| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Cellular and Molecular Immunology Laboratory, Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, MA 02114
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-producing CD8+ T cells
recognizing donor MHC in a direct fashion. Surprisingly,
CD8+ T cells activated directly were not required for graft
rejection. In nonrejecting mice, no T cell responses were detected.
Strikingly, peripheral sensitization to allogeneic MHC molecules in
these mice induced acute rejection of corneal grafts. We conclude that
only CD4+ T cells activated via indirect allorecognition
have the ability to reject allogeneic corneal grafts. Although
alloreactive CD8+ T cells are activated via the direct
pathway, they are not fully competent and cannot contribute to the
rejection unless they receive an additional signal provided by
professional APCs in the periphery. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ag presentation by both recipient- and donor-derived APCs contributes to T cell alloresponse and initiation of graft rejection. Indeed, alloantigen recognition occurs via two distinct mechanisms, referred to as the direct and indirect pathways. The direct alloresponse results from the stimulation of T cells by intact allogeneic MHC molecules displayed on donor cells. This response is dictated by the migration of donor passenger leukocytes out of the graft to the host’s lymphoid tissues (11, 12, 13). Additionally, it has been demonstrated that donor Ags are presented in peptide form during acute graft rejection (14, 15, 16). The T cell recognition of processed alloantigens in association with self-MHC molecules at the surface of recipient APCs has been called indirect allorecognition. While the direct response is polyclonal, the indirect response is oligoclonal and usually restricted to the recognition of a few immunodominant donor-derived peptides (17). Based upon this and despite the presence of both types of alloresponses at early stages after transplantation, a direct alloresponse is often viewed as the only driving force in acute rejection. In turn, presentation of alloantigens by recipient MHC class II+ APCs is thought to play a critical role in late acute and chronic graft rejection processes (18, 19, 20).
Under certain circumstances, the contribution of the direct response to acute rejection has been shown to be minimal. Abrogation of the direct CD4+ T response has been achieved by depletion of donor MHC class II+ cells from grafted tissues or by treatment of recipient with immunosuppressive drugs. In these situations, graft rejection is presumably initiated by the indirect response. In support of this view, Auchincloss et al. (21) have reported that the CD4+ T cell-mediated indirect response alone could trigger the rejection of skin grafts from MHC class II-deficient mice. It is noteworthy that in this model, anti-donor cytolytic activity mediated by CD8+ T cells was thought to be the effector mechanism (22, 23). Other lines of evidence have been provided suggesting that in the absence of a direct pathway, an indirect alloresponse is sufficient to ensure rejection of allografts (5, 24). Valujskikh et al. (24) have reported that reconstitution of SCID mice with a CD4+ T cell line specific for a single donor MHC class II peptide is sufficient to ensure the rejection of skin allografts. Furthermore, nude mice bearing an MHC class I-disparate skin graft have been shown to reject their graft after adoptive transfer of CD4+ T cells (5). However, in many instances, efficient destruction of MHC class I-disparate allografts requires the presence of a CD8+ T cell-mediated direct recognition of the alloantigens (25).
Recent studies using genetically engineered mice have shed some light on the roles of direct and indirect allorecognition in graft rejection. However, the exact contribution of each of these pathways to the alloresponse and the rejection process in normal individuals remains unclear. Corneal transplantation represents a useful model to address this issue. Corneal allografts enjoy high rates of spontaneous acceptance compared with other types of transplantation (40–50%) (26). This feature is particularly helpful to analyze the mechanisms underlying the graft rejection process. Convergent studies have shown that CD4+ T cells play an essential role in the rejection of orthotopic corneal allografts (7, 27, 28). In this regard, corneal transplantation does not differ from other types of tissue transplantation (6). In turn, a number of characteristics clearly distinguish corneal transplantation from other models (29): 1) normal corneas are devoid of MHC class II+ APCs (30); 2) the cornea expresses low amounts of MHC class I molecules (31); and, 3) minor histocompatibility Ags are more potent than MHC Ags in mediating corneal allograft rejection (32). Based upon these observations, it has been proposed that the indirect alloresponse represents the driving force in corneal graft rejection, while the direct response does not occur (33). However, the precise nature of the T cell alloresponse and the actual contributions of direct and indirect pathways in corneal transplantation remain to be determined.
