February 2003
Volume 44, Issue 2
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Immunology and Microbiology  |   February 2003
Survival in High-Risk Eyes of Epithelium-Deprived Orthotopic Corneal Allografts Reconstituted In Vitro with Syngeneic Epithelium
Author Affiliations
  • Junko Hori
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • J. Wayne Streilein
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Investigative Ophthalmology & Visual Science February 2003, Vol.44, 658-664. doi:10.1167/iovs.02-0399
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      Junko Hori, J. Wayne Streilein; Survival in High-Risk Eyes of Epithelium-Deprived Orthotopic Corneal Allografts Reconstituted In Vitro with Syngeneic Epithelium. Invest. Ophthalmol. Vis. Sci. 2003;44(2):658-664. doi: 10.1167/iovs.02-0399.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. In low-risk eyes of mice, most of the composite corneal grafts composed of syngeneic epithelium layered on allogeneic stroma and endothelium are accepted indefinitely. The study was undertaken to determine the fate of similar composite corneal grafts placed in high-risk mouse eyes.

methods. Epithelium-deprived allogeneic corneas (C57BL/6) were reconstituted in vitro with BALB/c epithelium, and then transplanted orthotopically into high-risk eyes of BALB/c mice. Graft survival was assessed clinically and evaluated histologically. Acquisition of donor-specific delayed hypersensitivity (DH) was also assessed in recipient mice. Recipients bearing healthy composite grafts were immunized subcutaneously with injected C57BL/6 spleen cells at 2 or 8 weeks after grafting, after which the fate of the grafts was evaluated.

results. Virtually all epithelium-deprived corneal allografts reconstituted in vitro with normal BALB/c corneal epithelium survived indefinitely when placed in high-risk eyes of BALB/c mice. Recipients of these composite grafts failed to acquire donor-specific DH when tested at both 2 and 8 weeks after grafting. Moreover, these recipients did not acquire the capacity to actively suppress donor-specific DH. Within 1 to 3 weeks of sensitization of recipient mice with spleen cells of donor origin, healthy composite grafts in residence for 2 or 8 weeks were rejected.

conclusions. Replacement of donor epithelium with syngeneic epithelium protects orthotopic allogeneic corneal grafts (stroma plus endothelium) placed in high-risk eyes from sensitizing their recipients and from immune-mediated rejection. Recipients of composite corneal grafts containing syngeneic epithelial layers act as though they are immunologically ignorant of the graft’s presence.

