Cytotoxic T Cells Play No Essential Role in Acute Rejection of Orthotopic Corneal Allografts in Mice

Abstract

purpose. To determine whether cytotoxic T cells of the direct alloreactive type are activated and responsible for early, acute failure of orthotopic corneal allografts observed in eyes of C57BL/6 but not of BALB/c mice.

methods. Corneas from BALB/c and BALB.B mice were placed orthotopically in eyes of C57BL/6 and β-2 microglobulin knockout mice (deficient in CD8⁺ cytotoxic T cells). Graft fates were assessed clinically, and the T lymphocytes of recipients were assayed for the capacity to lyse target cells bearing donor major (MHC) and/or minor histocompatibility (minor H) antigens (direct and indirect pathways, respectively).

results. Similar to BALB/c recipients, C57BL/6 mice with rejected cornea allografts acquired donor minor H–specific T cells. Unlike BALB/c recipients, C57BL/6 mice—both rejectors and acceptors—acquired donor MHC-specific T cells. β-2 Microglobulin knockout mice showed rejection of corneal allografts in a manner indistinguishable from C57BL/6 mice, including early, acute rejection, yet T cells from β-2 microglobulin knockout recipients of corneal allografts displayed no cytotoxic T cells specific for either donor MHC or minor H alloantigens.

conclusions. Although C57BL/6 mice acquired donor MHC-specific cytotoxic T cells (direct alloreactive cells), neither these cells nor donor minor H–specific cytotoxic T cells (indirect alloreactive cells) play any essential role in corneal allograft rejection, including the early acute failure uniquely observed in C57BL/6 eyes.

Unlike other types of solid tissue grafts, allografts of cornea placed orthotopically in the eyes of experimental animals often experience extended, sometimes even indefinite, survival. ^{1 2} Similarly, allografts placed in uncomplicated human eyes have a very high rate of success, even though only modest topical immunosuppression is typically used. ^{3 4 5} In many instances in which the cornea graft confronts the host with numerous strong transplantation antigens, the earliest signs of rejection may not appear until 3 to 5 weeks after engraftment, whereas other types of solid tissue allografts usually undergo acute rejection within 1 to 3 weeks. Based on these types of observations, orthotopic corneal allografts placed in eyes of experimental animals and humans are believed to experience immune privilege. ^{5 6 7} Despite the advantage displayed by orthotopic corneal allografts compared with other types of grafts, rejection of orthotopic corneal allografts in humans still represents a significant clinical problem. More corneal transplantations are performed per year (>50,000) in the United States and Eastern Europe than any other type of transplantation, and a success rate of 90% ⁴ means that more than 5,000 grafts fail per year. In addition, cornea allografts placed in so-called high-risk eyes experience an unacceptably high rate of rejection. ^{8 9} In experimental animals, the tempo of rejection in high-risk eyes is very fast (measured in days), ¹⁰ rivaling that of other solid tissue allografts. Therefore, understanding the pathogenesis of corneal allograft rejection has a high research and clinical priority. 

In the recent past, using a model of orthotopic corneal transplantation in mice, we have analyzed the fate of grafts in eyes of BALB/c and C57BL/6 mice. ^{11 12} In certain respects, the fate of the grafts and the immune responses of both types of recipients were similar. Thus, a significant number of allografts survived indefinitely in each, and the incidence of acceptance of major histocompatibility complex (MHC)–only disparate grafts was significantly higher than the incidence of acceptance of minor histocompatibility (minor-H)–only disparate grafts. All recipients of both strains acquired donor-specific delayed hypersensitivity (DH) within 4 weeks of engraftment, irrespective of whether the grafts were eventually rejected. Of particular interest is the finding that the systemic immunity (DH) displayed by graft recipients of both strains was directed at minor H–encoded, rather than MHC-encoded alloantigens. Moreover, all animals in which grafts were accepted for longer than 8 weeks (defined as indefinite survival) acquired donor-specific anterior chamber–associated immune deviation. ^{2 11}  

Despite these similarities, the fates of grafts in eyes of BALB/c and C57BL/6 mice were not identical. First, the incidence of rejection was considerably higher in C57BL/6 than in BALB/c mice. Second, a large proportion of grafts in eyes of C57BL/6 recipients displayed early acute rejection (within 2–3 weeks), whereas rejections of cornea grafts during this early period after grafting occurred very infrequently in BALB/c eyes. Third, in BALB/c rejector mice the ability to display donor-specific DH was first gained, then lost, whereas once the cornea grafts were rejected in C57BL/6 mice, a high level of donor-specific DH was retained indefinitely. ¹¹  