Here, we have used the sensitive ELISPOT technique to characterize the T cell alloresponse in a murine model of fully allogeneic corneal transplantation. We demonstrate that in the absence of MHC class II+ cells within the graft, allorecognition is mediated by two distinct T cell subsets that differ by their Ag specificities, cytokine profiles, and pathways of Ag recognition (direct vs indirect pathways). The implication of this finding in understanding the mechanisms underlying T cell allorecognition in vivo and its relationship to alloresponse and allograft rejection are discussed.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Six- to 8-wk-old female BALB/c (H-2d), B10.D2/nSnJ (H-2d), C57BL/10 (H-2b), C57BL/6 (H-2b), and BALB.B (C.B10-H2b; H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). They were maintained in our pathogen-free facility at the Schepens Eye Research Institute and treated according to the Association for Research in Vision and Ophthalmology resolution.
Orthotopic corneal transplantation and scoring
Donor central corneas were marked with a 2-mm diameter microcurette, excised by vannas scissors, and placed in PBS. Recipients were anesthetized by i.p. injection of ketamine and xylazine, and the right eye was excised from a 1.5-mm diameter piece in the central cornea to prepare the graft bed. The donor cornea was placed in the recipient bed and secured with interrupted 11–0 nylon sutures (Sharpoint, Vanguard, Houston, TX). After application of antibiotic ointment, the eye lids were closed for 3 days. Seven days later, the sutures were removed. The degree of opacity as well as the degree of neovascularization were assessed using a slitlamp biomicroscopy as previously described (34). Briefly, the opacification of the graft was quantified using a scoring opacity scale from 0 to 5+. The cornea was considered in a rejecting phase for a score equal to or greater than 2+.
Skin grafts
Tail to back allografts were performed according to the technique previously described by Billingham and Medawar (35). Tail was harvested from euthanized donor mice and placed in a graft bed prepared on the left side of a recipient mouse previously anesthetized with a cocktail of ketamine and xylazine. The graft was secured using Vaseline gauze and a bandage, which were removed 9 days later.
Preparation of responder cells
Spleen cells from naive, cornea-grafted, as well as skin-grafted BALB/c mice were used as a source of responder cells to measure the total alloresponse and the direct and indirect responses. RBC were lysed for 2 min in Tris-NH4Cl solution. Spleen cells were washed twice in AIM-V (Life Technologies, Grand Island, NY) medium containing 0.5% FCS and resuspended at 107 cells/ml with 0.5% FCS in AIM-V for further use.
T cells and T cell subsets isolation
T cells as well as CD4+ or CD8+ T cell subsets were isolated from mouse spleen cells by negative selection using commercially available T cell purification columns according to the manufacturer’s instructions (Accurate Chemical and Scientific, Westbury, NY; R&D Systems, Minneapolis, MN). Purified T cells were washed in HBSS and used at 5 x 105 cells/well in ELISPOT assays.
Preparation of APC
Mitomycin C (MMC)3-treated splenocytes from donor and recipient naive mice were used as allogeneic stimulator cells or syngeneic APCs, respectively. Single-cell suspensions of splenocytes devoid of RBC were prepared in AIM-V containing 0.5% FCS and treated with MMC (50 µg/ml) for 30 min at 37°C. The cells were washed once in HBSS, incubated for 10 min at 37°C, and washed once again and finally resuspended in AIM-V/0.5% FCS at 1–3 x 107 cells/ml.
Preparation of sonicates
Stimulator spleen cells were suspended at 3 x 107 cells/ml in AIM-V containing 0.5% FCS and sonicated with 10 pulses of 1 s each. The resulting suspension was frozen in a dry ice/ethanol bath, thawed at room temperature, and centrifuged at 1200 rpm for 10 min to remove intact cells.
