| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
METHODS. BALB/c mice were used as recipients of syngeneic, multiple minor
histocompatability (H)–disparate, or major histocompatibility complex
MHC-mismatched corneal transplants. Recipient beds were either
avascular (normal risk) or neovascularized (high risk). Mice were
randomized to receive either anti-CD154 antibody or control
immunoglobulin by intraperitoneal injection at surgery and once weekly
after surgery. After orthotopic corneal transplantation, all grafts
were evaluated for signs of rejection by slit lamp biomicroscopy over 8
weeks. The high-risk transplants were continuously observed until week
18 after the therapy was discontinued at week 8. Allospecific
delayed-type hypersensitivity (DTH) was evaluated after transplantation
in high-risk graft recipients. Frequency of interferon
(IFN)-
–secreting T cells in the hosts was measured by enzyme-linked
immunospot (ELISPOT) assay.
RESULTS. In normal-risk transplantation, the 8-week survival rate improved from
25% in control mice to 88% in anti-CD154–treated hosts of minor
H–disparate grafts (P = 0.0087) and from 78% in
control mice to 100% in anti-CD154–treated recipients of
MHC-mismatched transplants (P = 0.177). Of
particular significance, in high-risk transplantation, anti-CD154
therapy dramatically enhanced the survival of both minor H– and
MHC-disparate corneal transplants to 100% (P =
0.0001) and 92% (P = 0.0002), respectively. In
addition, the anti-CD154–treated mice did not exhibit allospecific
immunity. However, termination of anti-CD154 led to some loss in graft
survival, especially among high-risk minor H–disparate grafts. The
frequency of IFN-–producing T cells was significantly reduced in
anti-CD154–treated hosts.
CONCLUSIONS. Continuous suppression of the CD40-CD154 costimulatory pathway promotes the acceptance of corneal transplants, regardless of the degree of allodisparity or preoperative risk. The beneficial effect of anti-CD154 treatment may be due in part to inhibition of Th1-mediated responses.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CD154, also known as CD40 ligand (CD40L), is a 39-kDa type II membrane glycoprotein and member of the tumor necrosis factor (TNF) superfamily. It is preferentially expressed on activated CD4+ cells and mast cells. The counterreceptor for this ligand, CD40, is a 50-kDa integral membrane glycoprotein. It is found on a variety of antigen-presenting and mesenchymal cells, including B lymphocytes, dendritic cells, macrophages, monocytes, microglia, endothelial cells, and epithelial cells.3 4 5 Over the past several years, the central role of CD40-CD154 interaction in mediating T-cell–mediated immune responses has been firmly established.3 CD154-dependent activation of T cells occurs through signaling of CD40 at the level of antigen-presenting cells (APCs), which enhances requisite costimulatory pathways, including expression of B7.1/B7.2 (CD80/CD86).6 Ligation of CD40 on APCs also triggers production of high levels of interleukin (IL)-12,7 which is a potent stimulus for T-helper 1 (Th1) differentiation. Accordingly, it has been shown that blockade of the CD40-CD154 pathway is sufficient to induce tolerance to Th1-mediated contact hypersensitivity, and this is associated with inhibition of IL-12 mRNA expression and Th2 immune deviation.8
These data suggest that the CD40-CD154 pathway may serve as an ideal
candidate for molecular intervention for immunomodulatory therapy in
transplantation. In fact, consistent with its central role in
cell-mediated immunity, blockade of CD40-CD154 interaction by
anti-CD154 has been shown to prevent rejection of solid organ
allografts, such as cardiac, renal, pancreatic islet, and skin
grafts.9
10
11
However, the effect of blocking the
CD40-CD154 pathway on the fate of corneal transplants has not been
determined. In this series of experiments, we studied the effect of
systemic anti-CD154 monoclonal antibody (mAb) on the outcomes of
normal-risk and high-risk allogeneic corneal grafts and explored
whether this treatment can affect Th1-mediated allospecific
delayed-type hypersensitivity (DTH) responses and the frequency of
interferon (IFN)-–producing T cells in the spleen and draining
lymph nodes after transplantation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Anti-CD154 Administration
Hamster anti-CD154 mAb IgG was purified from culture supernatant
of MR1 hybridoma (American Type Culture Collection, Rockville, MD) by
using a protein A fast-flow column and was kindly provided by Linda
Burkly (Biogen, Cambridge, MA). Administration of anti-CD154 does not
functionally delete antigen-specific Th cells in mice.12
