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1 From the Kyoto Prefectural University and the 2 Osaka University School of Medicine, Japan; and 3 The Schepens Eye Research Institute, Boston, Massachusetts.
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Abstract |
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METHODS. Corneas were prepared from major histocompatibility complex (MHC)–only incompatible, minor histocompatibility (H)–only incompatible, and MHC-plus-minor H incompatible donors and grafted orthotopically to eyes of CD4 knockout (KO), CD8KO, and wild-type control mice. Graft survival patterns were assessed clinically and compared. Mice that retained healthy corneal allografts beyond 8 weeks were evaluated for evidence of donor-specific tolerance and anterior-chamber–associated immune deviation (ACAID) using local adoptive transfer reactions and challenge with orthotopic skin allografts.
RESULTS. Corneas grafted to CD8KO mice were rejected with an incidence and tempo indistinguishable from that in wild-type control animals. By contrast, MHC-only, and minor-H–only incompatible corneal grafts survived indefinitely in eyes of CD4KO mice. Approximately 50% of corneal grafts that confronted CD4KO recipients with both MHC and minor H alloantigens experienced delayed rejection, whereas similar grafts in wild-type recipients were rejected acutely. CD4KO mice with long-accepted grafts displayed neither donor-specific ACAID nor allograft tolerance.
CONCLUSIONS. CD8+ T cells play little or no role in acute rejection of orthotopic corneal allografts. Instead, acute rejection is mediated almost exclusively by CD4+ T cells. Moreover, when corneal allografts survive for 8 weeks without acute rejection, CD4+ T cells promote donor-specific ACAID thereby insuring long-term graft acceptance.
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Introduction |
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When allogeneic corneas are placed in mouse eyes with neovascularized corneas, a situation that resembles high-risk eyes in clinical ophthalmology, the incidence and vigor of graft rejection is enhanced, indicating that immune privilege has been compromised.9 However, even in this circumstance, in which rejection of the grafts occurs very rapidly (within 2 weeks), minor H, rather than MHC, antigens are more important, and DTH, rather than cytotoxic T cells, correlates better with graft rejection. Several laboratories have demonstrated that rejection of orthotopic corneal grafts can be more easily prevented by treating recipients with anti CD4+, rather than anti-CD8+, antibodies.10 In the aggregate, these various studies suggest that class II MHC-specific or MHC-restricted CD4+ T cells play a dominant role in acute rejection of orthotopic corneal allografts.
Because of this situation, we have recently completed a series of experiments in which ß2-microglobulin knockout (ß2mKO) mice, which are deficient in MHC class I expression and in CD8+ T cells, were used as recipients of allogeneic cornea grafts. Irrespective of the degree of immunogenetic disparity between graft donor and recipient (MHC alone, minor H alone, MHC plus minor H), the rate and incidence of rejection was identical in normal mice and in ß2mKO mice.11 These findings not only emphasize the importance of CD4+ T cells in cornea graft rejection, but they imply that CD8+ T cells are either unnecessary or are even incapable of rejecting orthotopic corneal grafts. This implication must be formally tested, however, because ß2mKO mice have cellular immune deficits beyond those of CD8+ T cells.12 For example, these mice also have no CD1-recognizing NK T cells,13 and cells of this type may have unsuspected roles to play in alloimmunity and rejection.14
To resolve the relative importance of class II-specific CD4+ and class I-specific CD8+ T cells in corneal allograft immunity, we have explored immune responses evoked by orthotopic corneal allografts in two strains of genetically manipulated mice, one of which is deficient in CD4 expression, the other in CD8 expression. We report that CD4-deficient mice were severely impaired in their ability to reject orthotopic corneal allografts. By contrast, corneal allograft rejection proceeded unimpaired in mice deficient in CD8-bearing T cells. Moreover, unlike normal mice, CD4-deficient mice with long-accepted corneal allografts did not acquire donor-specific immune deviation and therefore never accommodated to the cornea grafts by acquiring donor-specific tolerance. On the contrary, in some CD4-deficient mice, MHC-plus-minor H incompatible grafts that had been in residence for many months underwent delayed rejection. We suspect that, given sufficient time, donor-specific CD8+ T cells can eventually emerge as effectors of rejection of orthotopic corneal allografts.
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Materials and Methods |
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Orthotopic Corneal Transplantation
As described previously,15
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 72 hours with an 8-0 nylon tarsorrhaphy. All
grafted eyes were examined after 72 hours; no grafts were excluded from
analysis because of technical difficulties. Transplant sutures were
removed in all cases on day 7.