ELISPOT assays
Ninety-six-well ELISPOT plates (Polyfiltronics, Rockland, MA)
were coated with a capture mAb in sterile PBS overnight. Anti-IL-2,
-IFN-, -IL-4, and -IL-5 capture mAb were used at 3, 4, 2, and 5
µg/ml, respectively (PharMingen, San Diego, CA). On the day of the
experiment, the plates were washed twice with sterile PBS, blocked for
1.5 h with PBS containing 1% BSA, then washed three times with
sterile PBS. Responder cells or purified T cells were added to wells
previously filled with intact donor cells (direct response) or
syngeneic APCs together with donor sonicates (indirect response) as
previously described (36). Cells were incubated for
different periods of time depending on the cytokine measurement:
20 h for IL-2, 42 h for IFN- and IL-4, and 48 h for
IL-5. The plates were washed three times with PBS, then four times with
PBS containing 0.025% Tween. Biotinylated anti-lymphokine
detection mAbs were added at 2 µg/ml (PharMingen) and incubated
either for 5 h at room temperature or overnight at 4°C. After
washing three times with PBS containing 0.025% Tween,
avidin-horseradish peroxidase (1/2000) was added to each well for
1.5 h. Four washes with PBS were performed before the spots were
revealed by the addition of the developing solution composed of 800
µl of 3-amino-9-ethylcarbazole (Sigma; 10 mg dissolved in 1 ml
dimethylformamide) in 24 ml 0.1 M sodium acetate, pH 5.0, catalyzed by
12 µl H2O2. The resulting
spots were counted and analyzed on a computer-assisted ELISPOT image
analyzer (C.T.L., Cleveland, OH).
In vitro treatment with anti-CD4 and anti-CD8 mAb
Commercially available rat anti-mouse CD4 (GK1.5) and CD8 (53-6.72) mAbs were used in in vitro blockade experiments at 10 µg/ml (PharMingen).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although an allospecific delayed-type hypersensitivity (DTH)
response has been reported after allogeneic corneal transplantation
(7, 33, 34, 37), the precise nature of the T cells
activated in this model remains unclear. Here, we first investigated
the pattern of cytokines produced by T cells after allogeneic corneal
transplantation in mice. The total T cell response toward donor
alloantigens was analyzed in rejecting and nonrejecting BALB/c mice
grafted with fully allogeneic C57BL/6 (B6) corneas. In this
combination, 60% of the corneal allografts were rejected 4 wk after
transplantation. The rejection was estimated on the basis of the graft
opacity from 2 to 5 wk post-transplantation. The frequency of IL-2-,
IFN--, IL-4-, and IL-5-producing splenocytes activated in response
to allogeneic stimulation was measured in all cornea-transplanted mice.
To test this, recipient splenocytes (BALB/c) were cultured in vitro
with MMC-treated donor splenocytes (B6), a situation in which T cells
can be activated via both direct and indirect allorecognition pathways.
After 20–48 h (depending on the cytokine tested) the frequency of type
1 and 2 cytokine-producing cells was determined using the ELISPOT
technique as previously described (36). As expected, a
primary MLR, mainly characterized by the activation of IL-2 and IL-4
producers, was detected when lymphocytes from naive mice stimulated in
vitro with allogeneic cells (Fig. 1
). The
number of IFN-- and IL-2-producing cells was significantly increased
in mice undergoing corneal graft rejection compared with
nontransplanted animals. No IL-5 and a few IL-4-producing cells were
detected in the same conditions. Strikingly, in nonrejecting mice, the
frequencies of cytokine producers were equivalent to those found in
naive animals (data not shown). These results demonstrate that corneal
transplantation elicits a vigorous type 1 response in recipients that
is associated with the rejection.
|
cytokines are produced by two distinct T cell
subsets
Next, we investigated the contribution of
CD4+ and CD8+ T cells to
the cytokine production in mice undergoing corneal graft rejection. We
performed in vitro blockade experiments with mAbs against CD4 and CD8
molecules (Fig. 2). Activation of
IL-2-producing T cells during primary MLR was abrogated by anti-CD4
mAb. In addition, IL-2 release by activated alloreactive T cells
harvested from mice undergoing corneal transplant rejection was
significantly inhibited by in vitro treatment with anti-CD4, but
not anti-CD8 mAb. Conversely, the frequency of alloreactive
IFN--producing cells found in these mice was markedly decreased
after addition of anti-CD8 mAb. In turn, anti-CD4 mAb treatment
had negligible influence on IFN- production. We conclude that the T
cell response to corneal allografts is mediated by two phenotypically
and functionally distinct T cell subsets: CD4+ T
cells secreting IL-2 and CD8+ T cells producing
IFN-.