Recently, we analyzed the donor-specific cytotoxic T-cell responses of BALB/c mice to orthotopic corneal allografts. These experiments revealed that mice with rejected grafts (but not mice with accepted grafts) acquired primed minor-H–specific T cells. However, none of the mice displayed cytotoxicity directed at target cells bearing donor MHC alloantigens. ¹³ Thus, in BALB/c mice, direct alloreactive T cells (either CD4⁺ or CD8⁺) specific for donor MHC alloantigens are not activated. Instead, donor minor-H–specific DH and T cells are readily detectable, especially in mice in which grafts are rejected. Combined with evidence that sensitization of cornea allograft recipients to donor alloantigens correlates temporally with infiltration of recipient Langerhans’ cells into the graft, ¹⁴ we have deduced that only the so-called indirect pathway of allorecognition is activated by orthotopic corneal allografts. 

Because rejection of orthotopic corneal allografts in C57BL/6 recipients is more rapid and of higher frequency than in BALB/c recipients, we hypothesized that early acute rejection in C57BL/6 mice is caused by the acquisition of direct (MHC-specific) alloreactive T cells. To test this hypothesis, we examined whether primed direct and/or indirect alloreactive T cells are generated in C57BL/6 recipients of allogeneic cornea grafts. Our results indicate that C57BL/6 recipients resemble BALB/c recipients, in that rejectors acquired donor minor H–specific T cells. Unlike BALB/c mice, C57BL/6 recipients of BALB/c cornea grafts also acquired direct, donor MHC-specific T cells. However, this was equally true in mice in which grafts were or were not rejected. Finally, corneal allografts placed in eyes of β2-microglobulin–deficient mice (that are deficient in CD8⁺ T cells) displayed the same high incidence and rate of rejection, including early acute graft failure, that is found when wild-type C57BL/6 mice receive orthotopic corneal allografts. Thus, although corneal allografts in C57BL/6 eyes can invoke direct alloreactive T cells, these cells are not required for the vigorous immune rejections observed in this strain of mice. 

Materials and Methods

Mice

Adult male BALB/c (H-2 ^d) and C57BL/6 (H-2 ^b) were purchased from Taconic Farms (Germantown, NY). Adult male BALB.B (C.B10-H2^b/LilMcdJ, H-2 ^b), and β2-microglobulin–deficient (C57BL/6J-B2m^tm1Unc, H-2 ^b), referred to as β2μ KO mice in this article, were purchased from Jackson Laboratory (Bar Harbor, ME) ¹⁵ between 8 and 12 weeks of age and used as experimental subjects. All animals were treated according to the Association for Research in Vision and Ophthalmology Statement for the use of Animals in Ophthalmic and Vision Research. 

Induction of Corneal Neovascularization

Intrastromal sutures induce robust neovascularization of the normally avascular corneal stroma, ¹⁶ and untreated allografts placed in these high-risk eyes are rejected swiftly, as previously described. ¹⁰ Briefly, three interrupted 11-0 sutures were placed in the central cornea of one eye of a normal recipient mouse on day −14 under aseptic microsurgical technique using an operating microscope. The neovascularized corneas then served as high-risk beds for orthotopic corneal transplants on day 0. The sutures used to induce neovascularization were removed at the time of transplantation. 

Orthotopic Corneal Transplantation

The usual method for corneal transplantation has been described in detail elsewhere. ^{11 12} Briefly, each recipient was deeply anesthetized with an intraperitoneal injection of 3 mg ketamine and 0.0075 mg xylazine before all surgical procedures. The central 2-mm of the donor cornea was excised and secured in recipient graft beds with eight interrupted 11-0 nylon sutures (Sharppoint; Vanguard, Houston, TX). Antibiotic ointment was applied to the corneal surface, and the lids were closed for 24 hours by tarsorrhaphy (8-0 nylon sutures). All grafted eyes were examined after 72 hours; grafts with technical difficulties (hyphema, infection, or loss of anterior chamber) were excluded from further consideration. Transplantation sutures were removed in all cases on day 7. 

