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Characterization of Cell-Mediated Immune Responses Elicited by Orthotopic Corneal Allografts in Mice Yasushi Sonoda,* Yoichiro Sano,f Bruce Ksander,-\ andj. Wayne Streilein\ Purpose. Corneal allografts placed orthotopically induce a unique and unusual response in recipient mice. More orthotopic corneal allografts are accepted indefinitely than similar skin allografts. Of the rejected corneal grafts, class I major histocompatibility complex (MHC)incompatible grafts are rejected less frequently than grafts that express only minor histocompatibility complex (minor H) or MHC plus minor H alloantigens. To describe the spectrum of T cells activated (or not) by orthotopic corneal grafts, the authors examined the development of delayed hypersensitivity (DH) to donor-specific alloantigens. Methods. Recipient BALB/c mice received orthotopic corneal allografts from donor mice that were MHC incompatible at MHC loci only, multiple minor H loci only, or MHC plus multiple minor H loci. These groups of mice were examined to determine when alloantigen-specific DH developed. Results. The authors report that all mice, whether they accept or reject grafts, acquire donorspecific DH within 4 weeks of engraftment. This reactivity is primarily directed at minor H, rather than MHC-encoded, alloantigens. Through time, spontaneous DH reactivity disappears in all mice, and thereafter, donor-specific DH can be induced by cognate immunization only in mice that have rejected their cornea grafts.7 Conclusions. These results can be explained in the context of "direct" and "indirect" pathways of allorecognition. Because normal corneas lack passenger leukocytes, the potential for direct recognition of alloantigens on orthotopic corneal grafts is small. Therefore, T cells activated by orthotopic corneal allografts must recognize donor-derived antigens primarily on recipient antigen presenting cells, that is, through the indirect pathway of allorecognition. Because minor H antigens are the dominant cellular proteins in grafts, it is proposed that minor H determinants are the most immunogenic alloantigens in orthotopic corneal grafts because they are the major source of peptides that will be loaded onto recipient class II molecules for T-cell recognition. We further predict that long-term acceptance of corneal allografts is promoted when recipient mice acquire anterior chamber associated immune deviation (impaired and suppressed DH) directed at minor H alloantigens of the grafts. Invest Ophthalmol VisSci. 1995; 36:427-434. Although keratoplasty for the treatment of visual impairment secondary to corneal disease is often a suecessful clinical procedure, a significant number of operations fail. Frequently, immune rejection of the corneal graft is held to be responsible. For this reason, it is important to increase our knowledge and underFrom the the 'Department of Ophtludmoloin, Tokyo Medical College, Tokyo, japan, standing of the mechanisms by which the immune system recognizes and responds to histoincompatible corneas grafted orthotopically. Until recently, experimental analysis of immune responses to corneal grafts has been conducted in rabbits and other large laboratory animals. Maumanee was the first to point out that orthotopic corneal allografts performed in rabbits enjoyed prolonged survival, compared to solid tissue r » i j i_ u • • - I I J I S ed at Other U ^Medical S School, A l Boston, i ^ Massachusetts. L ^ D i l J g r a f tBased P l a Con inbeen the body.' Harvard studies ofOtthotOpiC this type, Sites it has suggested Supported by United Stales Public Hmlth Service grant EY05678. • a t l m m U n e • •• • • . c . ,, tU tth SuZittedforpublication March 16, 1994; r^sedSeptember 9, 1994; accepted privilege IS an important factor in the 1994. relative success experienced by orthotopic corneal al^ ^ r j . m^Streilein, Sevens Eye Search fnstitute, 20 Staniford 1 O S r a f t S i n m a n - Approximately 10 years ago, a method Street, Boston, MA 02114. was devised for accomplishing orthotopic corneal Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 Copyright © Association for Research in Vision and Ophlhalmology Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/18/2017 427 428 Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 2 grafts in rats, and within the past few years, this method has been adapted for use in laboratory mice.3 Using this model system, we have recently reported that immune privilege is extended to allogeneic corneas placed orthotopically in eyes of normal mice.4 Irrespective of the degree of immunogenetic disparity between donors and recipients of histoincompatible cornea grafts, a significant proportion of grafts was accepted by recipient mice, often for indefinite time