|
Traditionally, the immune response to allogeneic transplants is
initiated by the activation of MHC class II-restricted T cells. In
corneal transplantation, since MHC class II molecules have not been
detected in corneal tissues, it has been proposed that the
CD4+ T cell alloresponse is mediated exclusively
toward donor peptides presented by MHC class II+
recipient APCs, i.e., through the indirect pathway. However, no direct
evidence has been provided in support of this hypothesis. To address
this question, we measured the frequency of cytokine-producing T cells
activated through the indirect pathway in cornea-grafted mice using the
ELISA spot technique. This technique has been applied recently to
measure the indirect response in skin-grafted mice (36).
To detect indirect response in cornea-transplanted mice, T cells from
rejector animals were cultured with syngeneic APCs and donor sonicates.
As shown in Fig. 3A, a potent
indirect response was triggered in cornea-grafted mice. This response
was mediated predominantly by IL-2-producing cells, while a significant
number of IL-4-producers were also observed. We surmise that these
IL-4-producing CD4+ T cells may play an essential
role in the induction of DTH response after corneal transplantation, a
possibility supported by a recent study from Heeger’s laboratory
(38). The frequency of T cells producing IL-2 through the
indirect pathway of allorecognition averaged 8 x
10-5 (Fig. 3B). In contrast, the
number of IFN- producers was not significantly different in grafted
vs nongrafted mice (Fig. 3A), a result suggesting that only
CD4+ T cells were activated via indirect
allorecognition.
|
, the activation of IL-2-producing T
cells was blocked by anti-CD4 mAb, while treatment with
anti-CD8 mAb had marginal effect (Fig. 4A). To further
demonstrate that CD4+ T cells were activated
through the indirect pathway, the CD4+ T cell
population was purified from graft-rejecting mice and restimulated in
vitro with recipient APCs and donor sonicates. As shown in Fig. 4B, purified CD4+ T cells harvested
from cornea-grafted mice elicited a potent indirect response. Together,
these data show that CD4+ T cells producing IL-2
cytokine mediate indirect recognition of alloantigens in corneal
transplantation.
|
Both donor MHC and minor histocompatibility protein Ags are
potential sources of peptides in indirect alloresponses. Some evidence
suggests that in skin and heart transplantation models, the majority of
T cells activated via indirect allorecognition recognize processed
allo-MHC proteins. Here, since minor Ag-mismatched corneas are more
readily rejected than their MHC-disparate counterparts, it was crucial
to determine whether the same rules of immunodominance apply to
cornea-grafted mice. To test this, we compared indirect T cell
responses in MHC-disparate and fully allogeneic corneal transplant
models. BALB/c mice were grafted either with C57BL/10 corneas (MHC- and
minor histocompatibility-mismatched) or BALB.B corneas
(MHC-mismatched). Mice undergoing rejection were selected (60 and 20%
rejection rates, respectively). Recipient splenocytes were restimulated
with syngeneic APCs and BALB.B sonicates, and the frequency of
IL-2-producing cells activated against donor MHC
H-2b-derived Ags was measured. As shown in Fig. 5, we did not detect any indirect
response to allogeneic MHC molecules in mice grafted with fully
disparate or MHC-mismatched corneas. In contrast, vigorous indirect
responses to allo-MHC were found in the same donor-recipient mouse
combinations after skin grafting. We conclude that the indirect
recognition of MHC Ags by CD4+ T cells does not
contribute to corneal graft specific alloimmunity. It is noteworthy
that in mice rejecting MHC-mismatched corneas, the frequency of IL-2
producers was comparable to that recorded in naive mice, while some
alloreactive IFN--producing cells were detected in some recipient
mice only. Therefore, we cannot exclude that some T cell-independent
mechanisms may be involved in the rejection process.
|
Within days after transplantation, graft bone marrow-derived APCs
(B cells, dendritic cells, and macrophages) migrate to the host’s
lymphoid organs. Presentation of intact allogeneic MHC molecules on
these so-called passenger leukocytes is known to trigger a powerful
direct T cell response that plays a critical role in the rejection of
all forms of tissue allografts. Corneal transplantation may not follow
this rule, since at the time of transplantation corneal grafts are
devoid of professional APCs. Based upon this, it has been postulated
that these grafts cannot activate T cells through the direct pathway.
