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Departments of
*
Microbiology and Immunology,
Surgery,
Medicine, and
Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Oral tolerance has been suggested to be mediated by clonal
deletion/anergy (1, 4) or active suppression (4, 10, 11). In active suppression, regulatory cells are thought to play
a critical role by modulating the immune response through secretion of
Th2 cytokines and/or TGF-
(4, 12, 13). Such regulatory
cells have been shown to be capable of transferring tolerance to naive
recipients. In an experimental allergic encephalomyelitis
(EAE)3 model, the cells that
transferred this tolerance following oral administration of Ag were
CD8+ T cells (14). An important role
of CD8+ regulatory T cells in the generation and
maintenance of oral tolerance has also been suggested by
experimentation in other models (15, 16). Recently,
however, CD4+ T regulatory cells have been
suggested to play an important role in the development of oral
tolerance (17).
Although oral tolerance has been extensively studied in autoimmune disease models, it is only recently that oral tolerance has been demonstrated to prolong first-set transplant survival (7, 8, 9). As such, the mechanisms responsible for this graft prolongation by oral administration of alloantigen are still unclear. It has been reported that oral administration of alloantigen induces an Ag-specific reduction in delayed-type hypersensitivity responses (5, 9, 18), a decreased MLR (5, 9), and a reduction in allospecific CTL activity (18). Feeding alloantigen has also been suggested by some to induce a Th subset shift from Th1 responses to Th2 responses (5, 6, 9) that is associated with prolonged allograft survival (6, 9, 12). In this study we demonstrate the presence of active CD8+ regulatory cells infiltrating grafts in orally tolerized animals. Adoptive transfer of these CD8+ cells to naive rats transfers the graft tolerance seen in the original fed animals. The presence of high levels of mRNA for IL-4 in this CD8+ population suggests that Tc2 graft-infiltrating cells (GIC) result from oral transplantation tolerance and mediate the graft prolongation.
This is the first report to demonstrate that oral exposure to alloantigen generates intragraft regulatory cells that are capable of transferring prolongation of allograft survival to naive animals. The intragraft regulatory cells are CD8+ T cells, which may regulate rejection responses by Tc2-type activities or by Fas/Fas ligand (FasL) interaction with alloreactive T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Brown Norway (BN; RT1n) and Lewis rats (RT11) purchased from Harlan Sprague-Dawley (Indianapolis, IN) were used as donors and recipients, respectively. This strain combination is fully disparate at both major and minor histocompatibility complex loci. Animals were maintained in the Dalhousie University Faculty of Medicine animal care facility and were provided water and rodent chow ad libitum. All animal experimentation was undertaken in compliance with the guidelines of the Canadian Council on Animal Care.
Single-cell suspensions
Lymphocytes were isolated from the spleen or mesenteric lymph nodes (MLN) and single-cell suspensions were prepared following standard protocols (as we have described previously, Ref. 8). Cells were prepared, washed, and used for in vitro experiments in RPMI 1640 medium (Life Technologies, Burlington, Canada) supplemented with 20 mM HEPES (U.S. Biological Corp., Cleveland, OH), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS (Life Technologies). Spleen cells were purged of RBC by lysis for 5 min with ACK erythrocyte lysing buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.4). After lysis of RBC, spleen cells were washed in RPMI 1640.
Oral tolerance induction
Lewis rats were fed, without anesthetic, by intragastric intubation using a 3 1/2 Fr premature human infant feeding tube (Sherwood Industries, St. Louis, MO). All rats were fed 1 x 108 BN splenocytes suspended in 300 µl PBS (0.15 M NaCl and 0.05 M Na2PO4, pH 7.4) on days -14, -13, -12, -11, -10, and -1, with the day of kidney transplantation being day 0.
Kidney transplantation
Kidney transplantation was performed as described previously (8). Briefly, BN rats were anesthetized with sodium pentobarbital. After ligation of the abdominal aorta and vena cava proximal and distal to the renal artery and vein, the left kidney was perfused in situ with cold (4°C) heparinized 0.9% saline and removed with a cuff of the aorta and the renal vein as well as a long piece of the ureter. The donor kidney was kept in cold saline during preparation of the Lewis recipient. Following left nephrectomy of the recipient, the donor kidney was transplanted in an orthotopic position by end-to-side anastomosis of the cuff of the renal artery to the abdominal aorta, and the renal vein was connected to the inferior vena cava. The ureter was attached by end-to-end anastomosis. Three days after the transplant the right native kidney was removed, leaving rat survival dependent on the transplanted kidney.
MLR reaction
Lewis rat spleen and MLN responder cells were recovered from kidney transplant recipients on day 5 post-transplant. BN spleen cells were used as stimulators. Responder cells (1 x 105 cells/well) were cultured in 96-well round-bottom plates (Nunc, Naperville, IL) with or without equal numbers of mitomycin C (25 µg/ml)-treated stimulator cells in RPMI 1640 medium. The plates were incubated at 37°C for 72 h, then pulsed with [3H]thymidine (1 µCi/well) for 18 h and harvested with an automated cell harvester (Skatron, Sterling, VA). Proliferation was assayed by [3H]thymidine incorporation.