periods. In these studies, the fate of grafts expressing a diversity of alloantigens was examined, including multiple minor transplantation antigens, class I and class II antigens encoded by the murine major histocompatibility complex (MHC, H-2), and combinations thereof. Surprisingly, the results indicated that the immunogenetic rules that govern patterns of rejection for other solid tissue grafts do not appear to apply to orthotopic corneal allografts. For example, corneal grafts that displayed class I MHC alloantigens were rejected infrequently, whereas grafts that displayed minor transplantation antigens were rejected more often. In other solid tissue allografts, immunogenetic rules have been deduced5 that state that MHC-encoded antigens are more immunogenic than minor H antigens and that, therefore, MHC-disparate grafts are rejected more acutely than are grafts that only confront the recipient with minor H antigens. Although there are several well-documented exceptions to this rule,6 in general these rules apply to most solid tissue allografts. Based on our study of orthotopic corneal allografts in mice, we have proposed that the immunogenetic rules that govern the fate of orthotopic corneal allografts are inverted compared to other tissue grafts and that minor H antigens from the cornea are more immunogenic than antigens encoded within the MHC. In a recent study,7 we determined that indefinite acceptance of orthotopic corneal allografts in mice correlates positively with the development of anterior chamber associated immune deviation (ACAID). That is to say, mice that have accepted histoincompatible corneal allografts in excess of 2 months are unable to acquire delayed hypersensitivity (DH) to antigens expressed on the grafts, even when attempts are made to immunize the mice to donor antigens by nonocular routes. By contrast, mice that have rejected their corneal grafts readily develop DH when cells genetically identical to the graft are injected subcutaneously. The conclusion that mice with accepted corneal grafts have ACAID was bolstered by our demonstration that the spleens of these mice contain antigen-specific suppressor lymphocytes that inhibit the expression of DH in adoptive transfer recipients. The studies that form the basis of this report were initiated to gain insight into the early phases of host immune responses to alloantigens on orthotopic cor- neal graft. We wanted to determine whether and when alloantigen-specific, cell-mediated immunity developed in mice grafted with histoincompatible corneas and to discern whether delayed hypersensitivity might be implicated in early graft failure and rejection. MATERIALS AND METHODS Mice Six- to 12-week-old mice were obtained from the University of Miami breeding colony. All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The following inbred strains were used: BALB/c, C57BL/ 6, B10.D2, BALB.K. Method of Orthotopic Corneal Allografts As described elsewhere,3'4 donor corneas were excised by trephination by using a 2-mm bore and were placed in chilled phosphate-buffered saline before grafting. Recipients were deeply anesthetized with 0.66 mg ketamine hydrochloride (Vetalar; Parke-Davis; Detroit, MI) administered intramuscularly. The graft bed was prepared by trephining a 2-mm site in the central cornea of the right eye and discarding the excised cornea. The donor cornea was placed in the recipient bed and secured with 12 interrupted sutures (11-0 nylon, 50-//m diameter needle, Sharpoint; Vanguard, Houston, TX). Grafted eyes were examined after 72 hours; at that time, grafts with technical difficulties (hyphema, infection, loss of anterior chamber) were excluded from further consideration. At 9 days, the sutures were removed. Evaluation and Scoring of Orthotopic Cornea Transplants Grafts were examined by slit lamp microscopy at weekly intervals, as described elsewhere.4 A scoring system was devised to describe semiquantitatively the extent of opacity (0 to 5+), as follows: 0 = clear graft; 1+ = minimal superficial (nonstromal) opacity; 2+ = minimum deep stromal opacity; 3+ = moderate deep stromal opacity; 4+ = intense deep stromal opacity; 5+ = maximum stromal opacity. Grafts with opacity scores of 2+ or greater at 8 weeks were considered to have been rejected; grafts with scores of 4+ or greater at 2 weeks never cleared and were also regarded as rejected. Assay for Delayed Hypersensitivity Reaction in Mice Bearing Corneal Grafts At the appropriate time after grafting, 1 X 106 irradiated (2,000 rads) spleen cells from donors syngeneic with the corneal graft were injected in 10 y\ of Hanks' balanced salt solution into the right pinnae. The left Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/18/2017 429 T-Cell Immunity to Orthotopic Corneal Allografts ear pinnae