Here, we investigated whether a direct response occurs after fully
allogeneic corneal transplantation. To address this question, the T
cell response to intact donor cells was measured. Recipient T cells
were purified using negative selection columns and were mixed with
MMC-treated donor splenocytes. As expected, some IL-2 and IFN-
producers were activated via primary MLR. Strikingly, we detected very
high numbers of IFN--producing activated cells in mice experiencing
corneal graft rejection (Fig. 6). Indeed,
while some IL-2-producing cells were detected in the assay shown in
Fig. 6, this response was extremely low compared with that observed in
skin-grafted mice. In addition, we found that these few alloreactive
IL-2-producing cells were resistant to in vitro treatment with
anti-CD4 mAb in cornea-grafted mice (Fig. 2
; while in vitro
treatment with anti-CD4 mAb completely abolished the primary MLR
observed in naive mice). These observations suggest that a few T cells
other than CD4+ T cells might produce some IL-2.
The frequency of alloreactive IFN--producing T cells averaged
10-3 in cornea-grafted mice vs
10-4 in naive animals. To determine the
phenotype of these IFN--producing cells,
CD4+ and CD8+ T cells were
purified and tested for their ability to produce IFN- after
stimulation with intact donor cells. As shown in Fig. 7, CD8+ but not
CD4+ T cells secreted IFN- through the direct
pathway of allorecognition. To confirm that CD4+
T cells were not activated directly, purified
CD4+ T cells were cultured with intact donor
cells, and the frequency of IL-2-secreting cells was measured. We
found that the frequency of IL-2-secreting cells was identical in
cornea-grafted mice and naive animals (Fig. 8). This confirmed that
CD4+ T cells are not activated through the direct
pathway of allorecognition. Taken together these results show that
corneal transplantation elicits a vigorous direct response mediated
exclusively by CD8+ T cells secreting IFN-
cytokine.
|
|
|
Cytolysis of donor cells, resulting from the direct recognition of
allogeneic MHC class I molecules by CD8+ T cells,
is thought to be the effector mechanism of rejection in different
models of transplantation (4, 9, 10). Here, we determined
whether the direct activation of CD8+ T cells was
required for corneal graft rejection. For this purpose, BALB/c mice
were grafted either with wild-type (wt) B6 corneas or B6 corneas
lacking MHC class I expression (due to a disruption of
2-microglobulin gene). In the last
combination, no CD8+ T cell-mediated direct
recognition could occur. The survival of corneal grafts was monitored
after assessing the degree of opacity of the graft as previously
described (34). As shown in Fig. 9, BALB/c mice rejected B6 MHC
I-deficient and wt B6 corneas in similar fashions. This demonstrates
that the CD8+ T cell-mediated direct response
found in cornea-transplanted mice is not required for graft
rejection.
|
Apparently, CD8+ T cells activated directly
following corneal transplantation do not influence the fate of the
allograft. Two main factors may be responsible for the apparent
inability of activated CD8+ T cells to mediate
corneal graft rejection: 1) an improper stimulation of
CD8+ T cells by the corneal allograft, and 2) a
lack of recognition of MHC class I-positive target cells in the donor
cornea. To discriminate between these possibilities, we determined
whether corneal graft rejection could be induced following a potent
peripheral activation of T cells toward allogeneic MHC molecules. For
this purpose, mice that had permanently accepted allogeneic corneas
were grafted with allogeneic skin derived from the same donor. BALB/c
mice were engrafted with MHC-mismatched BALB.B corneas. We selected
recipient mice exhibiting no signs of rejection at 11 wk
post-transplantation (80% of the mice in this combination never reject
their grafts). These mice were then transplanted with BALB.B skin.
Strikingly, 15 days after skin transplantation all mice underwent
corneal graft rejection (Fig. 10B). No rejection of
corneal graft was observed in mice that had not received a skin graft
(Fig. 10A). We conclude that allogeneic MHC molecules
expressed on the skin graft had activated alloreactive T cells that
caused rejection of corneal allografts. Therefore, in acceptor mice,
the failure to reject was not due to the lack of target recognition.