Assessment of Graft Survival
Grafts were evaluated by slit lamp biomicroscopy twice a week. At
each time point grafts were scored for opacification. A previously
described scoring system2
was used to measure the degree
of opacification from 0 to 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 pupil margin visible; 4+, intense stromal opacity with the AC
visible; 5+, maximal corneal opacity with total obscuration of the AC.
Grafts with an opacity score of 2+ or greater after 3 weeks were
considered rejected (immunologic failure); grafts with an opacity score
of 3+ or greater at 2 weeks that never cleared by 8 weeks were also
regarded as rejected.2
Skin Grafts and Assessment of Skin Graft Survival
CD4+KO and C57BL/10 mice received an
orthotopic skin graft from BALB/c mice as described
previously.16
Briefly, skin grafts from body wall (8 x 8 mm) were placed on graft beds prepared on the thoracic wall of
anesthetized mice (halothane). Petrolatum gauze was placed over the
graft site followed by an application of mastisol. Antibacterial powder
(nitrofurazone) was then applied and the wound covered with dry gauze
followed by a plaster cast. One week later, the cast was removed, and
the grafts were examined every day. Grafts were regarded as rejected on
the day that the surface was judged by clinical inspection to be
completely denuded of epidermis.
Assay for DTH Reaction Elicited by Local Adoptive Transfer in Mice
Bearing Healthy Corneal Allografts
On day 14 after subcutaneous injection of 10 x
106 BALB.B spleen cells into naive C57BL/10 mice,
spleen cells were harvested as "responder" cells. Spleen cells
(1 x 106), obtained from
CD4+KO or C57BL/10 mice with long-accepted BALB.B
corneal allografts, were used as "regulator" cells. As a positive
control, naive CD4+KO spleen cells were used as
regulators. Regulators and responders were mixed with 1 x
106 irradiated (2000 rad) BALB.B spleen cells as
"stimulator" cells. Thus, 3 x 106 cells
in 10 µl were injected in 10 µl of Hanks’ balanced salt solution
into the right ear pinnae of naive C57BL/10 mice, as described
previously.17
After 24 hours and 48 hours, ear thickness
was measured with a low-pressure engineer’s micrometer (Mitutoyo–MTI,
Paramus, NJ). Ear swelling was expressed as follows: specific ear
swelling = (24-hour measurement of left ear - 0-hour
measurement of right ear) - (24-hour measurement of left ear - 0-hour measurement of left ear) x 10-3
mm. Ear swelling responses at 24 hours after injection are presented as
group mean ± SEM. All experiments were repeated at least twice
with similar results.
Statistical Methods
Statistical analyses were performed by using Student’s
t-test for comparison of DTH responses. We also constructed
Kaplan–Meier survival curves and used the Breslow–Gehan Wilcoxon test
to compare the probability of corneal graft survival. P < 0.05 was deemed significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Survival of Orthotopic Skin Allografts in Mice Deficient in CD4
Expression
To reach the second conclusion, it was necessary to determine
whether CD4KO mice are capable of rejecting orthotopic skin allografts,
and if so, with what incidence and tempo. Accordingly, full-thickness
skin grafts were prepared from BALB/c donors and placed on the thoracic
wall of CD4KO mice. In positive control animals, BALB/c skin was
grafted orthotopically to wild-type C57BL/10 mice. The survival of
these grafts was observed clinically. The median survival time of skin
allografts on CD4KO mice was found to be 32.2 ± 4.1 days, whereas
the median survival time of skin grafts on C57BL/10 recipients was
significantly lower (9.3 ± 0.4 days). These results indicate that
a deficit of CD4+ T cells attenuated the ability
of mice to reject skin allografts. Because all skin allografts placed
on CD4KO recipients were rejected by 40 days, alternative immune
effectors (presumably CD8+ T cells) were still
able to execute complete graft rejection. The finding that more than
90% of orthotopic corneal allografts on CD4KO mice were still healthy
at 40 days strongly suggests that CD8+ T cells
are largely unable to substitute as immune effectors in the eye when
CD4+ T cells are missing.