Assessment of Graft Survival

Grants were evaluated by slit lamp biomicroscopy twice a week. At each time point grafts were scored for opacification between 0 and 5+: 0, clear and compact graft; 1+, minimal superficial opacity; 2+, mild deep (stromal) opacity with pupil margin and iris vessels (iris structure) visible; 3+, moderate stromal opacity with only the pupil margin visible; 4+, intense stromal opacity with the anterior chamber visible; 5+, maximal corneal opacity with total obscuration of the anterior chamber. Early acute rejection was diagnosed if grafts received a clinical score of 3+ or more at 2 weeks. Grafts were also judged to be rejected if a score of 2+ or more was achieved at any time thereafter, and this score was maintained through the 8-week observation interval. Corneal grafts that undergo transient opacification followed by clearing are not considered rejected. ¹²  

Restimulation of Cytotoxic T Cells In Vitro

To determine whether alloantigens expressed on donor cornea grafts lead to priming of specific T cells, four or more recipients in each group were killed at 4 weeks after grafting and the ipsilateral cervical lymph nodes and spleens removed. Potential effector cells from recipients (5 × 10⁶) and irradiated (2000 R) stimulator lymphoid cells from appropriate donors (5 × 10⁶) were plated together in 24-well plates in 2 ml of culture medium containing RPMI 1640 (Gibco, Grand Island, NY) with the following additives: 10% heat-inactivated fetal bovine serum (Hyclone, Logan, UT), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine, and 100 U and 100 μg/ml penicillin and streptomycin, respectively (Gibco), 5 mM HEPES (Gibco), and 2 × 10⁻⁵ M 2-mercaptoethanol. Cells were cultured at 37°C in a humidified 5% CO₂ atmosphere for either 3 days (to assay primed, direct, MHC-specific cytotoxicity) or 6 days (to assay primed, indirect, minor H–specific cytotoxicity). When cultured for 6 days, 1 ml of medium was removed from the culture and 1 ml of fresh medium was added on day 3. 

Cytotoxicity Assay

To assess direct, MHC-specific cytotoxicity, ⁵¹chromium (Cr)-release assays were performed on day 3 after restimulation in vitro. P815 cells, ¹⁷ tumor cells derived from DBA/2 mice, which share the H-2 ^d alleles of BALB/c, served as targets. To assess indirect, minor H–specific cytotoxicity, ⁵¹Cr-release assays were performed on day 6 after restimulation in vitro. Spleen cells from BALB.B donors (share H-2 ^b allele with C57BL/6, but display multiple minor H antigenic disparities) were placed in 10 ml culture medium for 3 days before assay in the presence of 5 μg/ml concanavalin A (ConA). On the day of assay, ConA-target cells were labeled with ⁵¹Cr and mixed (1 × 10⁴ target cells) with effector cells in triplicate at ratios of 6:1, 12:1, 25:1, 50:1, and 100:1. Some experiments described in Table 1 have a maximum E:T ratio of 75:1. Six wells containing only medium and target cells were used to measure spontaneous release, and six wells containing 5 N HCl and target cells were used to measure maximal radioisotope release. After 4 hours’ incubation at 37°C in 5% CO₂, 25 μl of culture supernatant was removed from each well, and radioactivity was measured. Specific lysis was determined according to the formula: percentage of specific lysis = (cpm mean experimental − cpm mean spontaneous release)/(cpm mean maximal release − cpm mean spontaneous release) × 100. Spontaneous release ranged between 5% and 15%. All experiments were repeated at least three times with similar results, and the results of representative experiments are presented in the figures. For each data point shown in the figures, lymph nodes, or spleens were harvested and pooled from at least three mice. 

Statistical Methods

Comparison of cytotoxic activity was made by the Student’s t-test. We constructed Kaplan–Meier survival curves to compare the probability of graft survival over the follow-up period. We obtained the Mantel–Haenszel summary χ² statistic to compare the proportion of rejected transplants in the two groups. P < 0.05 was deemed significant. 