Alternatively, our results support the view that in the majority of
mice with MHC-mismatched corneal allografts, improper/suboptimal
activation of alloreactive T cells accounted for the lack of
rejection.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-producing CD8+ alloreactive T cells.
Thus, IL-2 and IFN- were produced by two distinct T cell subsets in
these mice. Indeed, recent studies by others also measuring cytokine
production at a single-cell level have demonstrated that the vast
majority of individual activated T cells secrete only one lymphokine
(39, 40). In addition, it has been reported that
alloreactive CD4+ Th1 cells from skin-grafted
mice produce IL-2, but no IFN- (38). Here, we provide
the first demonstration that in corneal transplantation, alloreactive
CD8+ T cells produce IFN- exclusively (Fig. 2). It is noteworthy that while alloreactive CD8+
T cells are known to be the main providers of IFN-, they also
normally secrete some IL-2. In turn, the absence of IL-2 production by
CD8+ T cells is thought to be associated with
split anergy (41).
Our study demonstrates that allogeneic MHC class I molecules expressed
on corneal allografts, devoid of MHC class II+
APCs, elicit a vigorous CD8+ T cell-mediated
direct response. Auchincloss et al. (22, 23) have
previously reported that the expression of allogeneic MHC class II
molecules on skin allografts was not required to initiate a CD8 direct
response. In this situation, Langerhans cells were shown to carry MHC
class I molecules to lymphoid organs and activate alloreactive
CD8+ T cells. Here, we found that MHC class
II+ passenger leukocytes (i.e., dendritic cells
and macrophages) are not required to prime alloreactive
CD8+ T. Strikingly, the frequency of
CD8+ T cells stimulated through the direct
pathway was similar in cornea- and skin-transplanted mice
(36). These observations suggest that donor APCs are not
critical for the differentiation of naive CD8+ T
cells into IFN--producing alloreactive CD8+ T
cells. Previous studies have pointed out that
CD8+ T cell activation requires the presence of
very few MHC class I molecules on APCs (42). It is also
believed that proper costimulation delivered by professional APCs is
necessary for lymphokine production and CTL activity by
CD8+ T cells (41, 43). Several
groups, however, have recently reported that under defined
circumstances naive CD8+ T cells can be activated
in the absence of professional APCs. For instance, bypass of
costimulation can be achieved when the density of TCR ligands is high
enough on presenting cells (44, 45). Lanzavecchia et al.
(46) have demonstrated that the number of triggered TCRs
required to reach the T cell activation threshold is much higher in the
absence of costimulation than under normal conditions. In turn, we
surmise that under physiological circumstances, costimulation is likely
to be critical to achieve full T cell activation. In support of this,
there is a markedly reduced Ag-primed population in people suffering
from mutations in the gene encoding for CD40 ligand (47).
In corneal transplantation, some evidence has been provided suggesting
that the cytotoxic activity of CD8+ T cells is
impaired (48). We conclude that while
CD8+ T cells from cornea-grafted mice secrete
IFN-, they apparently fail to mature into cytotoxic T cells, a
phenomenon also previously observed with HIV-specific
CD8+ T cells (49).