BALB/c mice were randomly selected to receive, in a masked fashion,
either anti-CD154 or control hamster IgG immunoglobulin (Ig; Biogen) at
a dose of 250 µg/mouse by intraperitoneal injection. Because studies
of other solid organ transplantations in animal models have reported
that extended treatment with anti-CD154 is more effective in preventing
graft rejection than is short-term treatment,11
13
14
a
sustained regimen of antibody therapy was applied in our studies on
days -1, 0, and 1 and once weekly. The treatment was terminated at
week 8, a time point at which donor-specific anterior
chamber–associated immune deviation (ACAID) normally develops in hosts
bearing accepted grafts, thereby sustaining long-term graft
acceptance.15
Induction of CNV and High-Risk Graft Beds
Suture-induced corneal neovascularization (CNV) is a
standardized system of inducing neovascularization to create high-risk
graft beds.16
Three interrupted sutures (11-0 nylon,
50-µm diameter needle; Sharpoint; Vanguard, Houston, TX) were placed
in the central cornea of one eye of normal BALB/c mice. As described
previously, neovascular growth into the normally avascular corneal
stroma can be appreciated from the limbus as early as 3 days after
suture placement. Neovessels occupy more than two quadrants of the
central cornea after 14 days. These mice with neovascularized graft
beds therefore served as high-risk recipients of orthotopic corneal
transplants.
Corneal Transplantation
BALB/c mice (n = 106) were used as recipients of
syngeneic (BALB/c, n = 10), minor histocompatibility
(H)–disparate (B10.D2, n = 48), or major histocompatibility
complex (MHC)–mismatched (BALB.b, n = 48) corneal
transplants. Mice bearing syngeneic grafts received anti-CD154
treatment. All other hosts were randomized to receive either anti-CD154
or control hamster Ig. Syngeneic grafts were transplanted to avascular
(normal-risk) recipient beds; minor H–disparate and MHC-disparate
grafts were transplanted to either avascular or neovascularized
(high-risk) beds. Corneal transplantation was performed according to
our well-established protocol.17
Briefly on day 0, the
central 2-mm area of the donor cornea was excised with Vannas scissors
and secured in the host graft bed of 1.8-mm diameter with eight
interrupted 11-0 nylon sutures (Sharpoint, Vanguard). Antibiotic
ointment was applied to the corneal surface, and the eyelids were
closed for 3 days with a tarsorrhaphy using 8-0 nylon sutures. All
grafted eyes were examined 3 days after surgery, and transplant sutures
were removed in all mice on day 7.
Evaluation and Scoring of Orthotopic Corneal Transplant Rejection
All grafts were evaluated in a masked fashion for the signs of
rejection by slit lamp biomicroscopy twice weekly over 8 weeks. The
high-risk transplants were continuously observed until week 18 after
the therapy was discontinued at week 8. At each time point, the grafts
were scored for opacity. A previously defined and standardized scoring
system was used to grade the degree of opacification from 0 to 5+ (0,
clear graft; 1, minimal superficial opacity; 2+, mild stromal opacity
with pupil margin and iris vessels visible; 3+, moderate stromal
opacity with only pupil margin visible; 4+, intense stromal opacity
with the anterior chamber visible; and 5+, maximal corneal opacity with
total obscuration of the anterior chamber).17
Grafts with
an opacity score of 2+ or higher after 3 weeks were considered to be
rejected, and grafts with an opacity score of 3+ or higher at 2 weeks
that never cleared were also regarded as rejected.17
Assessment of Donor-Specific DTH
Based on the finding that donor-specific DTH can be detected in
high-risk recipients as early as 2 to 3 weeks after
surgery,18
19
we evaluated allospecific DTH responses in
high-risk hosts (n = 5/group) bearing minor H–mismatched or
MHC-disparate corneal grafts treated with anti-CD154, 2 to 3 weeks
after transplantation. The regimen of anti-CD154 or hamster control Ig
treatment was the same as the aforementioned except that antibody
administration was terminated 1 week before ear challenge. Irradiated
(2000 rad) splenocytes (1 x 106) in 10 µl
of Hanks’ balanced salt solution from BALB.b donors syngeneic with the
corneal graft were injected into the right pinnae, as described
previously.18
BALB/c mice serving as positive controls
were immunized by subcutaneous injection of 10 x
106 BALB.b splenocytes 1 week before ear
challenge. BALB/c mice serving as negative controls were challenged
only with splenocytes but without prior immunization. At 24 and 48
hours after ear challenge, ear swelling was measured in a masked
fashion with a low-pressure micrometer (Mitutoyo; MTI, Paramus, NJ).