Results

Acquisition of Indirect Minor H–Specific Cytotoxic T Cells by C57BL/6 Recipients of Orthotopic Corneal Allografts

Panels of C57BL/6 mice underwent orthotopic transplantation of corneas obtained from eyes of either BALB/c (MHC plus minor H disparate), or BALB.B (minor H–only disparate) donors. After 4 weeks, cervical lymph nodes ipsilateral to the graft-containing eye and spleens were harvested. For positive control animals, panels of C57BL/6 mice received a subcutaneous (SC) injection of 10 × 10⁶ BALB/c or BALB.B lymphoid cells 2 weeks before death. Putative effector cells (5 × 10⁶) were cultured for 6 days in the presence of x-irradiated (2000 R) lymphoid cells from BALB.B donors. At the end of the culture interval, the cells were harvested and assayed for cytotoxic activity directed at ⁵¹Cr-labeled BALB.B lymphoid cells that had been stimulated in vitro with ConA. The results of a representative experiment are presented in Table 1 . In vitro stimulated lymphocytes from lymph nodes of C57BL/6 mice that rejected BALB/c cornea grafts (MHC plus minor H disparate) displayed a high level of target cell lysis (comparable to positive control animals), whereas similar lymphocytes from mice with healthy cornea grafts displayed insignificant levels of cytolysis. In the same manner, in vitro-stimulated lymphocytes from C57BL/6 mice that rejected BALB.B corneas (minor H-only disparate) lysed BALB.B target cells (comparable to positive control animals), although lymphocytes from mice with accepted grafts were incapable of lysing BALB.B. Similar findings emerged from studies in which recipient spleen cells were used (data not shown). These results indicate that C57BL/6 mice that reject orthotopic corneal allografts acquire T cells that are minor H–specific and therefore have been activated through the indirect pathway of allorecognition. A similar T-cell response has been reported for BALB/c mice bearing orthotopic corneal allografts. 

Acquisition of Direct, MHC-Specific Cytotoxic T Cells by C57BL/6 Recipients of Orthotopic Corneal Allografts

C57BL/6 mice underwent orthotopic transplantation of corneas obtained from BALB/c donors. At 4 weeks, ipsilateral cervical lymph nodes, as well as spleens, were harvested and stimulated in vitro for 3 days with x-irradiated BALB/c lymphoid cells. Positive control animals were immunized SC with BALB/c lymphoid cells 2 weeks before in vitro stimulation. The cultured cells were then harvested and assayed for cytotoxic activity against ⁵¹Cr-labeled P815 cells (a mast cell tumor derived from DBA/2 mice, expressing H-2^d alloantigens identical with BALB/c). The results of a representative experiment are presented in Table 1 . As anticipated, in vitro stimulated lymphoid cells from positive control mice caused significant release of radioisotope from P815 target cells. Similarly, in vitro stimulated lymphoid cells from C57BL/6 mice in which BALB/c grafts were rejected also lysed P815 target cells. In an unexpected finding, in vitro stimulated lymphoid cells from C57BL/6 mice bearing healthy (accepted) BALB/c grafts caused lysis of P815 targets. Results of a similar nature were obtained with spleen cells from C57BL/6 recipients of orthotopic corneal allografts. These findings indicate that the direct pathway of allorecognition is open in C57BL/6 mice receiving orthotopic grafts of allogeneic corneas. Although direct T cells were detectable in both rejector and acceptor mice, these results encouraged us to determine whether direct alloreactive T cells might be important or required for early, acute rejection of BALB/c cornea grafts in eyes of C57BL/6 mice. 

Fate of Orthotopic Corneal Allografts in Eyes of Mice Deficient in CD8⁺ T Cells

Mice with the gene for β-2 microglobulin rendered dysfunctional (β2μ KO) are deficient in CD8⁺ T cells, and their lymphoid cells are unable to lyse target cells in an antigenically specific manner. We turned to these mice as suitable subjects to test the hypothesis that CD8⁺ cytotoxic T cells are responsible for early, acute rejection of orthotopic corneal allografts in C57BL/6 mice. C57BL/6 and β2μ KO mice received orthotopic corneal grafts from BALB/c donors. In one panel of mice, the grafts were placed in normal eyes. In another panel, the grafts were placed in eyes rendered high risk—that is, neovascularized secondary to sutures placed through the central cornea 2 weeks previously. As a negative control, syngeneic grafts were placed on normal eyes (n = 5). The fate of the grafts was assessed clinically, and the results are summarized in Figures 1A 1B and 1C . The evidence indicates that the tempo and incidence of rejection of BALB/c cornea grafts were highly similar in wild-type C57BL/6 (n = 30) and β2μ KO mice (n = 11) and that significant numbers of grafts in both types of recipients were rejected early and acutely (Fig. 1A) . As revealed in Figure 1B , allografts in high-risk eyes were rejected in both wild-type (n = 10) and β2μ KO mice (n = 12) in a comparably acute and vigorous fashion. 