The precise role of CD8+ T cells in allograft
rejection is still controversial. Activation of a
CD8+ T cell response is usually sufficient, but
not always necessary, to ensure rejection. This is underscored by a
number of observations made in BALB/c mice engrafted with B6 skins
devoid of MHC class II expression (5). In the absence of
direct CD4 alloresponse, CD8+ T cells recognizing
donor MHC class I in direct fashion mediated acute rejection. In turn,
after depletion of these CD8+ T cells, the
remaining CD4+ T cell indirect response alone was
sufficient to ensure the rejection process. A direct
CD8+ T cell response is not required to ensure
the rejection of orthotopic corneal allografts. Indeed, the rejection
of fully allogeneic corneas grafted in either CD8 knockout recipients
or CD8+ T cell-depleted recipients is not
impaired (7, 27). It is important to note that many
CD8+ T cells are regularly found in the graft
tissue at the time of rejection (50). This observation
rules out the possibility that alloreactive T cells cannot reach their
targets. The inability of CD8+ T cells to mediate
corneal rejection could be related to the environment of the graft
itself and/or to the incomplete maturation of these cells into effector
T lymphocytes. While orthotopic corneal allografts induce a DTH
response, the emergence of CTL activity has been inconsistently
reported in mice (34, 48). Conversely, it is clear that
corneal allografts placed heterotopically (under the skin) stimulate
vigorous allospecific CTL activity (51). Therefore,
corneal allograft has the intrinsic ability to stimulate CTL, but this
process might not be fully achieved when the graft is placed in the
eye. This presumably explains why MHC-disparate orthotopic corneas are
poorly rejected. In support of this, we showed that sensitization
of T cells to allogeneic MHC molecules by placing an MHC-disparate
skin graft resulted in the rejection of initially accepted
MHC-mismatched corneas. While an indirect recognition of allogeneic
MHC molecules was observed in skin-grafted mice (Fig. 5), there was no
indirect response to MHC-derived peptides in cornea-grafted mice. It is
therefore unlikely that this response was necessary and sufficient to
induce the rejection of corneal grafts. After skin grafting, a vigorous
CTL response to donor MHC proteins is normally induced
(52). In our experimental model it is likely that the
induction of this response accounted for the destruction of the corneal
graft.
It has been reported that mice that did not reject corneal allografts 8 wk after transplantation were tolerant to the graft due to an active suppression mechanism (53). In this article, we have described a mechanism by which tolerance can be broken and rejection induced in recipients that totally accepted their grafts. Presumably, both induction of direct and indirect CD4+ T cell responses to donor MHC accounted for the activation/differentiation of functional alloreactive CD8+ T cells. This finding may have interesting implications in corneal transplantation. First, it shows that in mice with long term graft survival, CD8+ T cells against allogeneic MHC were still available. Second, our findings demonstrate that these T cells have retained the capacity to reject the graft when provided with proper stimulation. Therefore, T cell tolerance to corneal allografts is not absolute and can be broken when T cells are challenged with appropriate Ag and costimulation in the peripheral host’s lymphoid organs.
Convergent studies have shown that the CD4+ T
cell response is required for initiating corneal graft rejection
(7, 27, 28). Corneal transplantation represents an
interesting model, in that the graft is naturally devoid of MHC class
II+ passenger leukocytes and cannot theoretically
elicit CD4+ T cell-mediated direct alloresponse
(30). In this paper we have clearly identified a CD4
indirect response, but could not detect any CD4 direct response after
fully mismatched corneal transplantation. Together, these results
demonstrate that the recognition of alloantigens through the indirect
pathway is essential to corneal graft rejection. Since a potent DTH
toward minor Ags is induced following corneal transplantation,
CD4+ T cells activated indirectly could also be
the provider of cytokines required for graft destruction. Even though
IFN- is thought to be essential to mediate the rejection in this
situation, it has been shown recently that CD4+ T
cells deficient in IFN- production are still able to initiate the
destruction of an allogeneic skin graft via an indirect allorecognition
pathway (54).
In conclusion, we have shown that an allograft naturally devoid of MHC
class II+ APCs at the time of transplantation is
able to activate T cells through both indirect and direct
allorecognition pathways. Each alloresponse is mediated by different T
cell subsets, displaying different phenotypes and alloantigen
specificities (Table I). Even if the
direct CD8 response can induce graft destruction when proper
costimulation is provided, the CD4 indirect response is essential in
the rejection observed under normal circumstances. This supports the
idea that in the absence of MHC class II on the graft and presumably in
the case of MHC-matched transplants, indirect alloresponse represents
the main driving force in the rejection process. This implies that in
these types of transplantation, strategies designed to block the
indirect alloresponse, such as donor-peptide tolerization, may be
effective at preventing/delaying allograft rejection.