Ear-swelling responses are presented as mean ± SE. Because
results at 24 and 48 hours were similar, only 24-hour data are
presented.
ELISPOT Cytokine Measurement
The enzyme-linked immunospot (ELISPOT) assay was performed 2
weeks after high-risk transplantation of fully disparate corneal
grafts. ELISPOT plates (Polyfiltronics; Rockland, MA) were coated with
4 µg/ml of rat anti-mouse IFN- (R4-6 A2) capturing mAb
(PharMingen, San Diego, CA) in sterile phosphate-buffered saline (PBS)
overnight. The plates were then blocked for 1.5 hours with sterile PBS
containing 1% bovine serum albumin (BSA) and washed with sterile PBS.
Responder cells, either 8 x 105 draining
lymph node cells or 0.5 to 1 x 106 spleen
cells, were then placed in each well in the presence or absence of
mitomycin C–treated allogeneic splenocytes (as stimulator APCs; ratio
1:1). The cells were cultured at 37°C in 5%
CO2 for 42 hours. For detection of spots, 2
µg/ml biotinylated rat anti-mouse IFN- (XMG 1.2) mAb was used,
followed by 1.5 hours of incubation with streptavidin D horseradish
peroxidase (Vector, Burlingame, CA) diluted at 1:2000 in PBS with
0.025% Tween. All mAbs were purchased from PharMingen. After they were
washed, the plates were developed using 0.8 ml of
3-amino-9-ethylcarbazole (AEC; Sigma, St. Louis, MO; 10 mg dissolved in
1 ml dimethylformamide) mixed with 24 ml of 0.1 M sodium acetate (pH
5.0), containing 12 µl
H2O2. The resultant spots
were counted on a computer-assisted ELISPOT image analyzer (T Spot;
Cellular Technology, Cleveland, OH).20
Statistical Analysis
The rates of corneal graft survival were plotted as
Kaplan–Meier survival curves and compared by using the log rank
(Mantel–Cox) test. DTH data are presented as mean ± SE and were
compared by using analysis of variance (ANOVA). Statistical
significance was defined as a P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Fate of Normal-Risk Corneal Grafts
Minor H–disparate grafts treated with hamster Ig exhibited
vigorous and prompt rejection (Fig. 1)
, beginning as early as 17 days after transplantation. The cumulative
survival rates of this group at 4 and 8 weeks were 64% and 25%,
respectively. In contrast, minor H–disparate grafts treated with
anti-CD154 displayed significantly increased survival. The only
allograft rejection in this group occurred on day 35. The cumulative
survival rates in this group at 4 and 8 weeks were 100% and 87%,
respectively—significantly higher than rates seen among the hamster
mAb–treated control mice (P = 0.0087). The data
demonstrate that systemic anti-CD154 administration can profoundly
reduce the rejection rate of minor H–disparate corneal allografts.
|
|
|
|
|
|
|
Profile of Corneal Graft Recipients in Response to Donor
Alloantigen–producing alloreactive T cells in the cervical
draining lymph nodes and in the spleen 2 weeks after corneal
transplantation. As shown in Figure 8A
, the frequencies of alloreactive IFN-–producing recipient cells in
the draining lymph nodes was dramatically lower in anti-CD154–treated
hosts than in hamster Ig–treated hosts. Similar result was also
obtained for the splenocytes (Fig. 8B)
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Anti-CD154 monotherapy has been shown to effectively prevent acute rejection of cardiac, renal, and pancreatic islet allografts in both primates and rodents.9 10 11 12 Our current findings in corneal transplants are in agreement with these studies. The reported timing and duration of anti-CD154 administration in the previously reported studies is variable, ranging from only once at surgery to once or twice weekly for 2 to 7 weeks followed by once monthly for maintenance. In the aggregate, it appears that the timing of initial administration is critical. When treatment is delayed until 5 days after surgery in the mouse model of cardiac transplantation, no prolongation of graft survival has been observed.10 Moreover, an extended regimen is significantly more effective in preventing graft rejection than brief (less than 2 weeks) perioperative treatment. For example, a preliminary study has shown that administration of anti-CD154 only during the first postoperative week delays corneal graft rejection but improves overall graft survival only minimally.22 The disparity between the results of this report and ours is probably due to the different regimens used. Because sensitization to corneal allografts occurs within the first several weeks after transplantation,18 19 coverage of the host with anti-CD154 at least during this period appears to be critical in promoting graft success.
