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1From the Department of Immunology, Division of Medicine, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, London, United Kingdom; the 2Institute of Ophthalmology, University College London, London, United Kingdom; and 3Moorfields Eye Hospital, London, United Kingdom.
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Abstract |
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METHODS. CD28 knock-out (CD28KO) and wild-type BALB/c control mice received corneal grafts from fully major histocompatibility complex (MHC)–mismatched C3H donors and were treated with CTLA4-Ig and/or anti-CD154 Ab on days 0, 2, and 4 after transplantation. Proliferation of BALB/c and CD28KO T cells in response to C3H stimulators was examined in a mixed lymphocyte reaction (MLR) in the presence of CTLA4-Ig or anti-CD154 Ab.
RESULTS. Corneal allograft survival in wild-type BALB/c mice (median survival time [MST] 14 days) was significantly prolonged by blockade of the costimulatory pathways with CTLA4-Ig or anti-CD154 Ab (MST 21 days and 25 days respectively). MST in recipients treated with CTLA4-Ig and anti-CD154 Ab in combination was 29 days, not significantly longer than graft survival in single-treatment groups. MST in CD28KO recipients was 46 days and was not prolonged after treatment with anti-CD154 Ab (MST, 43 days). A similar result was found in the MLR, in which anti-CD154 Ab had no effect on proliferation of CD28KO compared with wild-type T cells. In CTLA4-Ig–treated CD28KO, grafts were rejected at an accelerated tempo, similar to that in wild-type BALB/c recipients (MST 16 days). More severe graft injury after the onset of rejection in untreated allograft recipients was accompanied by a higher number of graft-infiltrating CD45+ cells, but similar proportions of CD4+ and CD8+ cells.
CONCLUSIONS. CD28- and CD154-mediated costimulation have significant functional roles in corneal allograft rejection. Agents that modulate CD28 and CD154 pathways delay onset and reduce the severity of observed allograft rejection. However, their use in combination did not have an additive effect, MLR data indicating that the CD40–CD154 system depends on a functioning CD28 costimulatory pathway.
Allograft rejection is primarily dependent on activation of alloreactive T lymphocytes. After interaction of antigen-major histocompatibility complex (MHC) with the T-cell receptor–CD3 complex, which gives specificity to the immune response, costimulation through one or more additional receptor-ligand pairs has been shown to be necessary for full activation of naive T cells.5 The interaction of CD28 with its antigen-presenting cell (APC) ligands CD80/CD86 is one of the dominant costimulatory pathways, blockade of which has been demonstrated in many studies to result in prevention of allograft rejection and other immune responses.6 7 8 9 Although survival is prolonged in allograft models in many species, induction of tolerance has been more difficult to demonstrate, suggesting that costimulation can be effected by one of the CD28-independent pathways now recognized. In this respect, interaction of CD154 (CD40L) with the CD40 receptor on APCs is a likely candidate. On activation, T cells express CD154, and the CD40 receptor has been shown to upregulate several costimulatory activities of APC of importance in a range of T-cell–dependent immune responses. This pathway also plays a critical role in allograft rejection, as inhibition with an anti-CD154 antibody significantly prolongs murine cardiac,10 and primate islet11 allograft survival. Larsen et al.12 reported long-term acceptance of both of cardiac and skin grafts, indicating additive effects of blockade of both the CD28 and CD154 costimulatory pathways.
Selective blockade of costimulation through either CD28 or CD154 has been shown to prolong corneal allograft survival.13 14 In the study of Qian et al.,14 anti-CD154 antibody was administered weekly for 8 weeks, and at the termination of the treatment period, graft survival was examined. We examined the effect of blockade of the CD28 and CD154 pathways individually and in combination in murine corneal transplantation. Because loss of donor corneal transparency is the end point in corneal allograft rejection and can be graded by direct observation, comparison could readily be made between rapidity of rejection onset and severity of rejection parameters in different recipient treatment groups. This is not possible in other transplantation models, in which the interval to graft rejection is the sole end point. This model could therefore be used to examine the effects of costimulatory blockade on phenotypic correlates of rejection.