Despite the existence of immune privilege, 1 2 orthotopic corneal allografts sometimes fail, and immune rejection is the primary cause of graft failure. 3 Although the ability of immunity to break through the mechanisms of immune privilege is of interest to researchers, the rejection that overcomes immune privilege and causes corneal allograft failure in human beings is more than interesting: It causes blindness. Because of this unwanted outcome, it is important to develop strategies that have the potential to interfere with antigraft immunity and thereby promote graft acceptance. To that end, our laboratory has recently reported a novel procedure that virtually eliminates rejection of orthotopic corneal allografts in normal eyes of laboratory mice. 4 The procedure involves, first, removal of donor epithelium from the donor cornea, and second, replacement of the denuded anterior surface of the donor graft (now composed solely of stroma plus endothelium) with epithelium genetically identical with the intended recipient. When such composite grafts, comprising recipient-type epithelium and allogeneic donor stroma plus endothelium, were placed orthotopically in normal eyes of recipient mice, virtually all the grafts survived indefinitely and remained perfectly clear. Companion experiments demonstrated that an intact layer of recipient epithelium on these composite grafts had the remarkable ability to inhibit the development of inflammation (infiltration with inflammatory cells and angiogenesis) within the graft stroma. 4 The inference was drawn that corneal allografts that fail to become inflamed may also fail to sensitize their recipients to donor alloantigens. 
Although a significant proportion of orthotopic corneal allografts survive indefinitely when transplanted into normal eyes of mice and rats, 5 6 virtually no such grafts survive when transplanted into so-called high-risk eyes. Thus, full-thickness allogeneic corneas that are placed in eyes with suture-induced neovascularized corneas of otherwise normal mice are typically rejected within 14 days by an intense inflammatory and destructive reaction. 7 8 9 A similarly poor outcome has been reported for corneal transplants performed in high-risk human eyes. 3 In fact, the failure rate of corneas transplanted into human high-risk eyes is at least as high as the failure rate of other solid tissue grafts in humans (kidney, heart, liver, islets of Langerhans). Moreover, clinically available immunosuppressive therapy intended to reverse corneal graft rejection in high-risk eyes is too frequently inadequate. 3 7 8  
Because composite corneal grafts containing recipient epithelium that covers donor stroma plus endothelium has high acceptance rates in normal eyes of mice, 4 it seemed worthwhile to determine whether corneal allografts similarly covered with recipient epithelium would enjoy enhanced survival when placed in high-risk mouse eyes. The results of experiments described in this article indicate that corneal allografts covered with recipient epithelium had a very high rate of acceptance in high-risk eyes of otherwise normal mice. Moreover, recipients of these grafts showed no evidence of donor-specific sensitization, implying that graft acceptance may result from immunologic ignorance. 
Materials and Methods
Mice and Anesthesia
Male BALB/c (H-2d) and C57BL/6 (B6, H-2β) mice were purchased from Taconic Farms (Germantown, NY). All mice were used at 8 to 12 weeks of age and treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Each mouse was anesthetized by intramuscular injection of a mixture of 3.75 mg ketamine and 0.75 mg xylazine before all surgical procedures. 
Preparation of Epithelial Sheets and Epithelium-Deprived Corneas
The corneal epithelial cell layer was peeled off, as an intact sheet, from full-thickness corneas of normal BALB/c mice after a 1-hour incubation in 20 mM EDTA at 37°C. The epithelial sheets were then washed with PBS. The epithelium-deficient stroma plus endothelium component was also washed with PBS and then used as a graft. 
Composite Grafts Created by Reconstitution of the Cornea In Vitro
Corneal epithelium was prepared as an intact sheet from full-thickness normal corneas of BALB/c mice, as just described. With the aid of a dissecting microscope, these sheets were floated in PBS to avoid sticking and making folds and then gently placed to cover the stromal surface in PBS of 2-mm diameter corneal stroma plus endothelium components that had been similarly prepared from eyes of C57BL/6 mice. Immediately after the coverage, the composite cornea was gently removed from the PBS and placed on the high-risk recipient bed, and sutures were applied for orthotopic transplantation. 
Creation of High-Risk Eyes in Recipients
Three sutures of 11-0 nylon were placed in the central cornea of one eye in BALB/c mice. Fourteen days later, these eyes with intensely neovascularized corneas were regarded as high-risk and prepared as recipient beds. 
Orthotopic Corneal Transplantation and Graft Evaluation
Penetrating keratoplasty was performed as described previously. 4 5 Briefly, 2-mm diameter donor corneas (intact full-thickness or composite as described earlier) were placed in the same sized recipient bed with eight interrupted sutures (11-0 nylon). Sutures were removed at 8 days after grafting. Orthotopic grafts were observed with slit lamp microscopy at weekly intervals, and judgment of orthotopic corneal graft survival was performed in masked fashion according to a published scoring system 4 5 : 0, clear graft; 1+, minimal superficial nonstromal opacity; 2+, minimal deep stromal opacity with pupil margin and iris vessels visible; 3+, moderate deep stromal opacity with only pupil margin visible; 4+, intense deep stromal opacity with the anterior chamber visible; and 5+, maximum stromal opacity with total obscuration of the anterior chamber. Grafts with opacity scores of 2+ or higher after 3 weeks were considered to have been rejected. 
Histology of Reconstituted Corneal Grafts in High-Risk Eyes
Eyes bearing composite corneal grafts were removed for histologic assessment at 8 weeks after transplantation, fixed with 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. 
Assessment of Delayed Hypersensitivity
Two or 8 weeks after orthotopic corneal transplantation (using either composite grafts reconstituted in vitro or control full-thickness C57BL/6 corneal allografts), 1 × 106 irradiated (2000 rad) spleen cells/10 μL from C57BL/6 mice were injected into the right ear pinnae of all BALB/c recipient mice. As a positive control, a similar number of irradiated spleen cells were injected into the ear pinnae of normal BALB/c mice that had been immunized 1 week earlier by subcutaneous (SC) injection of 10 × 106 C57BL/6 spleen cells. As a negative control, 1 × 106 irradiated spleen cells were injected into ear pinnae of mice that were not previously immunized were not graft recipients. Twenty-four and 48 hours after intrapinnal injection, ear thickness was measured with a low-pressure engineer’s micrometer (Mitsutoyo; MTI Corp., Paramus, NJ). Ear-swelling was expressed as follows: Specific swelling = [(24-hour numerical values of right ear − 0-hour numerical values of right ear) − (24-hour numerical values of left ear − 0-hour numerical values of left ear)] × 10−3 mm. Ear-swelling responses at 24 hours after ear injection are presented as the group mean ± SEM. 
Test of Active Suppression of Delayed Hypersensitivity
One week after injection of 1 × 106 irradiated (2000 rad) C57BL/6 spleen cells/10 μL into the right ear pinnae of BALB/c mice bearing composite grafts reconstituted in vitro or control full-thickness corneal allografts, the same recipient mice received a second injection of 1 × 106 irradiated (2000 rad) C57BL/6 spleen cells/10 μL into the left ear pinna. As a positive control, a similar number of irradiated spleen cells was injected into the left ear pinnae of BALB/c mice that were immunized 1 week earlier by an intrapinnal injection of 1 × 106 C57BL/6 spleen cells (these mice were negative control mice in the original DH assessment). As a negative control, 1 × 106 irradiated C57BL/6 spleen cells were injected into the left ear pinnae of mice not previously grafted or immunized. At 24 and 48 hours after injection into the left ear pinna, ear thickness was measured with a low-pressure engineer’s micrometer (Mitutoyo, MTI Corporation, Paramus, NJ). Ear-swelling was expressed as follows: Specific swelling = [(24-hour numerical values of left ear − 0-hour numerical values of left ear) − (24-hour numerical values of right ear − 0-hour numerical values of right ear)] × 10−3 mm. Ear-swelling responses at 24 hours after ear injection are presented as the group mean ± SEM. 