BALB/c corneas confront C57BL/6 and β2μ KO recipients with both MHC and minor H–encoded alloantigens, rendering it possible that both direct and indirect alloreactive T cells could contribute to graft rejection. To eliminate the possible role of direct alloreactive T cells, panels of β2μ KO (n = 11) as well as C57BL/6 mice (n = 15) received minor H–only disparate BALB.B corneas in normal eyes. In this combination, donor-specific T cells can only recognize donor alloantigens through the indirect pathway. The clinical fate of these grafts is summarized in Figure 1C . In both panels of mice, the majority of the orthotopic corneal allografts were rejected, and the tempo of rejection was virtually identical. Early, acute graft rejection was observed with equal frequency in the two test strains. Together, these data suggest that mice with deficiencies of CD8⁺ T cells and the capacity to generate alloreactive cytotoxic cells are fully capable of rejecting orthotopic corneal allografts with a vigor similar to normal control animals. 

Search for Residual Cytotoxic T-Cell Activity in CD8⁺ T-Cell–Deficient Mice Bearing Orthotopic Corneal Allografts

The deficit of CD8⁺ T cells in β2μ KO mice is not absolute. ¹⁸ Therefore, it is possible that the conclusions drawn from the previous grafting experiments are not justified and that the small numbers of CD8⁺ T cells present in these mice are sufficient to cause rejection of orthotopic corneal allografts. To test this possibility, lymphoid cells were harvested from β2μ KO mice 4 weeks after they received orthotopic corneal grafts from BALB/c donors. Other β2μ KO mice, which were immunized SC with BALB/c lymphoid cells, served as conventionally primed control animals. Lymphoid cells were also harvested from naive C57BL/6 and β2μ KO donors and from C57BL/6 mice immunized SC with BALB/c spleen cells 2 weeks previously. Suspensions of lymphoid cells were stimulated in vitro with x-irradiated cells from BALB/c donors for 3 days or from BALB.B donors for 6 days. The responding cells were then harvested and assayed on ⁵¹Cr-labeled cells appropriate to determine whether direct or indirect alloreactive T cells were present. The results of representative experiments are presented in Figure 2 . When BALB/c cells were used as stimulators and P815 cells were used as targets, T cells from SC primed C57BL/6 donors were cytotoxic (see Fig. 2A ). BALB/c-stimulated T cells from none of the other sources lysed P815 targets at a high level, although T cells from in vivo primedβ 2μ KO donors appeared to cause minimal radioisotope release at the highest effector-to-target (E:T) cell ratios (100:1). When BALB.B donor cells were used to stimulate T cells from wild-type mice bearing BALB/c grafts or immunized with BALB/c lymphoid cells, only T cells from mice primed SC with lymphoid cells displayed any cytotoxicity directed at BALB.B ConA blast cells (see Fig. 2B ). Finally, when BALB.B donor cells were used to stimulate wild-type T cells from mice bearing BALB.B grafts (or that had been immunized SC with BALB.B lymphoid cells), SC-primed T cells were efficient killers of BALB.B target cells (see Fig. 2C ). Of the other cell sources, only a barely detectable increase in release of radioisotope was observed with T cells from β2μ KO mice primed by SC injections of BALB.B lymphoid cells. It is important to point out that in none of these experiments did T cells harvested from β2μ KO mice bearing orthotopic corneal allografts display any evidence of donor-specific cytotoxic activity. However, it is still possible that β2μ KO mice may in some as yet undetermined way compensate for the absence of CD8⁺ T cells and therefore mask the role of CD8⁺ T cells in corneal allograft rejection. We conclude that corneal allografts fail to prime the few residual CD8⁺ T cells present inβ 2μ KO mice, and that the ability of these mice to reject orthotopic corneal allografts in a manner indistinguishable from wild-type control animals is unrelated to CD8⁺ cytotoxic T cells. 

