the isotype plus complement and the storage medium–only groups. However, the degree of CD3⁺ cell infiltration was comparable among the three different groups. 

Discussion

The concept that depletion of class II-expressing “passenger” leukocytes could promote long-term survival of allografts by reducing immunogenicity was established more than 20 years ago ^{24 25 26} and has played a central role in solid organ (e.g., heart, kidney) transplantation. For this reason, and because we (and subsequently many others) showed that the cornea also bears bone marrow–derived cells, ^{11 12} we hypothesized that their depletion would promote graft survival. Distinct ex vivo strategies of CD45⁺ cell depletion from the corneal buttons were successively developed in the present study, and the most efficient method was assessed for HR corneal transplantation. The fact that the manipulation did not improve survival suggested that perhaps these bone marrow–derived cells may also be relevant in the promotion of graft tolerance. 

Our CD45⁺ cell density data in the normal corneal buttons (day 0) are comparable to those reported by Brissette-Storkus et al., ²⁷ despite the different mouse strains. Our data for CD45⁺ cell numbers in the cornea over time in the 4°C medium show slightly lower values (Fig. 1C , dotted line) than do those in Figure 2Bat corresponding time points, probably because of the different staining reagents. Alexa 488 anti-CD45 seems to be more sensitive than anti–CD45-FITC. Both sets of data, however, show similar trends. CD45⁺ cell density decreased over time in the medium at 4°C. Indeed, the increased apoptotic CD45⁺ cell density concomitant with decreased CD45⁺ cell density, detected by two different staining reagents, provides evidence that the reduction in CD45⁺ cells in the wholemount corneal buttons over prolonged storage time is not artifact. 

The decrease in the number of CD45⁺ cells assayed in the medium after 7 days of storage (Fig. 3B)may be accounted for by the short lifespan of the cells in the medium. Chinnery et al. ²⁸ have shown a turnover rate for bone marrow–derived corneal cells of approximately 24% by 2 weeks in an in vivo model. Our study, however, investigated cell viability/lifespan in an in vitro system, which may explain the shorter lifespan observed. Because of the delay in complete loss of CD45 expression after cell death (Fig. 4D) , we postulate that the actual number of viable CD45⁺ cells in the corneal buttons after the depletion manipulations is lower than the staining data alone may suggest. 

Because CD45 is a transmembrane molecule found on the surfaces of all nucleated hematopoietic cells, ¹⁰ it offers a means of targeting all leukocyte populations, including passenger APCs in the graft tissue, with the aim of reducing graft alloimmunity. All professional (bone marrow–derived) APCs are CD45⁺; hence, targeting CD45⁺ would allow the depletion of APC populations. Anti-CD45 mAbs have been successfully applied to deplete renal allografts of passenger leukocytes in allogeneic transplantation. ²⁹ In contrast, the CDC method used in this study provides a more precise way of targeting intragraft leukocytes because the complement system can be activated by specific complement-fixing-antibodies directed at specific cell surface (e.g., CD45) epitopes. The anti-CD45 antibody used in the present study, rat IgG2b, clone 30-F11, has also been previously used to readily lyse CD45⁺ cells in the presence of complement. ³⁰ A clinical trial of ex vivo perfusion of two complement-fixing anti-CD45 mAbs into kidneys (for the purpose of depleting donor-derived CD45⁺ cells, which include APCs) before transplantation achieved significant reductions in the incidence of rejection and improvements in long-term renal function. ¹⁵ The present study provides the first data regarding the feasibility of ex vivo CD45⁺ cell depletion in corneal graft tissue. 

Reductions in MHC class I and minor H antigen expression in the preserved corneal grafts have been found to improve the acceptance in corneal allografts in a normal-risk corneal transplantation model. ³¹ However, it is not known whether the approach would have the same effect in HR transplantation, in which graft-derived leukocytes are thought to be particularly relevant for alloimmunization. ⁶ Furthermore, preservation of corneal buttons requires a longer waiting time and can be detrimental to endothelial cells. Comparison of the various methods for CD45⁺ cell depletion in the present study showed that the corneas stored at 37°C underwent faster depletion than those stored at 4°C for prolonged periods. However, anti-CD45 antibody plus complement-mediated targeting of donor buttons is an even more efficient way to deplete passenger leukocytes ex vivo because most CD45⁺ cells can be depleted within 1 day. This reduces the length of time required for prolonged storage, which can be deleterious for the viability of the epithelial and the endothelial layers (the latter are especially essential for corneal transplant clarity). ^{22 23 32}  

In the study reported by Huq et al., ⁶ corneal transplantation using MHC class II knockout donor tissue led to significantly improved survival of HR allografts in mice. That experimental setting completely eliminated all donor MHC class II expression, thereby preventing not only direct sensitization but also indirect sensitization to donor MHC class II antigens because host APCs would also be unable to process and present donor MHC class II to T cells. In the present study, which relied on passenger leukocyte depletion, however, only the direct pathway was suppressed, with a reduced capacity of donor APCs to present donor MHC molecules to host T cells. In contrast, host APCs would still be able to process donor MHC class II molecules expressed by nonleukocyte populations. Thus, the fact that we did not observe an improved transplantation survival in our in vivo transplantation study may be accounted for by at least two factors leading to graft rejection, the lack of effect of this donor passenger leukocyte depletion strategy on the indirect pathway mediated by host APCs and the suppression of a possible tolerogenic effect of graft (donor)-derived cells on host immunity. Indeed, strong evidence is accumulating that will indicate immature dendritic cells of either donor or host origin play a fundamental role in transplant tolerance by perhaps activating regulatory T cells (Tregs). ^{33 34 35} In fact, a growing number of studies are focused at the therapeutic use of immature dendritic cells on the induction of Tregs in transplantation, raising the specter that graft passenger leukocyte depletion may also have deleterious consequences in terms of Treg induction. Further experiments are required to investigate this provocative hypothesis. 

 Supported by National Institutes of Health Grant EY-R01-12963.

 Submitted for publication February 18, 2008; revised July 24, December 12 and 19, 2008; accepted April 21, 2009.

 Disclosure: X. Zhang, None; L. Shen, None; Y. Jin, None; D.R. Saban, None; S.K. Chauhan, None; R. Dana, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Enumeration of CD45⁺ cells in corneal buttons stored in medium. Representative confocal micrograph of wholemount corneal buttons, fresh (A) or stored for 14 days (B) in medium at 4°C. Scale bars, 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 afterward) 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 quality of the fit was calculated as R ².

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 wholemount corneal buttons stored in 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 a 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; P < 0.01 in the 21-day and 28-day groups).

Figure 3.

 

Detection of CD45⁺ cells in 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 CDC on corneal buttons. (A) Percentage reduction of CD45⁺ cells in the corneal buttons with CDC treatment compared with 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 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 versus CD45 expression of bone marrow–derived dendritic cells stored in medium 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 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 after transplantation.

The authors thank Lu Chen, Andrew Taylor, Sharmila Masli, Dong-feng Chen, and Rita Barcia for helpful discussions and Don Pottle (Confocal Microscopy Unit) and Qiang Zhang for excellent technical assistance.