Allosensitization in Recipients Bearing Accepted Grafts
BALB/c mice bearing healthy composite corneal grafts for 8 weeks received an SC injection of 10 × 106 C57BL/6 spleen cells. Separate panels of BALB/c mice received intrapinnal challenge with 1 × 106 irradiated C57BL/6 spleen cells to assay for delayed hypersensitivity (DH) at 2 weeks after grafting. The fate of the resident composite grafts was evaluated clinically as described herein. Allosensitization leading to positive DH is induced by both 1 × 106 and 10 × 106 allogeneic spleen cells. For cognate sensitization, the 10 × 106 dose is routinely used in this laboratory. When sensitization was tested after intrapinnal injection of allogeneic spleen cells to assay DH, 1 × 106 cells were injected because of technical limitations of this intradermal site. The sensitization evoked by these two sensitizing doses was similar. 
Statistical Analyses
Corneal graft survival in panels of recipient mice were compared with Kaplan-Meier survival curves and Breslow-Gegan Wilcoxon test. Ear-swelling measurements were evaluated by using a two-tailed Student’s t-test. P < 0.05 was deemed significant. 
Results
Fate in High-Risk Eyes of Epithelium-Deprived Orthotopic Corneal Allografts Reconstituted In Vitro with Syngeneic Epithelium
Nylon sutures (three 11-0) were placed in the central cornea of right eyes of groups of BALB/c mice. Two weeks later, the stroma of these corneas was intensely neovascularized and inflamed. At this time, corneas were excised from normal eyes of BALB/c and C57BL/6 donors. The corneas were incubated in EDTA for 1 hour, after which the epithelium was gently removed as an intact sheet. Composite corneas were then created by gently layering an epithelial sheet over the anterior surface of a denuded stroma plus endothelium. Composite corneas containing BALB/c epithelium and C57BL/6 stroma plus endothelium are referred to as composite corneal allografts. Composite corneal allografts and intact, full-thickness C57BL/6 corneas (positive control) were then grafted orthotopically onto high-risk eyes of BALB/c recipients. The fate of these grafts was assessed clinically, and the results are presented in Figure 1 . As anticipated, all full-thickness C57BL/6 corneal grafts were rejected within 3 weeks in high-risk eyes. By contrast, only 1 of 10 composite corneal allografts was rejected at 3 weeks. Moreover, the remaining composite grafts survived and retained their optical clarity for the entirety of the 12-week observation interval. 
Eyes bearing these composite allografts were removed at 8 weeks and subjected to histologic examination. As the photomicrographs presented in Figure 2 indicate, composite corneal allografts displayed no evidence of inflammatory infiltrates in the stroma and no evidence of neovessels. Epithelial, stromal, and endothelial cells were present and displayed a normal appearance in these grafts. By contrast, rejected full-thickness C57BL/6 corneal allografts showed intense stromal inflammation and neovessels, with loss of identifiable keratocytes. No corneal endothelial cells were observed. These findings indicate that an intact layer of syngeneic epithelium covering the surface of an allograft of stroma plus endothelium protects these grafts from immune rejection, even if the composite graft is placed in a high-risk eye. 
Capacity to Induce DH in Epithelium-Deprived Orthotopic Corneal Allografts Reconstituted In Vitro with Syngeneic Epithelium Placed in High-Risk Eyes
The next experiments examined whether epithelium-deprived corneal allografts reconstituted in vitro with syngeneic epithelium would sensitize recipients to donor-specific alloantigens. BALB/c mice that received in their high-risk eyes epithelium-deprived corneal allografts reconstituted in vitro with normal syngeneic epithelium or control full-thickness C57BL/6 corneal allografts were tested for donor-specific DH, by an intradermal injection into the ear pinna of 1 × 106 x-irradiated C57BL/6 spleen cells, at 2 and 8 weeks after grafting. Positive control BALB/c mice were immunized 1 week before ear challenge with an SC injection of 10 × 106 C57BL/6 spleen cells. Ear-swelling was assessed 24 and 48 hours after intrapinnal injection. The results of this experiment are presented in Figure 3 . As reported previously, 10 full-thickness C57BL/6 corneas placed in high-risk eyes induced donor-specific DH in all recipients when tested at both 2 and 8 weeks after grafting. By contrast, none of the recipients of epithelium-deprived corneal allografts reconstituted in vitro with normal syngeneic epithelium placed in high-risk eyes displayed donor-specific DH, whether tested at 2 or 8 weeks after grafting (Fig. 3) . These results have two possible explanations: On the one hand, absence of donor-specific DH may indicate that recipients of composite corneal allografts never became sensitized to donor antigens. On the other hand, the absence of donor-specific DH may reflect the emergence of immune suppression directed at donor alloantigens. The following experiment was designed to discriminate between these two possibilities. 
Capacity to Induce Suppression of Donor-Specific DH of Epithelium-Deprived Orthotopic Corneal Allografts Reconstituted In Vitro with Syngeneic Epithelium Placed in High-Risk Eyes
In the preceding experiment, BALB/c recipients of epithelium-deprived corneal allografts reconstituted in vitro with normal syngeneic epithelium were tested for DH at 8 weeks after grafting by an intradermal injection into the right ear pinna of 1 × 106 x-irradiated C57BL/6 spleen cells. This dose of allogeneic spleen cells is sufficient to induce donor-specific DH. Because these mice displayed insignificant ear-swelling responses (indicating that DH to donor alloantigens had not yet developed), it was possible to use them in a subsequent experiment to determine whether immune suppression was present. Thus, 1 week after the first injection of donor spleen cells into the right ear pinna, the left ear pinna of these mice received an injection of 1 × 106 x-irradiated C57BL/6 spleen cells. Positive control BALB/c mice were immunized 1 week before the left ear challenge with a right ear injection of 1 × 106 C57BL/6 spleen cells. Ear-swelling responses were once again determined at 24 and 48 hours. The prediction of this experiment is that if donor-specific suppression is responsible for the absence of donor-specific DH when the mice were challenged at 8 weeks, the repeat injection of donor lymphoid cells 1 week later should also fail to elicit significant ear-swelling. The results are presented in Figure 4 . All recipients of epithelium-deprived corneal allografts reconstituted in vitro with normal syngeneic epithelium that received an intrapinnal injection of donor spleen cells 1 week earlier mounted significant ear-swelling responses to a second intrapinnal challenge with donor spleen cells. The intensity of these swelling responses was quantitatively similar to that of the positive control groups. These results indicate that mice bearing healthy composite corneal allografts do not acquire the capacity to suppress immunity directed at donor alloantigens. Therefore, we conclude that orthotopic composite corneal allografts covered with recipient epithelium are not immunogenic and fail to sensitize their recipients. We infer that failure of sensitization accounts for why these grafts are accepted indefinitely. 
Fate in High-Risk Eyes of Healthy Composite Allografts after Systemic Allosensitization