Discussion

It is important to understand the immunopathogenesis of rejection when orthotopic corneal allografts fail. In general, solid tissue allografts elicit a diverse spectrum of alloantigen-specific immune effectors, ranging from T cells that mediate DH and cytotoxic T cells to antibodies of both the complement-fixing and non–complement-fixing types. ^{19 20} There is now compelling evidence that this range of effector modalities is evoked by orthotopic corneal allografts. Our laboratory has focused in a mouse model on studying the pathogenesis of acute rejection of orthotopic corneal allografts. For other types of solid tissue allografts, except in previously primed recipients, acute graft rejection is mediated by alloreactive T cells, rather than antibodies. Recent evidence indicates that antibodies play no role in acute or subacute rejection of orthotopic corneal allografts in rodents. ²¹ Controversy surrounds the putative roles of CD4⁺ and CD8⁺ T cells in acute and subacute rejection of orthotopic grafts of solid tissue grafts. ²² On the one hand, CD4⁺ T cells (of the DH types) and CD8⁺ T cells have each been found to be capable of mediating acute rejection of orthotopic skin, heart, and kidney grafts. ²³ On the other hand, the weight of evidence suggests that, of the two T cell types, CD4⁺ cells are the more important. A similar situation applies to orthotopic corneal allografts. Studies in both rat and mouse models have implicated CD4⁺ T cells as central to cornea graft rejection, and a role for CD8⁺ T cells remains in doubt. ^{24 25 26 27 28}  

The evidence presented in this report and previously ²⁴ demonstrates, at least in a model using C57BL/6 and BALB/c mice, that CD8⁺ cytotoxic T cells play no essential role in acute or subacute rejection of orthotopic corneal grafts. The tempo and incidence of rejection of allogeneic corneas grafted to eyes of β2μ KO mice were indistinguishable from that of similar grafts in eyes of wild-type C57BL/6 mice. Because our results reveal that orthotopic corneal allografts elicit no detectable T cells in β2μ recipients, we conclude that neither early acute nor subacute cornea graft rejection involves T cells. 

Recipients of solid tissue grafts can become sensitized through two distinctly different pathways of allorecognition. One set of alloreactive T cells (direct) recognizes donor MHC class I or II alloantigens directly (without benefit of processing by antigen-presenting cells [APCs]). Direct alloreactive T cells are capable on their own of rejecting solid tissue grafts, and rejection of this type is typically acute (within 1–2 weeks). Another set of alloreactive T cells (indirect) recognizes donor MHC as well as minor H alloantigens only after they have been taken up by recipient antigen presenting cells and presented by recipient MHC molecules. Depending on the number of MHC and minor H alloantigenic differences, indirect alloreactive T cells can mediate acute (many antigenic differences) as well as subacute (fewer antigenic differences) graft rejection. We have previously shown that orthotopic corneal allografts in BALB/c mouse eyes elicit sensitized CD4⁺ and CD8⁺ T cells of the indirect type only. Because corneal allografts in BALB/c mice are not typically rejected until 3 or more weeks after grafting and because a significant proportion of corneal allografts in eyes of C57BL/6 mice are rejected before 3 weeks, we hypothesized that direct T cells, specific for donor MHC alloantigens, mediate early, acute rejection in the latter mice. 

Our results clearly indicate that C57BL/6 mice differ from BALB/c mice in their ability to acquire direct, donor MHC-specific T cells after receiving orthotopic corneal allografts. However, two pieces of data argue against these cells’ being responsible for early acute rejection in this instance. First, direct T cells were detected readily in both acceptor and rejector C57BL/6 mice. Second, no direct (or indirect) donor-specific T cells were detected in β2μ KO mice, yet these mice also displayed the capacity to reject orthotopic corneal allografts in an early, acute fashion. Thus, our hypothesis is incorrect, and early acute rejection of cornea grafts by C57BL/6 mice, compared with BALB/c mice, is not due to activation of donor MHC-specific T cells of the direct type. 

In one sense, this conclusion strengthens the thesis that acute rejection of orthotopic corneal allografts in mice is mediated solely by CD4⁺ T cells, presumably of the DH type. But in another sense, we are at a loss to explain the apparent irrelevancy of donor-specific T cells evoked by orthotopic cornea allografts. Donor-specific T cells of the direct type have only been detected in normal C57BL/6 recipients, not in normal BALB/c mice. However, in both BALB/c and C57BL/6 recipients of cornea grafts, donor minor H–specific T cells of the indirect type are easily detected. Moreover, in both strains, only mice that reject their orthotopic grafts acquire indirect T cells. T cells from mice that accept their cornea grafts display no evidence that minor H–specific indirect T cells are activated. The paradox is that there is a tight correlation between graft rejection and acquisition of indirect T cells, yet graft rejection proceeds normally in the complete absence of CD8⁺ T cells! 