The exact mechanism of action of anti-CD154 monotherapy in the prevention of corneal allograft rejection remains to be fully elucidated. Our findings indicate that the Th1-mediated allospecific DTH response is suppressed in animals receiving anti-CD154 treatment. To distinguish whether anti-CD154 therapy suppresses the induction or expression of the DTH response, we have evaluated the effect of anti-CD154 on allospecific DTH response in BALB/c mice presensitized by subcutaneous injection of B6 spleen cells. The mice were given either anti-CD154 antibody or hamster Ig 1 week after immunization and 1 week before ear challenge. We found that anti-CD154 antibody–treated mice mounted a strong DTH response that was comparable to that in hamster Ig–treated mice as well as that in DTH-positive control animals. Thus, the data suggest that anti-CD154 treatment is effective through its inhibitory function on induction of the DTH response. Because alloreactive responses of the DTH type correlate strongly with graft rejection, as reported by many laboratories,17 23 24 25 anti-CD154 may therefore prolong corneal graft survival through suppression of Th1-type sensitization.
In addition to testing for the effect of anti-CD154 therapy on
induction of allospecific DTH, we additionally evaluated the effect of
such treatment on the frequency of IFN-–producing T cells in the
hosts. Consistent with the DTH data, we observed a significant
reduction of IFN-–secreting Th1 cells in hosts of grafts receiving
anti-CD154 treatment. Similar to our data, it has been shown that the
profile of cytokine expression in murine cardiac allografts changes
from a Th1-biased (IFN- and IL-2) to a Th2-biased (IL-4 and IL-10)
pattern in anti-CD154–treated animals.26
Although our
data in regard to CD40-CD154 blockade and suppression of the IFN-
response support the in vivo observation of suppressed DTH, we cannot
at this point comment on the Th2 cytokine response. Because deviation
away from a Th1 response in the corneally grafted host prevents corneal
graft rejection,27
it would be of additional interest to
examine the Th2 cytokine response in these anti-CD154–treated hosts,
or to block Th2 cytokine activity, such as IL-4 or IL-10, to observe
whether anti-CD154 treatment is still effective in preventing graft
rejection.
Although anti-CD154 suppresses the induction of allosensitization, studies suggest that inhibition of allosensitization alone may not be sufficient to maintain long-term graft acceptance in high-risk eyes.28 In fact, contrary to studies on renal and pancreatic islet transplantation in which long-term graft survival has been reported even after cessation of anti-CD154 therapy,9 11 40% of the accepted high-risk grafts in our study were rejected 4 weeks after withdrawal of therapy among the minor H–disparate grafts. The reasons for this observation remain unclear. In some CD4+-deficient mice, fully mismatched corneal allografts are rejected within 10 weeks of engraftment29 or undergo delayed rejection after long-term acceptance.30 The CD4+ T-cell knockout mice that have rejected allogeneic grafts do not generate significant DTH responses,29 suggesting a mechanism involving CD8+ T cells. Based on these findings, it is possible that donor-specific CD8+ T cells will eventually emerge as effectors of rejection of corneal allografts. Of note, unlike our results with minor H–disparate high-risk grafts, the majority of high-risk MHC-disparate transplants enjoyed prolonged acceptance, even after the therapy was withdrawn. This may be due to the less critical role of MHC disparity in corneal allograft rejection.21
It is important to emphasize that we cannot state with certainty whether the prolongation of graft survival by the treatment was due entirely to suppressed allosensitization or is also due to early acquisition of allospecific tolerance. Previous reports indicate that mice bearing accepted corneal allografts for 8 weeks or more show development of allospecific ACAID,31 which is one form of tolerance. We have found that anti-CD154–treated acceptors of high-risk grafts acquire ACAID to donor alloantigens at week 18, whereas hamster Ig–treated control mice universally do not to acquire ACAID at the same time point (data not shown). However, we cannot address at this time whether blockade of the CD40-CD154 costimulatory pathway induces early development of ACAID, even before 8 weeks after surgery.