|
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Gilles Benichou, Cellular and Molecular Immunology Laboratory, Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114. E-mail address: gilles{at}vision.eri.harvard.edu
3 Abbreviations used in this paper: MMC, mitomycin C; DTH, delayed-type hypersensitivity; wt, wild type.
Received for publication October 24, 2000. Accepted for publication May 25, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-2-producing cells in the CD8+ T cell subset. Eur. J. Immunol. 28:3630.[Medline]
. Transplantation 69:1016.[Medline]This article has been cited by other articles:
|
|
 |
|
  X. Zhang, L. Shen, Y. Jin, D. R. Saban, S. K. Chauhan, and R. Dana Depletion of Passenger Leukocytes from Corneal Grafts: An Effective Means of Promoting Transplant Survival? Invest. Ophthalmol. Vis. Sci., July 1, 2009; 50(7): 3137 - 3144. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Gelman, M. Okazaki, J. Lai, C. G. Kornfeld, F. H. Kreisel, S. B. Richardson, S. Sugimoto, J. R. Tietjens, G. A. Patterson, A. S. Krupnick, et al. CD4+ T Lymphocytes Are Not Necessary for the Acute Rejection of Vascularized Mouse Lung Transplants J. Immunol., April 1, 2008; 180(7): 4754 - 4762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Kuffova, M. Netukova, L. Duncan, A. Porter, B. Stockinger, and J. V. Forrester Cross Presentation of Antigen on MHC Class II via the Draining Lymph Node after Corneal Transplantation in Mice J. Immunol., February 1, 2008; 180(3): 1353 - 1361. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Shen, Y. Jin, G. J. Freeman, A. H. Sharpe, and M. R. Dana The Function of Donor versus Recipient Programmed Death-Ligand 1 in Corneal Allograft Survival J. Immunol., September 15, 2007; 179(6): 3672 - 3679. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Joseph, D Raj, V Shanmuganathan, R J Powell, and H S Dua Tacrolimus immunosuppression in high-risk corneal grafts Br J Ophthalmol, January 1, 2007; 91(1): 51 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Sagoo, G. Chan, D. F. P. Larkin, and A. J. T. George Inflammatory Cytokines Induce Apoptosis of Corneal Endothelium through Nitric Oxide Invest. Ophthalmol. Vis. Sci., November 1, 2004; 45(11): 3964 - 3973. [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]
|
|
|
|
|
|
A Vitova, M Filipec, A Zajicova, M Krulova, and V Holan Prevention of corneal allograft rejection in a mouse model of high risk recipients Br J Ophthalmol, October 1, 2004; 88(10): 1338 - 1342. [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. Andrassy, S. Kusaka, E. Jankowska-Gan, J. R. Torrealba, L. D. Haynes, B. R. Marthaler, R. C. Tam, B. M.-W. Illigens, N. Anosova, G. Benichou, et al. Tolerance to Noninherited Maternal MHC Antigens in Mice J. Immunol., November 15, 2003; 171(10): 5554 - 5561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Hamrah, S. O. Huq, Y. Liu, Q. Zhang, and M. R. Dana Corneal immunity is mediated by heterogeneous population of antigen-presenting cells J. Leukoc. Biol., August 1, 2003; 74(2): 172 - 178. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Chalermskulrat, I. P. Neuringer, W. J. Brickey, N. J. Felix, S. H. Randell, J. P. Ting, and R. M. Aris Hierarchical Contributions of Allorecognition Pathways in Chronic Lung Rejection Am. J. Respir. Crit. Care Med., April 1, 2003; 167(7): 999 - 1007. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Liu, P. Hamrah, Q. Zhang, A. W. Taylor, and M. R. Dana Draining Lymph Nodes of Corneal Transplant Hosts Exhibit Evidence for Donor Major Histocompatibility Complex (MHC) Class II-positive Dendritic Cells Derived from MHC Class II-negative Grafts J. Exp. Med., January 22, 2002; 195(2): 259 - 268. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or
together with blocking mAbs (10 µg/ml): anti-CD4 (
),
anti-CD8 (
), or
anti-CD4 plus anti-CD8 mAbs
(
). The
frequency of IL-2 (A) and IFN-
) or anti-CD8 (
) blocking mAbs
(10 µg/ml). B, Frequency of CD4+ T cells
activated indirectly. CD4+ T cells were first purified from
splenocytes collected in cornea-grafted, naive (negative control), and
skin-grafted BALB/c mice (positive control) and then mixed with
MMC-treated BALB/c APCs (ratio 1:5) and C57BL/6 sonicate. The frequency
of IL-2-producing CD4+ T cells activated through the
indirect pathway was determined. Data are the mean ± SEM from
four mice.