was inoculated with an equal volume of Hanks' balanced salt solution only. As a positive control, a similar number of spleen cells was injected into the ear pinnae of mice immunized by subcutaneous injection of 10 X 106 spleen cells of the appropriate allogeneic strain. After 24 hours, ear thickness was measured with a low-pressure engineer micrometer (Mitutoyo, MTI, Paramus, NJ). Ear swelling was expressed as follows: specific ear swelling = (24 hours measurement of right ear - 0 hours measurement of Syngenic MHC + Minor MHC Only Minor Only right ear) — (24 hours measurement of left ear — 0 3 Corneal Graft Disparity hours measurement ofleft ear) X 10~ mm. Ear swellFIGURE l. Fate of orthotopic corneal allografts in BALB/c ing responses at 24 hours after injection are presented mice. Each data point indicates the clinical score of a graft as individual values (10~3 mm) for each tested animal, at 8 weeks after engraftment. The clinical basis for assigning and as group mean ± SEM. The DH data were obscores of i + to 4+ is described in Materials and Methods. tained from groups of mice ear challenged at the postBALB/c mice received allografts from either MHC plus migrafting time designated in the Results section. Earnor (C57BL/6), MHC only (BALB.K), or minor only challenged mice in which DH responses were deter(B10.D2) disparate mouse strains. Accepted grafts are identimined were sacrificed; no mice were rechallenged. fied by the symbol (•), and rejected grafts are shown as (+). Statistical Analyses Ear swelling measurements were evaluated statistically by using a two-tailed Student's West. All P values < 0.05 were deemed significant. Corneal graft rejection was evaluated using a two-tailed Fisher's exact test. All P values of <0.05 were deemed significant. RESULTS Normal adult BALB/c mice received orthotopic grafts of corneas obtained from donors representing different degrees of immunogenetic disparity: C57BL/6 grafts confront BALB/c recipients with MHC plus multiple minor H incompatible alloantigens; B10.D2 grafts present only minor H alloantigens to BALB/c; BALB.K grafts display only MHC-encoded alloantigens to BALB/c. A large series of orthotopic grafts was performed, and Figure 1 displays a summary of the fate of these grafts 8 weeks after grafting, as judged by clinical examination with a slit lamp. As reported previously, graft failure (defined as corneal opacity >2+) can be determined as early as 4 weeks after grafting. Irreversible failure was diagnosed when an orthotopic allograft displayed 2+ or greater opacity for 2 or more weeks and was opaque at 8 weeks after engraftment. BALB/c mice failed to reject syngeneic corneal grafts (n = 15, 0% rejected). BALB/c mice that received MHC plus minor H disparate (n = 18, 44% rejected) or minor H only disparate grafts (n = 17, 41% rejected) rejected these grafts at a frequency significantly greater than syngeneic controls (P = 0.018 and 0.031, respectively). By contrast, BALB/c mice that received MHC only disparate corneal grafts rejected these grafts at a reduced frequency (n = 15, 20% rejected) that was not significantly different from syngeneic control grafts (P= 0.233). The following series of experiments examined panels of orthotopic corneal graft recipients subjected to various experimental protocols to determine whether and when antigenspecific DH developed. Onset of Delayed Hypersensitivity in Mice Bearing Orthotopic Corneal Allografts BALB/c mice bearing corneal allografts from C57BL/ 6 donors were first tested for DH at 2 weeks after grafting. At this time, approximately 30% of grafts displayed opacity of 2+ of• greater, whereas the remainder displayed little evidence of inflammation.4 It was too early to predict at 2 weeks which grafts would eventually succumb to rejection; we previously found that some grafts with 2+ opacity scores at 2 weeks cleared within the next few weeks, whereas grafts clear at 2 weeks became opaque during the next few weeks and remained so indefinitely (considered to be rejected). DH reactions of grafted mice were compared with responses of the positive control group, specifically with sensitized normal BALB/c mice that received subcutaneous injections of C57BL/6 spleen cells (10 X 106) and ear challenged with C57BL/6 alloantigens 2 weeks later. As displayed in Figure 2 and Table 1, the positive control group demonstrated significant DH (specific ear swelling of 57 X 10~3 mm ± 9). When the ears of cornea-grafted mice were challenged with irradiated C57BL/6 spleen cells at 2 weeks after grafting, none of the ears developed significant swelling (11 X 10~3 mm ± 3) compared with naive mice (35 X 10~3 mm ± 6). Thus, if orthotopic corneal allografts are capable of inducing delayed hypersensitivity, these results indicate that at 2 weeks after grafting, the sensitization process had not yet reached levels that could be detected by this assay. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/18/2017 Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 430 standing (>8 weeks) healthy orthotopic corneal allografts display ACAID—that is, they are specifically unable to mount donor-specific DH responses when primed against donor alloantigens. 80 _, 60 . Time Course and Eventual Outcome of Delayed Hypersensitivity Reactivity in Mice Bearing Orthotopic Corneal Allografts o> 40 . Naive Primed Grafted FIGURE 2. Donor-specific delayed hypersensitivity in BALB/ c mice bearing orthotopic C57BL/6 corneal allografts for 2 weeks. Ears of grafted and primed, and naive mice received injections of 1 X 106 irradiated (2000R) C57BL/6 spleen cells. Ear swelling responses were measured by micrometer after 24 hours. Each data point represents the response of a single animal. The bar indicates mean ear swelling response for the group. Delayed hypersensitivity response of grafted mice was not significantly different from naive control mice. The next time at which DH was assessed in grafted mice was 4 weeks after keratoplasty. Based on our previous experience with murine orthotopic corneal allografts, we anticipated that approximately 25% of C57BL/6 grafts on BALB/c recipients at this time would be opaque (score of >2+). Almost invariably, these grafts can be expected to remain opaque and never resolve, that is, they are irreversibly rejected. The remaining grafts (75%) at 4 weeks after grafting express little or no evidence of opacity or edema, even though a few of these grafts may undergo failure and rejection during the subsequent weeks. Two groups of mice were assayed for DH at 4 weeks after grafting: one panel consisted of mice with clear corneas (termed "acceptors"), and the other panel consisted mice with opaque corneas (scores of , termed "rejectors"). The ear swelling responses to intrapinnae challenge with irradiated C57BL/6 cells are presented in Figure 3 and are summarized in Table 1. Mice that we categorized as rejectors at 4 weeks displayed intense C57BL/6-specific DH (88 X 10~3 mm ± 8) compared with naive mice (25 X 10~3 mm ± 6). Thus, mice that rejected their corneal grafts became immunized systemically by their orthotopic corneal grafts. Surprisingly, DH responses were also detected in the challenged ears of mice that we categorized as acceptors (74 X 10~3 mm ± 14). It would appear, therefore, that recipients of orthotopic corneal allografts become systemically sensitized to alloantigens on the grafts, irrespective of whether the grafts are rejected. This is of considerable interest because we previously reported that acceptor mice bearing long- Additional panels of grafted BALB/c mice were ear challenged for C57BL/6-specific DH at 8 weeks and 20 weeks after keratoplasty. Mice with rejected corneas were tested, as were mice with accepted corneas. The results are presented in Figures 4 and 5 and are included in summary Table 1. The findings indicate that DH reactivity to donor alloantigens was lost in mice that accept their corneal grafts at 8 and 20 weeks after grafting. By contrast, mice with rejected corneal grafts displayed an intermediate DH response at 8 weeks, in which some individual mice still displayed significant DH activity whereas others lost the ability to generate graft-specific DH (Fig. 4). Graft-specific DH reactivity waned over the next 3 months. At 5 months after keratoplasty, all but a single mouse failed to display DH, whether the graft was judged to be healthy or was rejected at the time. Thus, the DH reactivity evoked by orthotopic allogeneic corneal grafts proves to be short-lived and correlates poorly with clinical assessment of graft outcome. Specificity of Donor-Directed Delayed Hypersensitivity Displayed by Mice Bearing Orthotopic Corneal Allografts C57BL/6 corneas confront BALB/c recipients with both MHC and minor H alloantigens. To determine TABLE i. Time Course of Donor-Specific Delayed Hypersensitivity After Orthotopic Corneal Transplantation Delayed Hypersensitiwty Responses Time After Grafting Acceptors Rejectors Positive Controls* 2 weeks 4 weeks 8 weeks 20 weeks —f ++ — — — ++ —/ +t — ++ ++ "*""*" ++ C57BL/6 corneas were grafted orthotopically to BALB/c mice; recipients were ear challenged with C57BL/6 (10 X 10°) spleen cells at appropriate times afte grafting. Ear swelling responses (DH) are listed as positive or legative, based on data presented in Figures 2, 3, 4, and 5. * DH response of mice immui ized for the corresponding length of time by subcutaneous inoo lation of allogeneic spleen cells. t At 2 weeks after grafting, it >vas not possible to distinguish "acceptor" from "rejector" mice. X At this time, some mice were positive for DH, whereas others were negative. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/18/2017 431 T-Cell Immunity to Orthotopic Corneal Allografts 140 -, * 120- 100 - CO elling (x1 b CO 40 20 - o E E 0 -r, —• T* • • 60 - t —•— t Accepted Ilin 60 - <u CO CO LJJ • • # & 40 * — 20 - • • t 0. Naive • • 40• • Primed Accepted Primed Rejected O 80 - • 60- 0- Primed 120 - 5, • 80- 20- Naive 100 - —r~ * 100- • 80 - CO UJ • 140- • 120 - Rejected • • t• • Accepted Rejected FIGURES 3, 4, and 5. Donor-specific delayed hypersensitivity in BALB/c mice bearing orthotopic C57BL/6 corneal allografts for 4 (Fig. 3, above left), 8 (Fig. 4, above right), and 20 weeks (Fig. 5, left). Ear challenge and measurements were performed as described in the legend to Figure 2. "Accept" refers to mice with clear corneal grafts; "reject" refers to mice with corneal opacity of a: +2. Primed mice (positive control group) were immunized by subcutaneous inoculation of allogeneic spleen cells for 4, 8, and 20 weeks, respectively. *Mean delayed hypersensitivity is significantly different from naive mice at P < 0.05. 20 weeks the relative contributions these different types of alloantigens make to the acquisition of DH in grafted mice, the following experiments were performed. First, a panel of BALB/c mice received orthotopic cornea grafts from BALB.K donors; the latter confront the recipients only with MHC alloantigens. The ears of these mice were challenged with BALB.K spleen cells 4 to 10 weeks later, and the DH response was measured (Fig. 6). The positive control group consisted of an equal number of mice primed to the alloantigens for the corresponding length of time (4 to 10 weeks). The results indicate that mice that accept MHC-bnly disparate corneal allografts fail to generate MHC-specific DH (32 X 10~3 mm ± 3) compared with the positive control group (80 X 10~3 mm ± 11). However, among mice that rejected corneal grafts, although 2 of 6 displayed MHC-specific DH, the mean response (50 X 10~3 mm ± 1 2 ) was not significantly different from naive mice (24 X 10~3 mm ± 3). Second, a panel of BALB/c mice received corneal allografts from C57BL/6 donors. Four weeks after grafting, the ears of these mice were challenged with B10.D2 spleen cells, which only share minor H antigens with C57BL/6. The DH responses of these mice are presented in Figure 7. The results indicate that all grafted mice (rejector and acceptor) mount minor H antigen-specific DH responses. The magnitude of the responses by rejector (103 X 10 3 mm ± 12) and acceptor (75 X 10~3 mm ± 9) mice were equivalent in intensity to the response displayed by positive control mice (67 X 10"3 mm ± 8). Third, BALB/c mice received orthotopic grafts from B10.D2 mice, a circumstance in which the graft presents only minor H anti120 -, 100 80 •=- 6 0 - f 40 20 Naive Primed Accepted Rejected FIGURE 6. Donor-specific delayed hypersensitivity in BALB/ c mice bearing orthotopic BALB.K corneal allografts (MHC only; disparate allografts). Ear challenges and measurements were performed as described in the legend to Figure 2. *Mean delayed hypersensitivity is significantly different from naive mice a t P < 0.05. MHC = major histocompatibility complex. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/18/2017 Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 432 140 -, 1 •£• 80 - 100 60 - 80 - ing 3 O X 100 -, 120 - 60 - 5 n 40 - ra 20 - 20 - JJ -I- 40 - 0 Naive Primed Accepted Naive Rejected Primed Accepted Rejected FIGURES 7 and 8. Donor-specific delayed hypersensitivity in BALB/c mice bearing orthotopic corneal allografts (minor H only disparate allografts). Recipients of C57BL/6 grafts (Fig. 7, left) or B10.D2 grafts (Fig. 8, right) were ear challenged with irradiated B10.D2 spleen cells (1 X 106), and their ears were measured. Results are presented as described in the legend to Figure 2. *Mean DH is significantly different from naive mice at P < 0.05. gens to the recipient. When the ears of these mice were challenged with B10.D2 spleen cells 4 weeks later (Fig. 8), positive DH response were detected in mice that had accepted their minor H-disparate grafts (64 X 10"3 mm ± 9) and in mice that had rejected their grafts (60 X 10~3 mm ± 14). These responses were significantly greater than negative control mice (24 X 1O~3 mm ± 8) and were equivalent to the positive control group (49 X 10~3 mm ± 5). These results are summarized in Table 2. In the aggregate, these findings imply that minor H antigens, rather than MHC-encoded antigens, are more successful inducers of DH in mice bearing orthotopic corneal allografts. DISCUSSION Unlike orthotopic, MHC-disparate skin allografts, orthotopic corneal allografts are riot uniformly rejected in mice. In the former, substantial experimental evidence indicates that full-thickness