The preceding results indicate that mice bearing healthy composite grafts failed to become sensitized to donor alloantigens. Two possible explanations exist for this situation: composite grafts may lose their capacity to express donor transplantation antigens, or recipients of composite grafts may be immunologically ignorant of the graft’s presence. To distinguish between these two possibilities, two panels of recipient mice were studied. In the first, recipients bearing healthy composite grafts at 2 weeks after grafting received an intradermal injection into the ear pinna of 1 × 106 irradiated C57BL/6 donor spleen cells. Unlike uninjected counterparts, all these mice rejected their composite grafts within 2 weeks (Fig. 5A) . In the second, recipients bearing healthy composite allografts for 8 weeks received an SC injection of 10 × 106 C57BL/6 spleen cells. Nine of 10 established composite allografts died because of rejection within 1 to 3 weeks of systemic sensitization (Fig. 5B) . We conclude that orthotopic composite corneal allografts covered with recipient epithelium retain their alloantigenicity, but prevent recipients from becoming sensitized to the donor alloantigens—a form of immunologic ignorance. 
Discussion
Full-thickness (epithelium-containing) allogeneic corneas induce donor-specific sensitization when grafted orthotopically, 10 11 12 when implanted into the anterior chamber, 13 14 and when placed beneath the kidney capsule. 15 16 Corneal epithelium also expresses class I major histocompatibility complex (MHC) antigens more strongly than do either keratocytes or corneal endothelial cells. 17 18 19 Findings of this type have led to the proposal that the primary immunogenicity of the cornea as an allograft resides within the epithelium. The validity of this proposal is challenged, however, by the observation that corneal allografts from which the epithelial layer has been removed are much more immunogenic and vulnerable to rejection than full-thickness allogeneic corneas. 4 Moreover, as we have reported, the simple artifice of covering an epithelium-deprived allogeneic cornea graft (stroma plus endothelium) with epithelium genetically identical with the intended recipient virtually eliminates the vulnerability of that graft to rejection when it is placed in low-risk graft beds. 4 In the present experiments, similar composite grafts (syngeneic epithelium, allogeneic stroma plus endothelium) were placed in neovascularized (high-risk) eyes of mice, and most of these grafts survived indefinitely. Moreover, mice bearing healthy composite allografts in high-risk eyes failed to acquire donor-specific DH, suggesting that the covering of syngeneic epithelium on these grafts somehow shielded the recipient immune system from donor antigens expressed on keratocytes and endothelium. Together, the results suggest that corneal epithelium possesses a graft-survival-promoting action that is revealed if the epithelium itself confronts the recipient with no alloantigens. 
Support for this suggestion is provided by our experiments that examined the fate of healthy composite allografts after systemic immunization of recipients with donor alloantigens. Whether composite allografts were in place in high-risk eyes for 2 or 8 weeks at the time of immunization, virtually all the grafts became opaque and were rejected. These results make three important points. First, they indicate that the lack of donor-specific DH in mice bearing healthy composite allogeneic corneal grafts is due to an absence of allosensitization, because cognate systemic immunization produced relatively acute rejection of the composite grafts. Second, these results indicate that healthy composite corneal allografts continue to express transplantation antigens of the original donor. 20 We showed, by using enhanced green fluorescence protein transgenic mice, that donor stromal keratocytes and endothelial cells are retained virtually intact in accepted corneal allografts. Cells of the composite grafts bearing donor antigens must have served as the targets of the alloimmune effector T cells that were induced by systemic sensitization with donor antigens. Third, these results indicate that a covering of syngeneic epithelium is powerless to protect allogeneic stroma and endothelium from immune rejection if sensitization is achieved systemically. For these reasons, we are convinced that the reason composite corneal allografts are accepted, even in high-risk mouse eyes, is that the syngeneic epithelial surface arranges immunologic ignorance within the recipient. This ignorance is passive, however, because active sensitization after the composite graft had been accepted evoked graft failure. 
It was important for this series of experiments to be conducted in high-risk, neovascularized eyes of mice, because it is known that the immune privilege that is normally present in low-risk eyes is abolished in high-risk eyes. Conventional corneal allografts placed in high-risk eyes are summarily rejected within 2 weeks of engraftment, 9 10 a brutal reminder of the what absence of immune privilege can mean. Moreover, high-risk eyes cannot support induction of anterior-chamber-associated immune deviation (ACAID), 21 a corollary of immune privilege that arises within 8 weeks of corneal grafting and is important in long-term survival of accepted corneal allografts. 11 22 23 Yet, mice bearing healthy composite corneal allografts for 8 weeks in high-risk eyes displayed no evidence of impaired capacity to acquire donor-specific DH—evidence against the presence of ACAID. Thus, the immunologic ignorance that prevents these composite graft-bearing mice from acquiring donor-specific DH also prevents them from acquiring ACAID. 
It is worth considering what property or properties of epithelium allow it, when genetically identical with the recipient, to prevent the recipient immune system from recognizing and rejecting the allogeneic stroma and endothelium with which the epithelium forms a composite graft. We have shown that orthotopic grafts of allogeneic corneas deprived of epithelium rapidly induce neovascularization (both heme and lymph angiogenesis) in the recipient bed and in the graft stroma. 4 Such grafts rapidly acquire recipient bone-marrow-derived dendritic cells and macrophages, class II MHC-expressing cells that capture donor alloantigens and migrate, presumably through neolymph vessels, 24 25 26 to the draining cervical lymph nodes, where they induce rapid and intense donor alloimmunity. 27 28 29 A similar angiogenic response coupled with infiltration of bone-marrow-derived cells is observed when full-thickness corneal allografts are placed in either low- or high-risk beds. 30 By contrast, our present experiments indicate that allogeneic stroma-plus-endothelium grafts covered with an epithelial layer genetically identical with the recipient incited little evidence of angiogenesis within the graft bed, and infiltration by recipient leukocytes was also at a low level. We infer that syngeneic epithelium layers produce factor(s) that suppress angiogenesis and inflammation within the graft and its bed, and we further speculate that the ability to produce these factors is compromised when the epithelium is allogeneic with respect to the recipient. Presumably, the high expression of transplantation antigens on the epithelium alerts the recipient’s immune system quickly to the presence of the allogeneic graft, and the rapid immune response eliminates the epithelium’s capacity to suppress angiogenesis and inflammation. Experiments to identify the putative factors produced by corneal epithelium are the subject of current investigations. 
The ability of syngeneic corneal epithelial sheets to suppress orthotopic corneal allograft rejection, even in high-risk eyes, is remarkable and unprecedented. If it is possible to grow sheets of corneal epithelium in vitro from small samples of recipient cornea and if these sheets possess the same antiangiogenic and anti-inflammatory properties as do their in vivo counterparts, a novel strategy can be envisioned in which long-term survival can be achieved in high-risk eyes of composite grafts composed of syngeneic epithelium and allogeneic stroma plus endothelium. 
 