We were surprised that direct activation of CD8⁺ cytotoxic T cells occurred in C57BL/6 mice but not BALB/c mice. The absence of direct T cells in BALB/c mice in our previous experiments is more easily explained than the presence of these cells in the current series of experiments. The absence of direct CD8⁺ T cells in BALB/c mice is most likely due to the absence of donor-derived class I–positive APCs in the corneal allograft. These cells are believed to be responsible for activating direct CD8⁺ T cells. However, this suggests these APCs must be present in the donor corneas when direct CD8⁺ T cells are activated in the C57BL/6 recipients receiving BALB/c donors. There are at least two possible explanations for these data. First, there are some, as of yet undetermined, class I–positive APCs present in corneal tissue and either the frequency or immunogenicity of these allo-APCs are different in these two strains of mice. Second, direct CD8⁺ T cells are not activated by donor APCs, but through class I–positive donor corneal cells that migrate from the eye to the draining lymph node. The frequency of these cells migrating from the eye to the lymph node could be different in these two strains of mice. The latter possibility seems less likely, because there is little evidence that cells other than professional APCs can prime specific T cells. However, currently there are no data indicating APCs are present in the normal cornea. 

In their report of 1991, He et al. ²⁴ came to conclusions similar to ours, using anti-CD4 and anti-CD8 antibodies to demonstrate that CD4⁺, but not CD8⁺, T cells are required for rejection of orthotopic corneal grafts. At the time of their study, the question of relative contributions of direct and indirect pathways of allorecognition to rejection of orthotopic corneal allografts was not formally considered. Moreover, the strain combination they used—C57BL/6 corneas to BALB/c recipients—precluded an analysis of this issue. Our findings, using corneal allografts in eyes of C57BL/6 recipients, add at least two important new dimensions to our understanding of corneal allograft rejection. First, unlike BALB/c mice, C57BL/6 recipients of MHC-disparate orthotopic corneal allografts acquire direct alloreactive T cells, making it possible to test for the participation of these cells in graft rejection. Second, by using β2μ KO mice (C57BL/6 background), we were able to exclude formally a role for T cells of either the direct or indirect type in acute rejection of orthotopic cornea grafts. 

When allogeneic corneas are grafted into high-risk eyes of BALB/c and C57BL/6 mice, they are subjected to an early, vigorous alloimmune response. Most of these grafts are destroyed within 7 to 10 days. In both instances, the lymphoid organs of the recipients acquire donor MHC-specific T cells of the direct alloreactive type. Similarly, direct alloreactive T cells that mediate DH are also activated. Yet, corneas grafted into high-risk eyes of β2μ KO mice showed the same high and rapid rate of rejection as did grafts in high-risk eyes of C57BL/6 mice. These results suggest that even the direct T cells activated by grafts in high-risk eyes are irrelevant to graft outcome. We conclude that whether corneas are grafted into normal eyes or into high-risk eyes, the rejections that are observed as early as 1 week after grafting or as late as 6 weeks after grafting are mediated solely by CD4⁺ T cells. 

The dissociation between activated donor-specific T cells and the absence of a role for these cells in rejection of orthotopic corneal allografts can be at least partly explained. With respect to T cells of the indirect type (directed at peptides of minor H antigens displayed on recipient MHC class I molecules), in fully allogeneic corneas of the type used in our experiments (MHC mismatched), the cells of the graft are not capable of serving as targets, in that none express recipient MHC class I molecules. BALB/c grafts on C57BL/6 recipients represent this type of situation. However, when minor H–disparate donor and recipient share the same MHC, cells of graft can serve as targets of indirect T cells. BALB.B grafts on C57BL/6 recipients represent this type of situation. Regarding T cells of the direct type (directed a donor MHC class I antigens), parenchymal cells of the graft can express target class I molecules. Because graft epithelium is rapidly replaced after keratoplasty in mice (Hori J, unpublished observation, 1999), expression of MHC molecules by corneal epithelial cells may be irrelevant to graft outcome. The fate of the graft seems to be determined primarily by the fate of its endothelium, and it is relevant that constitutive expression of class I molecules on corneal endothelium is much lower than other types of cells, ²⁹ rendering them relatively weak as targets of class I–specific T cells. Moreover, corneal endothelial cells constitutively express CD95 ligand (CD95L). ³⁰ Perhaps when activated (CD95⁺), direct alloreactive T cells contact graft endothelium, interaction of CD95 with its ligand induces apoptosis of the T cells before they can deliver a lethal hit to the endothelium. The reports of Stuart et al. ³¹ and Yamagami et al ³² provide strong evidence that CD95L expression on corneal endothelium is important in protecting the graft against acute rejection. 