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Methods |
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Generation of Anti-CD154 Antibody
A B-cell hybridoma producing hamster mAb against mouse CD154 (MR1, IgG2a) was obtained from American Type Culture Collection (ATCC, Manassas, VA). Hybridoma cells were cultured in RPMI 1640, 1% IgG-depleted FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Antibody was purified by protein G chromatography (Sigma-Aldrich).
ELISA for CTLA4-Ig and Anti-CD154 Ab Concentration
A 96-well microtiter plate (Nalge Nunc International, Taastrup, Denmark) was coated with 2.5 µg/mL rabbit anti-human IgG (Fc-specific; Dako, Glostrup, Denmark) or 2.5 µg/mL mouse anti-hamster IgG (BD Pharmingen, Cowley, UK) capture Ab in 0.1 M Na2HPO4 (pH 9.6). After overnight incubation at 4°C, the plate was washed with PBS and 0.1% Tween and blocked with 2% milk for 45 minutes at 37°C. Plates were washed again, and the standards (human IgG; Zymed, Cambridge, UK; hamster IgG, Serotec, Kidlington, UK) or samples were added in 100-µL volumes to each well in triplicate, incubated for 2 hours at room temperature, and washed again. The secondary Ab, biotin-conjugated goat anti-human IgG (1:2000; Sigma-Aldrich) or biotin-conjugated mouse anti-hamster IgG (2 µg/mL; BD Pharmingen) was then added and incubated for 1 hour at room temperature. In the case of CTLA4-Ig, the capture Ab was preincubated with 5% rabbit and 5% goat serum (Sigma-Aldrich) in PBS and 0.1% Tween for 30 minutes at room temperature to block a nonspecific cross-reaction. After four washes, 100 µL of streptavidin-horseradish peroxidase (HRP) 1:10,000 (Dako) in PBS and 0.1% Tween was added to each well for 15 minutes at room temperature. After four additional washes, 50 µL of TMB substrate (Zymed) was added to the wells and incubated for 5 minutes at room temperature. The reaction was stopped by the addition of 50 µL of 1 M H2SO4. Plates were read at 450 nm with a microplate reader. The ELISA detection limit was 4 ng/mL for CTLA4-Ig and 1 ng/mL for anti-CD154 Ab.
Animals
Inbred adult female adult BALB/c mice (H-2d; Harlan Olac, Bicester, UK) and female BALB/c CD28-deficient (H-2d) mice,15 obtained from The Jackson Laboratory (Bar Harbor, ME) were used as graft recipients. Targeted deletion was confirmed by PCR analysis for the CD28 sequence in wild-type BALB/c and knock-out (KO) mice. Female C3H/He (H-2k; Harlan Olac) mice were used as allogeneic and BALB/c as syngeneic donors. Animals were maintained in a specific pathogen-free facility and treated in accordance to the UK regulations for care of experimental animals. Animal studies were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Mixed Lymphocyte Reaction
The MLR was performed as previously described,16 with some modification. In brief, the spleen and major lymph nodes (cervical, inguinal, and abdominal) of BALB/c CD28KO mice and C3H mice were used to collect APCs and T cells, which were separated with a nylon wool column. For purification, T cells were resuspended with 1 mL M5/114 supernatant (anti-I-Ad antibody) and 1 mL ADH4 supernatant (anti-CD8 antibody) for 30 minutes on ice. The cells were then washed twice (635g for 5 minutes) and incubated with goat anti-rat magnetic beads (Dynabeads; Dynal, Bromborough, UK) twice for 30 minutes a on rotator at 4°C. Stimulator cells (APCs) were irradiated with 30 Gy. The stimulator (C3H) and responder (CD4+ cells from BALB/c mice) cells were resuspended at 2 x 105 cells at a 1:1 ratio. The plate was cultured at 37°C in 5% CO2 for 72 hours before being pulsed with 1 µCi/well [3H]-TdR for 16 hours.