skin allografts activate a diverse spectrum of immune T-cell effector modalities. As a consequence, recipients of these grafts acquire donor-specific T cells that mediate delayed hypersensitivity, T cells that are primed to lyse approTABLE priate target cells, and memory T cells. The results presented in this article, along with previously reported data,7 indicate that a distinct, incomplete, and temporally limited spectrum of T-cell effectors is activated by orthotopic corneal allografts. T cells that me^ diate delayed hypersensitivity and regulatory T cells that suppress expression of delayed hypersensitivity are detected. Donor-specific DH was detectable in corneal allograft recipients as early as 4, but not 2, weeks after engraftment. This is. of interest because mice that are grafted orthotopically with allogeneic skin display donor-specific DH within 7 days (ref. 7 and unpublished observations, 1993). It is thought that the primary mode of sensitization after skin grafting, and an important reason for the rapidity with which sensitization is evoked, is the release of so-called "passenger leukocytes" from the skin at the time of grafting.8"10 The passenger cells of skin include Langerhans cells within the epidermis and dendritic cells and macrophages in the dermis. The superior sensitizing ability of "passenger cells" within skin grafts has been ascribed, on the one hand, to their ability to migrate from the graft and travel to draining regional lymph nodes10 and, on 2. Specificity of Delayed Hypersensitivity Induced by Orthotopic Corneal Allografts Delayed Hypersensitivity* Graft Donor Recipient Ear Challenge Type of Alloantigen C57BL/6 C57BL/6 B10.D2 BALB.K BALB/c BALB/c BALB/c BALB/c C57BL/6 B10.D2 B10.D2 BALB.K MHC + Minor H Minor H only Minor H only MHC only Acceptors Rejectors Positive^ Controls * Ear challenge conducted at 4 weeks after grafting. t DH response of mice immunized by subcutaneous inoculation of allogeneic spleen cells of the appropriate alloantigen. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/18/2017 T-Cell Immunity to Orthotopic Corneal Allografts the other hand, to the powerful array of accessory signals with which dendritic cells activate T cells.10 This pathway of allorecognition has been called "direct" because recipient T cells recognize donor alloantigens directly on donor-derived cells.11 Comparing skin to cornea as a graft, the central region of the normal cornea possesses virtually no passenger cells, neither Langerhans cells within the corneal epithelium nor dendritic cells or macrophages in the stroma.'2 We suspect, but have no direct evidence, that the delay in onset of donor-specific DH in corneagrafted mice is due to the lack of passenger cells (i.e., Langerhans cells) in the donor cornea. In support of this suspicion, Peeler and Niederkorn have demonstrated that the capacity of heterotopic cornea grafts to induce DH in mice is related to the graft's content of Langerhans cells.13 When normal corneas, which lack Langerhans cells, are grafted to the thoracic wall they fail to elicit graft-specific DH, whereas cornea grafts, forced by experimental artifice to contain Langerhans cells, evoke vigorous DH when grafted to the thoracic wall of allogeneic recipients. Despite the fact that the normal corneas we used for grafting were deficient in Langerhans cells, recipients of these allografts eventually acquired donor-specific DH. Sensitization in these mice indicates that allorecognition of corneal antigens can occur by the "indirect" pathway, that is, recipient T cells recognize donor alloantigens after they have been processed and presented as peptides on host MHC molecules.1415 We have examined orthotopic C57BL/6 corneal grafts 21 to 28 days after engraftment into BALB/c eyes and have found numerous Iad+ mononuclear and dendritic cells within the central epithelium and stroma (unpublished observations, 1994). This suggests that recipient antigen-presenting cells have invaded the graft, which raises the possibility that these cells can provide the cellular basis for indirect pathway recognition of minor H and MHC alloantigens on the graft.1617 Based on their considerations, it is tempting to speculate that the immune system can only recognize alloantigens from orthotopic corneal grafts through the indirect allorecognition pathway. If true, it would be worthwhile to devise experimental strategies that can preferentially inhibit this pathway, rather than the direct pathway. We were surprised to learn that all cornea-grafted mice displayed donor-specific DH at 4 weeks and that this reactivity subsequently disappeared spontaneously whether the cornea grafts were accepted or rejected. Moreover, it was of interest to learn that the evanescent DH reactivity displayed at this time was