Figure 1.
 
Fate in high-risk eyes of epithelium-deprived C57BL/6 corneal allografts reconstituted in vitro with BALB/c (syngeneic) epithelium. Three 11-0 nylon sutures were placed in the central cornea of eyes of BALB/c mice. After 2 weeks, these eyes were used as graft beds (high-risk). At that time, composite corneal allografts (BALB/c epithelium layered onto denuded C57BL/6 stroma-endothelium) were prepared in vitro and placed in the high-risk beds. As a positive control, full-thickness C57BL/6 corneas were placed in high-risk BALB/c eyes. The survival of the grafts was assessed clinically. (⋆) Survival pattern significantly better than that of full-thickness allografts (P < 0.0001).
Figure 1.
 
Fate in high-risk eyes of epithelium-deprived C57BL/6 corneal allografts reconstituted in vitro with BALB/c (syngeneic) epithelium. Three 11-0 nylon sutures were placed in the central cornea of eyes of BALB/c mice. After 2 weeks, these eyes were used as graft beds (high-risk). At that time, composite corneal allografts (BALB/c epithelium layered onto denuded C57BL/6 stroma-endothelium) were prepared in vitro and placed in the high-risk beds. As a positive control, full-thickness C57BL/6 corneas were placed in high-risk BALB/c eyes. The survival of the grafts was assessed clinically. (⋆) Survival pattern significantly better than that of full-thickness allografts (P < 0.0001).
Figure 2.
 