At 16 weeks after grafting, orthotopic corneal allografts remained healthy and clear in six CD4KO mice, and two such grafts remained clear in wild-type C57BL/10 mice. To determine whether these recipients bearing long-standing healthy corneal allografts had become tolerant of donor alloantigens, each mouse received an orthotopic BALB/c skin allograft at this time. The fate of the skin grafts and the original corneal grafts was assessed clinically. C57BL/10 mice bearing healthy BALB/c skin grafts rejected the orthotopic skin grafts, but the corneal grafts remained clear and healthy. By contrast, CD4KO mice bearing healthy corneal allografts not only rejected orthotopic BALB/c skin grafts, but they also rejected long-standing corneal grafts (see Fig. 2 ). These findings indicate that a form of tolerance develops in normal (wild-type) mice bearing long-standing orthotopic corneal allografts that protects the graft from systemic, donor-specific alloimmunity. By contrast, cornea graft–bearing CD4KO mice display no such tolerance, because exposure to donor alloantigens on orthotopic skin grafts causes them to reject the corneal grafts. Thus, CD4+ T cells appear to be important, not only in generating corneal allograft immunity, but in generating tolerance in mice that accept orthotopic corneal allografts for prolonged time intervals.
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Discussion |
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Despite the absence of bone marrow–derived cells within the normal cornea, and the absence of class II MHC gene expression on corneal parenchymal cells, class II-restricted, CD4+ T cells play the prime role in mediating rejection of orthotopic corneal allografts. The evidence from the literature in support of this conclusion is considerable. Treatment of mice with anti-CD4 antibodies inhibits rejection of orthotopic corneal allografts.10 Rejection of corneal grafts correlates better with recipient acquisition of donor-specific DTH than cytotoxic T cells.8 Our current experimental results show that CD4KO mice possess virtually no capacity to reject either minor H–only or MHC-only disparate corneal grafts. In fact, the only corneal grafts that CD4KO mice eventually rejected were grafts expressing MHC-plus-minor H incompatibilities. Even so, only 50% of these grafts were destroyed, and rejection was significantly delayed (many between 56 and 112 days after grafting) compared with corneal grafts rejected by wild-type recipients.
We have previously provided evidence to support the view that corneal alloantigens are recognized by recipient T cells largely, albeit not exclusively, through the so-called indirect pathway of allorecognition.7 In this pathway, peptides derived from MHC and/or minor alloantigens are processed and presented by recipient antigen-presenting cells that infiltrate the graft. Conventional wisdom dictates that peptides generated in this manner are loaded predominately on recipient class II MHC molecules for presentation to CD4+ T cells. We presume that this is one reason why CD4+ T cells are so important in corneal allograft immunity. In fact, therapeutic strategies that prevent recipient APC from migrating into the graft not only promote corneal allograft acceptance,25 but they suppress induction of donor-specific DTH, a CD4+ T-cell–mediated response.25 The inability of CD4KO mice to reject minor H–only disparate corneal grafts can be explained by the absence of CD4+ T cells required to recognize donor alloantigens through the indirect pathway.
Approximately 30% of MHC-only disparate (B10.D2) corneal grafts were rejected by normal C57BL/10 mice. The absolute failure of CD4KO mice to reject B10.D2 corneal grafts tells us that CD4+ T cells that recognize donor class II alloantigens are the mediators. However, we cannot be sure whether the effector T cells recognized peptides derived from class II alloantigens presented in the context of self class II molecules (indirect pathway of allorecognition), or whether these CD4+ T cells directly recognize donor class II alloantigens (direct pathway). We favor the former (indirect) pathway because corneal cells do not normally express class II molecules. However, we are aware that Peeler and Niederkorn,26 and Nicholls et al.27 have demonstrated that grafted corneal tissue can upregulate the expression of class II molecules. Experiments to distinguish between the direct and indirect pathways of class II recognition in CD4KO mice grafted orthotopically with allogeneic corneas are under way.
It is more challenging to explain why CD4KO mice, which utterly failed
to reject MHC-only or minor H–only disparate grafts, mounted more
successful immune rejections against grafts expressing MHC-plus-minor H
disparities. At the very least, we suspect that
CD8+ T cells must be involved. It is generally
believed that activation of naive CD8+ T cells
requires help and that CD4+ T cells are usually
regarded as the source of helper factors. However, helper cells of this
type are missing in CD4KO mice. Therefore, alternative sources must be
considered. Activated CD8+ T cells are known to
secrete cytokines, including IL-2 and interferon-
, which can provide
help, and therefore should be considered. It may be relevant that
C57BL/6 recipients of orthotopic BALB/c corneal allografts acquire
activated CD8+ T cells that recognize class I
H-2d antigens directly. Although these cells were
detected in cytotoxicity assays, it is possible that they also secrete
helper factors in response to recognition of donor class I antigens. If
cells such as this were activated in CD4KO recipients of BALB/c corneal
grafts, then they may have been able to provide help for
CD8+ T cells that recognize BALB/c-derived minor
H alloantigens through the indirect pathway. Therefore, we propose that
CD4KO mice reject BALB/c corneal grafts through the actions of minor
H–specific cytotoxic T cells, and that these cells are promoted by
factors released from CD8+ T cells that recognize
H-2d class I antigens directly. It is of interest
that BALB/c corneas were rejected by CD4KO mice in a delayed fashion
compared with graft rejection in wild-type C57BL/10 mice. Perhaps the
tempo of orthotopic corneal allograft rejection is dictated by whether
CD4+ T cells participate or not. In their
absence, rejection is desultory and incomplete.