Orthotopic Corneal Transplantation and Definition of Corneal Graft Rejection
Orthotopic corneal transplantation was performed in the right eye of all animals (6–10 weeks), as described by Zhang et al.,17 with minor modifications. A 2.5-mm diameter donor corneal graft was sutured into a 2.0-mm recipient corneal bed with one continuous 11-0 nylon suture (Ethicon, Somerville, NJ). At the completion of the procedure, the anterior chamber was reformed with sterile saline and chloramphenicol ointment (Martindale Pharmaceuticals, Brentwood, UK) applied on the cornea. The eyelids were sutured closed to protect the cornea for the initial 48 hours. At that time and thrice weekly thereafter, the eye was examined by operating microscope. Technically satisfactory grafts had corneal opacity grade 0 to 1 at all times until suture removal on day 7. All other graft recipients, and those with intraocular hemorrhage or cataract were excluded.
Corneal transparency is an indicator of corneal endothelial function and of graft endothelial injury. The onset of graft rejection was diagnosed when corneal opacity increased to grade 3 (no iris vessels visible) in a graft previously clear after transplantation (Fig. 1) . Grading of corneal opacity was as described by Sonoda and Streilein18 with minor modifications, as follows: 0, completely transparent cornea; 1, minimal corneal opacity, but iris vessels easily visible; 2, moderate corneal opacity, iris vessels still visible; 3, moderate corneal opacity, only pupil margin visible; 4, complete corneal opacity, pupil not visible.
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Immunohistology
Rejection was confirmed by end point histology in all graft recipients. Corneas were excised on the day of observed onset of rejection. The tissue was snap frozen in liquid nitrogen and stored at -70°C or fixed in 10% formalin and embedded in paraffin wax before staining with hematoxylin and eosin (H&E) or immunohistochemistry as follows. Paraffin sections were incubated with 10 µg/mL rat anti-mouse CD45 mAb; cryostat sections were incubated with 1 µg/mL rat anti-mouse CD4 or 3 µg/mL rat anti-mouse CD8 monoclonal antibody (both from BD Pharmingen). Sections were then washed, incubated with biotinylated rabbit anti-rat IgG secondary Ab (2 µg/mL, Vector Laboratories, Orton Southgate, UK), washed, and incubated with avidin-biotin complex (ABC; Vector Laboratories). The chromogen 3,3'-diaminobenzidine (DAB; Dako) was then applied before hematoxylin counterstaining. Positive controls were sections of mouse spleen; negative controls were sections incubated with rat IgG2a or IgG2b (R&D Systems, Abingdon, UK) as the primary Ab.
Scoring of Positive Immunostaining in the Tissue
Positive-stained cells were counted in three adjacent light microscopic fields in the center of the corneal graft, using 100x objective magnification. Counts were made in three nonserial sections of each specimen in three corneas from each treatment group. Cryostat sections were evaluated semiquantitatively as follows: 0, no staining; 1, 10% or less of cells stained; 2, 11% to 30% stained; 3, 31% to 50% stained; 4, 51% to 80% stained; 5 more than 80% stained.
Statistical Analysis
Actuarial graft survival was analyzed using the Kaplan-Meier survival method.19 The log-rank test was used to examine for statistical differences between the groups. Student’s t-test was used to analyze immunohistochemistry data and corneal opacity time. P < 0.05 was defined as statistically significant and only significant probabilities are shown.
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Results |
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CD28 Pathway Modulation.
MST of allografts in CTLA4-Ig–treated BALB/c mice (n = 8) was 21 days, significantly prolonged compared with untreated (P < 0.003) and human IgG–treated recipients (n = 6, MST 12 days). MST in untreated CD28KO mice (n = 6) was 46 days, significantly longer than in wild-type mice (P < 0.001). In contrast, allograft survival in CTLA4-Ig–treated CD28KO mice (n = 6) was shortened (MST 16 days), consistent with blockade by CTLA4-Ig of negative signaling through CD152 (Fig. 2A) .