Histologic appearance of in vitro-generated composite corneal grafts. Epithelium derived from BALB/c corneas was layered in vitro onto the denuded surface of C57BL/6 stroma-endothelium corneas. These composite corneas were grafted orthotopically into high-risk eyes of BALB/c recipients. (A, B) Histologic appearance of composite grafts at 8 weeks; (C, D) full-thickness C57BL/6 corneal allografts. Magnification: (A, C) ×10; (B, D) ×40.
Figure 2.
 
Histologic appearance of in vitro-generated composite corneal grafts. Epithelium derived from BALB/c corneas was layered in vitro onto the denuded surface of C57BL/6 stroma-endothelium corneas. These composite corneas were grafted orthotopically into high-risk eyes of BALB/c recipients. (A, B) Histologic appearance of composite grafts at 8 weeks; (C, D) full-thickness C57BL/6 corneal allografts. Magnification: (A, C) ×10; (B, D) ×40.
Figure 3.
 
Induction of donor-specific DH after orthotopic transplantation of epithelium-deprived C57BL/6 corneal allografts reconstituted with normal BALB/epithelium in high-risk BALB/c recipients (syngeneic epi + allogeneic st-end). Composite grafts of epithelium-deprived C57BL/6 corneas with BALB/c epithelium were orthotopically grafted into high-risk eyes of BALB/c recipients. Positive control (Pos. C) animals received SC injection of 10 × 106 C57BL/6 spleen cells 1 week before assay. At 2 weeks (A) or 8 weeks (B) after grafting, mice received an injection of x-irradiated C57BL/6 spleen cells (1 × 106) into the right ear pinna, and ear-swelling responses were assessed 24 and 48 hours later. Negative control (Neg. C) received right-ear-pinna challenge only. Mean 24-hour ear-swelling responses are compared with the negative control. *Responses significantly greater than the negative control (*P < 0.05, ***P < 0.005).
Figure 3.
 
Induction of donor-specific DH after orthotopic transplantation of epithelium-deprived C57BL/6 corneal allografts reconstituted with normal BALB/epithelium in high-risk BALB/c recipients (syngeneic epi + allogeneic st-end). Composite grafts of epithelium-deprived C57BL/6 corneas with BALB/c epithelium were orthotopically grafted into high-risk eyes of BALB/c recipients. Positive control (Pos. C) animals received SC injection of 10 × 106 C57BL/6 spleen cells 1 week before assay. At 2 weeks (A) or 8 weeks (B) after grafting, mice received an injection of x-irradiated C57BL/6 spleen cells (1 × 106) into the right ear pinna, and ear-swelling responses were assessed 24 and 48 hours later. Negative control (Neg. C) received right-ear-pinna challenge only. Mean 24-hour ear-swelling responses are compared with the negative control. *Responses significantly greater than the negative control (*P < 0.05, ***P < 0.005).
Figure 4.
 
Absence of active suppression of DH in recipients of epithelium-deprived orthotopic corneal allografts reconstituted in vitro with syngeneic epithelium (syngeneic epi + allogeneic st-end). BALB/c recipients of epithelium-deprived corneal allografts reconstituted in vitro with normal syngeneic epithelium were immunized at 8 weeks after grafting by an intradermal injection into the right ear pinna of 1 × 106 x-irradiated C57BL/6 spleen cells. One week later, these mice received into the left ear pinna an injection of 1 × 106 x-irradiated C57BL/6 spleen cells. Ear-swelling was assessed 24 and 48 hours later. Positive control BALB/c mice were immunized 1 week before the left ear challenge with a right ear pinna injection of 1 × 106 C57BL/6 spleen cells. Negative control mice received a left-ear-pinna challenge only. Mean 24-hour ear-swelling responses are compared with the positive control. *Responses significantly greater than control (*P < 0.05, ***P < 0.01).
Figure 4.
 
Absence of active suppression of DH in recipients of epithelium-deprived orthotopic corneal allografts reconstituted in vitro with syngeneic epithelium (syngeneic epi + allogeneic st-end). BALB/c recipients of epithelium-deprived corneal allografts reconstituted in vitro with normal syngeneic epithelium were immunized at 8 weeks after grafting by an intradermal injection into the right ear pinna of 1 × 106 x-irradiated C57BL/6 spleen cells. One week later, these mice received into the left ear pinna an injection of 1 × 106 x-irradiated C57BL/6 spleen cells. Ear-swelling was assessed 24 and 48 hours later. Positive control BALB/c mice were immunized 1 week before the left ear challenge with a right ear pinna injection of 1 × 106 C57BL/6 spleen cells. Negative control mice received a left-ear-pinna challenge only. Mean 24-hour ear-swelling responses are compared with the positive control. *Responses significantly greater than control (*P < 0.05, ***P < 0.01).
Figure 5.
 