The idea that allogeneic MHC-specific T cells can act as adjuvants, promoting the immunogenicity of minor H antigens, is suggested by our experimental results. This idea has been advanced previously. In the early 1970s, rejection of skin grafts bearing weak transplantation antigens was promoted in Syrian hamsters by sensitizing the recipients simultaneously with weak antigens plus MHC-encoded alloantigens.28 The so-called allogeneic effect, originally described by Katz et al.,29 was formulated to describe the ability of MHC alloantigens to enhance humoral immune responses to soluble protein antigens. The proposed mechanism advanced to explain the allogeneic effect focused on T cells that recognize class II MHC alloantigens. Our evidence leads us to focus on class I–recognizing CD8+ T cells, but the outcome would be expected to be the same.
Although the evidence from studies of orthotopic corneal allografts emphasizes the key role CD4+ T cells play in immune rejection, evidence from experiments with other types of solid tissue allografts is less clear on this point. Experiments attempting to determine which effector cell (CD4+ or CD8+) is more important in graft rejection were popular in the 1980s.30 31 Monoclonal antibodies were used to negatively or positively select CD4+ and CD8+ T cells, which were then tested for relative capacity to cause graft rejection when adoptively transferred into appropriate recipients bearing skin, heart, or kidney grafts. In some studies, CD8+ T cells alone—without the participation of any CD4+ T cells—were sufficient to cause graft rejection. In other studies, CD4+ T cells provoked graft rejection independently of CD8+ T cells. In yet a third type of study, both CD4+ and CD8+ T cells were required to achieve acute, rather than delayed or chronic, graft rejection. More recently, skin allograft survival was assessed in CD4KO mice 32 . Depending on the genetic background of the strain, one group claimed that the grafts were not rejected, whereas another group observed that CD4KO mice can still reject skin allografts. Based on recent studies from other laboratories 10 , as well as our current results, we believe that CD4+ T cells play the central, if not the only, role as effector cells in orthotopic corneal allograft rejection. We suspect that this is the case, because the normal cornea has no "passenger leukocytes," a deficit that requires recipient antigen-presenting cells to process exogenous donor transplantation antigens for presentation through the class II pathway to T cells. CD4+ T cells are the responding cells in this situation.
In the aggregate, our experimental results confirm that CD4+ T cells determine the tempo and incidence of acute rejection of orthotopic corneal allografts. In addition, they illuminate a role for CD4+ T cells in regulating allograft immunity, thereby promoting the long-term survival of orthotopic corneal allografts. In normal mice that accept orthotopic corneal allografts for prolonged intervals (beyond 8 weeks), donor-specific ACAID emerges and secures the future integrity of the graft by preventing subsequent development of destructive allograft immunity. However, in CD4KO mice bearing corneal allografts for more than 8 weeks, no evidence of ACAID was detected. Instead, cornea graft–bearing CD4KO mice rejected orthotopic skin grafts acutely, and shortly thereafter, the long-standing cornea grafts were destroyed. Thus, CD4+ T cells have two antithetical roles to play in the immunobiology of corneal allografts. On the one hand, CD4+ T cells play a critical role in mediating acute rejection of orthotopic cornea grafts. On the other hand, CD4+ T cells are needed to promote allograft tolerance and ACAID, thereby ensuring the long-term survival of grafts that manage to survive acute graft rejection mechanisms. If this knowledge is to be used eventually to enhance the survival of corneal allografts in humans, it is important to understand the conditions that enable CD4+ T cells to promote tolerance without promoting acute graft rejection.
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Acknowledgements |
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Footnotes |
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Submitted for publication December 28, 1998; revised May 19, 1999; accepted June 23, 1999.
Commercial relationships policy: N.
Corresponding author: J. Wayne Streilein, The Schepens Eye Research Institute, 20 Staniford Street, Boston, MA 02114. E-mail: waynes{at}vision.eri.harvard.edu
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) and
wild-type C57BL/6 (
) mice after the application of orthotopic skin
grafts are presented.