 Supported in part by US Public Health Service Grants EY-10765 and EY-19765.

 Submitted for publication August 2, 1999; revised December 22, 1999; accepted February 2, 2000.

 Commercial relationships policy: N.

 

Table 1.

 

Percentage Specific Lysis

	Effector–Target Ratio
		6:1	12:1	25:1	50:1	100:1
Minor H–Specific CTL
Donor: BALB/c
Recipient: B6
Target cells: BALB.B
Negative Control (naive)	2 ± 1	8 ± 1	9 ± 1	11 ± 1	18 ± 1
Positive Control (primed)	23 ± 3	39 ± 2	58 ± 2	69 ± 5	66 ± 1
Accepted	7 ± 1	9 ± 1	15 ± 1	18 ± 2	23 ± 1
Rejected	14 ± 2	27 ± 1	32 ± 2	49 ± 4	57 ± 1
Minor H–Specific CTL
Donor: BALB.B
Recipient: B6
Target cells: BALB.B
Negative Control (naive)	8 ± 1	5 ± 1	5 ± 1	2 ± 1	4 ± 1
Positive Control (primed)	9 ± 1	13 ± 1	21 ± 1	37 ± 2	43 ± 2
Accepted	3 ± 1	8 ± 1	9 ± 1	15 ± 1	18 ± 1
Rejected	11 ± 1	72 ± 1	30 ± 2	46 ± 2	42 ± 1
Class I–Specific CTL
Donor: BALB/c
Recipient: B6
Target cells: P815
Negative Control (naive)	3 ± 1	3 ± 1	6 ± 1	9 ± 1	12 ± 1
Positive Control (primed)	11 ± 1	18 ± 1	28 ± 1	40 ± 2	54 ± 2
Accepted	4 ± 1	19 ± 1	22 ± 1	39 ± 1	46 ± 1
Rejected	11 ± 1	25 ± 2	37 ± 2	49 ± 1	54 ± 3

 Recipient mice received orthotopic corneal allografts from the respective donor mice. Positive controls (CTL) were mice immunized subcutaneously with donor spleen cells. Naive mice served as negative control animals. After the fate of the corneal allografts was determined, draining cervical lymph nodes were stimulated in vitro with donor splenic stimulator cells (6 days for minor specific CTL, 3 days for Class I specific CTL). Lymphocytes were harvested and assayed with ⁵¹Cr-labeled target cells in a standard assay. Results are displayed as the mean specific ⁵¹Cr release from triplicate culture wells (±SEM).

Figure 1.

 

Fate of allogeneic corneas in eyes of mice deficient in CD8⁺ T cells. MHC plus minor H–disparate BALB/c corneas (A, B), or minor H–only disparate BALB.B corneas (C) were placed in normal (A, C) or high-risk (neovascularized corneas; B) eyes of C57BL/6 andβ 2μ KO mice or syngeneic BALB/c mice. Cumulative graft survival (retention of corneal clarity) through 56 days is presented as Kaplan–Meier plot. Curves of C57BL/6 and β2μ KO recipients were statistically indistinguishable from each other in any plot.

Figure 2.

 

Cytotoxic T-cell activity from CD8⁺ T-cell–deficient mice bearing orthotopic corneal allografts. MHC plus minor H–disparate BALB/c (A, B), or minor H–only disparate BALB.B (C) corneas were placed in eyes of C57BL/6 and β2μ KO recipients. Positive control (primed) C57BL/6 and β2μ KO mice were immunized SC with BALB/c (A, B) or BALB.B (C) spleen cells. Normal C57BL/6 and β2μ KO mice served as negative control (naive). Four weeks (graft recipients) or 2 weeks (positive control animals) later, when the fate of the grafts was known, ipsilateral/draining cervical lymph node cells were stimulated in vitro for 3 days (A) or 6 days (B, C) with x-irradiated BALB/c (A, B) or BALB.B (C) spleen cells, harvested, and assayed on ⁵¹Cr-labeled target cells: P815 cells of DBA/2 origin (A) or ConA-activated splenocytes of BALB.B origin (B, C). Mean (± SEM) percentage of specific radioisotope release in 4-hour triplicate assays at differing E:T ratios are presented (*P < 0.05).