Fate in high-risk eyes of established epithelium-deprived C57BL/6 corneal allografts reconstituted in vitro with BALB/c (syngeneic) epithelium after allosensitization by C57BL/6 spleen cells. Composite corneal allografts (BALB/c epithelium layered onto denuded C57BL/6 stroma-endothelium) were orthotopically placed in high-risk BALB/c eyes. As a positive control, full-thickness C57BL/6 corneas were placed in high-risk BALB/c eyes. (A) At 2 weeks after grafting, when all composite grafts had survived, mice received an injection of x-irradiated C57BL/6 spleen cells (1 × 106) into the right ear pinna for DH assay. (B) At 8 weeks after grafting, when all composite grafts had survived, recipients were sensitized with an SC injection of C57BL/6 spleen cells (10 × 106). The subsequent survival of the resident composite grafts was assessed clinically.
Figure 5.
 
Fate in high-risk eyes of established epithelium-deprived C57BL/6 corneal allografts reconstituted in vitro with BALB/c (syngeneic) epithelium after allosensitization by C57BL/6 spleen cells. Composite corneal allografts (BALB/c epithelium layered onto denuded C57BL/6 stroma-endothelium) were orthotopically placed in high-risk BALB/c eyes. As a positive control, full-thickness C57BL/6 corneas were placed in high-risk BALB/c eyes. (A) At 2 weeks after grafting, when all composite grafts had survived, mice received an injection of x-irradiated C57BL/6 spleen cells (1 × 106) into the right ear pinna for DH assay. (B) At 8 weeks after grafting, when all composite grafts had survived, recipients were sensitized with an SC injection of C57BL/6 spleen cells (10 × 106). The subsequent survival of the resident composite grafts was assessed clinically.
The authors thank Jacqueline M. Doherty and Jian Gu for their support. 
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Figure 1.
 
Fate in high-risk eyes of epithelium-deprived C57BL/6 corneal allografts reconstituted in vitro with BALB/c (syngeneic) epithelium. Three 11-0 nylon sutures were placed in the central cornea of eyes of BALB/c mice. After 2 weeks, these eyes were used as graft beds (high-risk). At that time, composite corneal allografts (BALB/c epithelium layered onto denuded C57BL/6 stroma-endothelium) were prepared in vitro and placed in the high-risk beds. As a positive control, full-thickness C57BL/6 corneas were placed in high-risk BALB/c eyes. The survival of the grafts was assessed clinically. (⋆) Survival pattern significantly better than that of full-thickness allografts (P < 0.0001).
Figure 1.
 
Fate in high-risk eyes of epithelium-deprived C57BL/6 corneal allografts reconstituted in vitro with BALB/c (syngeneic) epithelium. Three 11-0 nylon sutures were placed in the central cornea of eyes of BALB/c mice. After 2 weeks, these eyes were used as graft beds (high-risk). At that time, composite corneal allografts (BALB/c epithelium layered onto denuded C57BL/6 stroma-endothelium) were prepared in vitro and placed in the high-risk beds. As a positive control, full-thickness C57BL/6 corneas were placed in high-risk BALB/c eyes. The survival of the grafts was assessed clinically. (⋆) Survival pattern significantly better than that of full-thickness allografts (P < 0.0001).
Figure 2.
 
Histologic appearance of in vitro-generated composite corneal grafts. Epithelium derived from BALB/c corneas was layered in vitro onto the denuded surface of C57BL/6 stroma-endothelium corneas. These composite corneas were grafted orthotopically into high-risk eyes of BALB/c recipients. (A, B) Histologic appearance of composite grafts at 8 weeks; (C, D) full-thickness C57BL/6 corneal allografts. Magnification: (A, C) ×10; (B, D) ×40.
Figure 2.
 
Histologic appearance of in vitro-generated composite corneal grafts. Epithelium derived from BALB/c corneas was layered in vitro onto the denuded surface of C57BL/6 stroma-endothelium corneas. These composite corneas were grafted orthotopically into high-risk eyes of BALB/c recipients. (A, B) Histologic appearance of composite grafts at 8 weeks; (C, D) full-thickness C57BL/6 corneal allografts. Magnification: (A, C) ×10; (B, D) ×40.
Figure 3.
 
Induction of donor-specific DH after orthotopic transplantation of epithelium-deprived C57BL/6 corneal allografts reconstituted with normal BALB/epithelium in high-risk BALB/c recipients (syngeneic epi + allogeneic st-end). Composite grafts of epithelium-deprived C57BL/6 corneas with BALB/c epithelium were orthotopically grafted into high-risk eyes of BALB/c recipients. Positive control (Pos. C) animals received SC injection of 10 × 106 C57BL/6 spleen cells 1 week before assay. At 2 weeks (A) or 8 weeks (B) after grafting, mice received an injection of x-irradiated C57BL/6 spleen cells (1 × 106) into the right ear pinna, and ear-swelling responses were assessed 24 and 48 hours later. Negative control (Neg. C) received right-ear-pinna challenge only. Mean 24-hour ear-swelling responses are compared with the negative control. *Responses significantly greater than the negative control (*P < 0.05, ***P < 0.005).
Figure 3.
 