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CD28+CD40 Pathway Modulation.
Combined treatment with CTLA4-Ig and anti-CD154 antibody (n = 7, MST 29 days) significantly extended allograft survival compared with untreated BALB/c recipients (P < 0.0035), but not compared with treatment with each agent alone. Longer allograft survival was found in CD28KO mice treated with anti-CD154 (n = 6, MST 43 days) compared with untreated wild-type BALB/c mice (P < 0.001), but not compared with untreated CD28KO mice (Fig. 2C) .
Duration of Corneal Graft Opacity before Diagnosis of Rejection
The interval from initial detection of corneal opacity (grades 1, 2), until diagnosis of rejection (grades 3 or 4) varied between groups. Whereas in untreated allograft recipients this interval was 2.7 ± 0.3 days, it was significantly prolonged to 15.6 ± 5.8 days in animals with CTLA4-Ig treatment (P < 0.05), 11.9 ± 1.5 days with anti-CD154 Ab (P < 0.001), and 16.6 ± 3.1 days with CTLA4-Ig and anti-CD154 Ab (P < 0.006). In untreated CD28KO mice, this interval was 32.8 ± 8.8 days and in CD28KO mice with anti-CD154 antibody treatment, 41 ± 8.9 days. CTLA4-Ig–treated CD28KO mice had a corneal opacity time before diagnosis of rejection similar to those with untreated allografts (4.2 ± 0.9 days; P < 0.31 vs. allografts; Fig. 3 ).
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Mixed Lymphocyte Reaction
As expected, addition of CTLA4-Ig or anti-CD154 antibody showed a dose-dependent inhibition of proliferation of wild-type BALB/c but not CD28KO T cells. Inhibition was observed at concentrations ranging from 1 to 20 µg/mL CTLA4-Ig; equivalent concentrations of anti-CD154 antibody had less effect on proliferation (Fig. 4A 4B) . However, addition of CTLA4-Ig or anti-CD154 Ab did not alter the proliferation profile of CD28KO T-cells (Fig. 4C) . Human and hamster IgG had negligible effects.
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Discussion |
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We found a significant difference in allograft survival between CD28KO mice compared with wild-type BALB/c mice. Allograft survival in CD28KO recipients was longer than in CTLA4-Ig–treated mice, though only one cornea survived long-term. Variably extended survival of allografts of other tissues has been found in CD28KO mice: minimum prolongation of skin and longer survival of cardiac grafts. Long-term graft acceptance has not been reported.23 24 25 Corneal allograft rejection in CD28KO mice treated with CTLA4-Ig was accelerated compared with that in untreated CD28KO animals, in accordance with reported results in cardiac transplants.23 26 This is probably the result of blocking negative signaling interactions between CD80/86 and CD152 (CTLA4) on activated T cells. Alternatively, because CD152 is also expressed on regulatory T cells,27 28 it is possible that the molecule is interfering with regulatory pathways controlling T-cell activation. In either case, these data highlight that CD28- T cells are capable of being activated but that CD80/86 interactions with CD152 normally prevent this from happening. These T cells must be activated in a CD28-independent manner. It is of interest that in a rat model, we have found rejection of corneal allografts, even in the presence of blocking levels of CTLA4-Ig,13 indicating that other costimulatory pathways are operational in this model.
One of the most striking findings was the absence of additive effects in agents blocking CD40- and CD28-mediated costimulation. Synergy between these two reagents has been observed in several transplantation models and induced autoimmunity models.12 21 29 Of particular interest therefore was the in vitro correlate that, in contrast to the predicted effect on proliferation of BALB/c responders, CD154 blockade had no effect on proliferation of CD28KO responders to C3H stimulators in an MLR. Thus, at least with respect to proliferation, the CD40–CD154 system depends on a functioning CD28 costimulatory pathway, and cross-regulation of the B7h-inducible costimulator (ICOS) and CD40–CD154 signaling is one possible explanation. Because CD28 signaling is essential for optimal T-cell ICOS expression,30 31 this may be a functional phenotype of CD28 deficiency. No ICOS signaling is available to induce CD154 expression,32 33 with the result that anti-CD154 Ab has little effect on the MLR.