The currently available preventive regimens for corneal transplant rejection are associated with significant complications, and it thus would be desirable to devise intervention strategies that can prolong graft survival by specifically targeting molecular mediators involved in generation of the alloimmune response. A variety of successful experimental strategies have been developed including induction of tolerance to donor corneal cells,32 macrophage depletion,33 deviation of recipient immune systems toward Th2 response,27 and intervention with the function or expression of adhesion molecules, cytokines, or T cells.34 35 36 37
Although further studies are needed to determine whether treatment with anti-CD154 can alter induction of tolerance to transplantation antigens and whether local (ocular) application of anti-CD154 can similarly promote allograft survival, our data demonstrating near-universal graft acceptance regardless of the degree of allodisparity or risk indicate that blockade of the CD40-CD154 costimulatory pathway holds significant promise as an effective modality for promoting corneal transplant survival. However, because it is suspected that CD8+ T cells may mediate delayed corneal allograft rejection, anti-CD154 therapy may not prevent delayed corneal rejection, because CD154 is not an important costimulatory molecule for CD8+ T cell activation.36 39 Accordingly, the long-term prevention of graft rejection may require alternative strategies after cessation of anti-CD154 therapy.
|
Footnotes |
|---|
Submitted for publication July 28, 2000; revised December 4, 2000; accepted December 20, 2000.
Commercial relationships policy: C (MRD), P (MRD), N (all others).
Corresponding author: M. Reza Dana, Laboratory of Immunology, Schepens Eye Research Institute, Harvard Medical School, 20 Staniford Street, Boston, MA 02114. dana{at}vision.eri.harvard.edu
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  L. Chen, S. Huq, H. Gardner, A. R. de Fougerolles, S. Barabino, and M. R. Dana Very Late Antigen 1 Blockade Markedly Promotes Survival of Corneal Allografts Arch Ophthalmol, June 1, 2007; 125(6): 783 - 788. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Watson, A. J. T. George, and D. F. P. Larkin Differential effects of costimulatory pathway modulation on corneal allograft survival. Invest. Ophthalmol. Vis. Sci., August 1, 2006; 47(8): 3417 - 3422. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Huq, Y. Liu, G. Benichou, and M. R. Dana Relevance of the Direct Pathway of Sensitization in Corneal Transplantation Is Dictated by the Graft Bed Microenvironment J. Immunol., October 1, 2004; 173(7): 4464 - 4469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Dana Corneal Antigen-Presenting Cells: Diversity, Plasticity, and Disguise The Cogan Lecture Invest. Ophthalmol. Vis. Sci., March 1, 2004; 45(3): 722 - 727. [Full Text] [PDF]
|
|
|
|
|
|
J. Yamada, K. Maruyama, Y. Sano, S. Kinoshita, Y. Murata, and J. Hamuro Promotion of Corneal Allograft Survival by the Induction of Oxidative Macrophages Invest. Ophthalmol. Vis. Sci., February 1, 2004; 45(2): 448 - 454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Ardjomand, J. C. McAlister, N. J. Rogers, P. H. Tan, A. J. T. George, and D. F. P. Larkin Modulation of Costimulation by CD28 and CD154 Alters the Kinetics and Cellular Characteristics of Corneal Allograft Rejection Invest. Ophthalmol. Vis. Sci., September 1, 2003; 44(9): 3899 - 3905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Silverman, D. O. Zamora, Y. Pan, P. V. Texeira, S.-H. Baek, S. R. Planck, and J. T. Rosenbaum Constitutive and Inflammatory Mediator-Regulated Fractalkine Expression in Human Ocular Tissues and Cultured Cells Invest. Ophthalmol. Vis. Sci., April 1, 2003; 44(4): 1608 - 1615. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