Induction of donor-specific DH after orthotopic transplantation of epithelium-deprived C57BL/6 corneal allografts reconstituted with normal BALB/epithelium in high-risk BALB/c recipients (syngeneic epi + allogeneic st-end). Composite grafts of epithelium-deprived C57BL/6 corneas with BALB/c epithelium were orthotopically grafted into high-risk eyes of BALB/c recipients. Positive control (Pos. C) animals received SC injection of 10 × 106 C57BL/6 spleen cells 1 week before assay. At 2 weeks (A) or 8 weeks (B) after grafting, mice received an injection of x-irradiated C57BL/6 spleen cells (1 × 106) into the right ear pinna, and ear-swelling responses were assessed 24 and 48 hours later. Negative control (Neg. C) received right-ear-pinna challenge only. Mean 24-hour ear-swelling responses are compared with the negative control. *Responses significantly greater than the negative control (*P < 0.05, ***P < 0.005).
Figure 4.
 
Absence of active suppression of DH in recipients of epithelium-deprived orthotopic corneal allografts reconstituted in vitro with syngeneic epithelium (syngeneic epi + allogeneic st-end). BALB/c recipients of epithelium-deprived corneal allografts reconstituted in vitro with normal syngeneic epithelium were immunized at 8 weeks after grafting by an intradermal injection into the right ear pinna of 1 × 106 x-irradiated C57BL/6 spleen cells. One week later, these mice received into the left ear pinna an injection of 1 × 106 x-irradiated C57BL/6 spleen cells. Ear-swelling was assessed 24 and 48 hours later. Positive control BALB/c mice were immunized 1 week before the left ear challenge with a right ear pinna injection of 1 × 106 C57BL/6 spleen cells. Negative control mice received a left-ear-pinna challenge only. Mean 24-hour ear-swelling responses are compared with the positive control. *Responses significantly greater than control (*P < 0.05, ***P < 0.01).
Figure 4.
 
Absence of active suppression of DH in recipients of epithelium-deprived orthotopic corneal allografts reconstituted in vitro with syngeneic epithelium (syngeneic epi + allogeneic st-end). BALB/c recipients of epithelium-deprived corneal allografts reconstituted in vitro with normal syngeneic epithelium were immunized at 8 weeks after grafting by an intradermal injection into the right ear pinna of 1 × 106 x-irradiated C57BL/6 spleen cells. One week later, these mice received into the left ear pinna an injection of 1 × 106 x-irradiated C57BL/6 spleen cells. Ear-swelling was assessed 24 and 48 hours later. Positive control BALB/c mice were immunized 1 week before the left ear challenge with a right ear pinna injection of 1 × 106 C57BL/6 spleen cells. Negative control mice received a left-ear-pinna challenge only. Mean 24-hour ear-swelling responses are compared with the positive control. *Responses significantly greater than control (*P < 0.05, ***P < 0.01).
Figure 5.
 
Fate in high-risk eyes of established epithelium-deprived C57BL/6 corneal allografts reconstituted in vitro with BALB/c (syngeneic) epithelium after allosensitization by C57BL/6 spleen cells. Composite corneal allografts (BALB/c epithelium layered onto denuded C57BL/6 stroma-endothelium) were orthotopically placed in high-risk BALB/c eyes. As a positive control, full-thickness C57BL/6 corneas were placed in high-risk BALB/c eyes. (A) At 2 weeks after grafting, when all composite grafts had survived, mice received an injection of x-irradiated C57BL/6 spleen cells (1 × 106) into the right ear pinna for DH assay. (B) At 8 weeks after grafting, when all composite grafts had survived, recipients were sensitized with an SC injection of C57BL/6 spleen cells (10 × 106). The subsequent survival of the resident composite grafts was assessed clinically.
Figure 5.
 
Fate in high-risk eyes of established epithelium-deprived C57BL/6 corneal allografts reconstituted in vitro with BALB/c (syngeneic) epithelium after allosensitization by C57BL/6 spleen cells. Composite corneal allografts (BALB/c epithelium layered onto denuded C57BL/6 stroma-endothelium) were orthotopically placed in high-risk BALB/c eyes. As a positive control, full-thickness C57BL/6 corneas were placed in high-risk BALB/c eyes. (A) At 2 weeks after grafting, when all composite grafts had survived, mice received an injection of x-irradiated C57BL/6 spleen cells (1 × 106) into the right ear pinna for DH assay. (B) At 8 weeks after grafting, when all composite grafts had survived, recipients were sensitized with an SC injection of C57BL/6 spleen cells (10 × 106). The subsequent survival of the resident composite grafts was assessed clinically.
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