directed primarily at minor H, rather than MHC-encoded, alloantigens. It is generally thought minor H antigens represent polymorphic cellular proteins that must be processed into peptides that are then loaded onto 433 class II molecules before they can be recognized by T cells.1819 Thus, by definition, minor H antigens are recognized through the indirect pathway. Because there may be more minor H-type proteins in cells than there are MHC-encoded antigens, minor H antigens may be the major source of peptides recognized through the indirect pathway. Perhaps this is why the immunogenetic rules of transplantation are inverted for orthotopic corneal allografts.4 According to this construction, T cells have little opportunity to recognize corneal alloantigens by direct pathways because the grafts lack passenger leukocytes. Therefore, we predict that when recipient antigen-presenting cells infiltrate the graft, minor H antigens represent the major source of donor-derived proteins processed and presented through the indirect pathway to T cells. If this prediction is correct, it is possible that minor H antigens may act as immunodominant alloantigens in orthotopic corneal allograft rejection. Experiments will be conducted to test this prediction directly. There may be a clinical counterpart to the proposal that corneal alloantigens are recognized by T cells through an indirect antigen presentation pathway, and it is related to the controversy in ophthalmology concerning the role of HLA antigens in corneal allograft failure. The evidence that HLA matching improves clinical corneal transplant survival is incomplete and contradictory.20"23 To resolve this controversy, a multi-center Collaborative Corneal Transplantation Study was recently conducted in the United States.24 One conclusion reached from this study is that HLA matching does not improve the survival rate of keratoplasties, even in so-called high-risk situations. Surprisingly, another conclusion reached by this study is that matching for the human ABO isohemagglutinin locus, /, does improve corneal graft survival. Although the ABO antigens are carbohydrates and can be detected by antibodies, the antigens are synthesized intracellularly by glycosyl transferases that are polymorphic. As such, these allotypic proteins can provide peptides that could be loaded onto MHC molecules and recognized by T cells. We speculate that corneal rejection may occur in humans because T cells recognize MHC-associated peptides derived from polymorphic glycosyl transferases encoded at locus /. In other words, an indirect pathway of allorecognition may be the basis of rejection of ABO-incompatible corneal allografts. We previously reported that mice with longstanding, accepted orthotopic corneal allografts have donor-specific ACAID; they cannot be induced to mount DH reactions when primed against donor alloantigens.7 By contrast, sensitization leading to donor-specific DH can be readily induced in mice bearing irreversibly rejected orthotopic corneal allografts. We report here that all cornea-grafted mice acquire donor- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933410/ on 06/18/2017 434 Investigative Ophthalmology & Visual Science, February 1995, Vol. 36, No. 2 specific DH and that this response disappears in mice that accept or reject their grafts. ACAID, the stereotypic, deviant immune response acquired by acceptor mice, has been closely linked to the mechanisms responsible for immune privilege in the eye.25'26 This suggests that ACAID induction is critical to the success of orthotopic corneal allografts. The observation that ACAID is detected after donor-specific DH has been induced in cornea recipients reveals that ACAID can be imposed on previously sensitized animals. Experiments that confirm this prediction were recently obtained.27 Key Words corneal transplantation, immune privilege, immunoregulation, transplantation, ACAID Acknowledgments The authors thank Debbie Bradley and Marie Ortega for expert technical assistance and for providing mice from the mouse breeding facility. References 1. Maumanee AE. The influence of donor-recipient sensitization on corneal grafts. Am J Ophthalmol. 1951; 34:142-152. 2. Williams KA, Coster DJ. Penetrating corneal transplantation in the inbred rat: A new model. Invest Ophthalmol Vis Set. 1985;26:23-30. 3. She SH, Steahly LP, Moticka EJ. A mediod for performing full-thickness, orthotopic, penetrating keratoplasty in the mouse. Ophthalmic Surg. 1990; 21:78107817. 4. Sonoda Y, Streilein JW. Orthotopic corneal transplantation in mice: Evidence that the immunogenetic rules of rejection do not apply. Transplantation. 1992;54:694-703. 5. Klein J. Natural History of the Major Histocompatibility Complex. 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