Download Characterization of cell-mediated immune responses elicited

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