Another striking feature of this model is the shorter survival of corneal allografts after treatment with CTLA4-Ig and anti-CD154 Ab, compared with other organs in the same strain combination using the same treatment regimens. Strain variation in allograft MSTs after CD40–CD28 blockade has been demonstrated.34 However, the shorter survival of corneas may be explained in part by the sensitivity of corneal tissue. Diagnosis of corneal graft rejection is by direct visualization of reduced transparency. Clinical diagnosis in this way allows early rejection to be detected, but also less extensive rejection in which a minor degree of transparency remains, equating in this report to graft clarity grade 3 rather than 4. Accordingly, the corneal model allows variations in the extent of allogeneic injury to be observed. We have shown that rejection led to submaximum loss of transparency in the CD28KO graft recipient groups treated with CTLA4-Ig and anti-CD154, but not untreated BALB/c. In contrast to cornea, the clinical diagnosis of rejection in heart transplantation is defined as cessation of heartbeat, and in skin transplantation as complete loss of visible graft epidermal tissue. Allogeneic graft injury insufficient to cause total loss of graft function in such models may not be categorized as rejection and account for some of the apparent greater survival of grafts in those models after costimulatory blockade.
The finding of altered clinical phenotype of rejection after costimulatory blockade reported herein was accompanied by reduction in the number of graft-infiltrating CD45+ cells. At the time of rejection, there were significantly reduced numbers of CD45+ cells in treated compared with untreated BALB/c mice. The number of CD45+ cells was also reduced in allografts in CD28KO and anti-CD154 Ab–treated CD28KO. Further supporting the correlation of rejection phenotype with the number of graft-infiltrating CD45+ cells, rejected grafts in CTLA4-Ig treated CD28KO mice contained a number of cells similar to that in rejected grafts in untreated BALB/c mice. The low number of CD45+ cells may be due both to the blockade of costimulation resulting in failure of activation and the promotion of the apoptotic death of graft-infiltrating lymphocytes.20
Özkaynak et al.35 have shown recently that blocking the CD40–CD154 pathway with an anti-CD154 mAb alone prolongs cardiac allograft survival, but does not prevent the development of transplant arteriosclerosis, interstitial fibrosis, or focal myocyte necrosis. These outcomes may be analogous to histologic features in rejected corneas. In those specimens from CTLA4-Ig- and/or anti-CD154–treated recipients in which a low number of CD45+ cells was found, graft sections showed an increase in thickness and fibrosis with loss of keratocyte orientation, possibly equivalent to chronic graft rejection.36 It is of interest that Özkaynak et al.35 could prevent such fibrosis and transplant arteriosclerosis with the combined inhibition of the CD40–CD154 and ICOS pathways.
In recent years there has been considerable interest in using molecules to block interactions of CD40–CD154 and CD28–CD80/86, to prevent allograft rejection. Studies in primates have suggested that this approach has considerable promise for solid organ transplantation in humans. In most cases a short course of treatment is highly effective, although repeated doses enhance efficacy.21 In this study we have shown that, although blockade of these pathways at the time of transplantation is effective in extending graft survival, it does not abolish allograft rejection. We suggest that this is in part because the fragile nature of the cornea allows the earliest effects of rejection to be readily observed. Corneal allografts therefore represent a highly sensitive model in which to assess the effectiveness of costimulatory blockade in preventing any degree of allogeneic rejection.
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Footnotes |
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Submitted for publication January 27, 2003; revised March 26, 2003; accepted April 1, 2003.
Disclosure: N. Ardjomand, None; J.C. McAlister, None; N.J. Rogers, None; P.H. Tan, None; A.J.T. George, None; D.F.P. Larkin, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Daniel F. P. Larkin, Cornea and External Diseases Service, Moorfields Eye Hospital, City Road, London EC1V 2PD, UK; f.larkin{at}ucl.ac.uk.
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