Isolation of GIC
GIC were isolated as we have described previously (8). Briefly, at 5 days post-transplantation, rats were sacrificed, and kidney allografts were flushed in situ with heparinized (100 U/ml) 0.9% saline and removed from the rat. The kidney was minced and then digested in 25 ml collagenase solution (50 µg/ml collagenase (Life Technologies) and 20% FBS in RPMI 1640). To clear the debris, including dead tubular epithelial cells, the cell suspensions were rapidly passed down a loosely packed glass wool column (300 mg sterile glass wool in a 10-ml syringe), then mixed with isotonic Percoll (Pharmacia, Piscataway, NJ) to a concentration of 30% Percoll and centrifuged at 460 x g for 20 min at 10°C. The pellet was resuspended in RPMI 1640, then loaded on top of a cushion of 70% Percoll (in RPMI 1640) and centrifuged as described above. The cells overlying the cushion were removed for GIC examination.
Flow cytometry
The leukocytes isolated from the kidney graft were incubated
with the following primary Abs: R73 (anti-
TCR; PharMingen,
San Diego, CA), OX-8 (anti-CD8; Serotec, Oxford, U.K.), and W3/25
(anti-CD4; Serotec) at a concentration of 10 µg/ml in PBS
solution containing 0.5% (w/v) BSA (Roche, Indianapolis, IN). The
secondary Ab was a polyclonal FITC-labeled anti-mouse IgG (Organon
Teknika, Durham, NC). Following incubation with the FITC-labeled Ab at
a 1/500 dilution, the cells were fixed with 1% paraformaldehyde in PBS
and examined on a FACScan (BD Biosciences, Mountain View, CA).
Purification of CD8+ or CD4+ T cells from GIC
Graft-infiltrating T cells were enriched by incubating GIC in warm nylon wool (Polysciences, Warrington, PA) columns (1 g nylon wool in a 10-ml syringe) with RPMI 1640 at 37°C for 1 h to remove the majority of the B cells, macrophages, and other accessory cells. The enriched population was usually 85–90% T cells as analyzed by flow cytometry. Enriched T cells were incubated with W3/25 or OX-8 (anti-CD4 or anti-CD8 Ab, supplied by Biotex, Edmonton, Canada) for 30 min before passage through immunoenrichment columns (Biotex), which enrich for CD8+ or CD4+ cells by negative selection. The purity of CD8+ or CD4+ cells obtained by this method was normally >97% (assessed by flow cytometry).
Direct CTL assay
Five days after transplantation, allograft kidneys were removed. Single-cell suspensions of CD8+ cells from the kidney GIC were prepared as effector cells. 51Cr (100 µCi for 1 h at 37°C)-labeled BN spleen Con A (2 µg/ml for 72 h) blasts were used as target cells. The effector and target cells were plated in V-bottom 96-well microtiter plates (1 x 104 target cells/well) at E:T cell ratios ranging from 100:1 to 12.5:1 in RPMI 1640 medium. After incubation at 37°C for 12 h, the plates were centrifuged, and lysis was measured by 51Cr release.
RT-PCR
Relative (rather than quantitative) RT-PCR was performed as we
have described previously (8). Briefly, total RNA was
obtained from the CD8+ GIC (1–2 x
106 cells) using TRIzol (Life Technologies) as
suggested by the manufacturer. RNA was reverse transcribed using
Moloney murine leukemia virus reverse transcriptase (Life Technologies)
and random hexamer primers (Life Technologies) according to the
manufacturer’s instructions. PCR amplification of the product of the
RT was achieved with a mixture containing 1x PCR buffer, 0.2 mM dNTP,
0.4 µm each of sense and antisense primers, and 2.5 U Taq polymerase
(BRL, Rockville, MD) for 40 cycles. Ten-microliter aliquots of PCR
products were analyzed in ethidium bromide-stained agarose gels.
Primers were developed from the published sequences for the respective
rat cytokines as we have previously described (8).
Experimental amplicons were compared with the housekeeping -actin
amplicon, and relative amplification within samples was assessed.
Cell transfer
To generate GIC for cell transfer, Lewis rats were fed 1 x 108 BN splenocytes on days -14, -13, -12, -11, -10, and -1 and given kidney transplants on day 0 as described above. For control GIC, unfed Lewis rats were used as recipients. On day 5 post-transplantation, whole GIC, CD8+, or CD4+ cell populations were isolated from kidneys of fed and unfed animals. GIC were transferred i.v. into naive rats that had received a kidney transplant 1 day before the GIC injection. The GIC population isolated from one kidney was transferred into a single paired naive Lewis rat. Three days after kidney transplantation, the recipient native right kidney was removed, leaving rat survival dependent only on the transplanted BN kidney.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five days after kidney transplantation, the spleen and MLN were
removed from the rats orally exposed or not exposed to alloantigen.
Cell proliferation was assessed by MLR using mitomycin C-treated donor
spleen cells as stimulators. In this experiment sensitization to
alloantigen occurs in vivo in response to a kidney transplant. The MLR
was used to assess whether prior oral exposure to alloantigen would
decrease this sensitization induced by the graft. Fig. 1
shows the results of the MLR using
spleen cells (Fig. 1A) or MLN cells (Fig. 1B)
derived from fed (orally exposed to alloantigen before kidney
transplantation) or unfed rats. As shown in Fig. 1, prior oral exposure
to alloantigen led to increased allosensitization, as seen by an
increased response to allochallenge. Data are shown for a
representative experiment and also shown is the mean percent increase
for five experiments. Previous alloexposure by the oral route does not
lead to reduced allorecognition and activation, which would be
indicated by a proliferative response lower than that seen in the
control MLR, where sensitization is generated by the kidney transplant.
In fact, in all experiments there was a significant increase over
control MLR with effector cells from the animals previously exposed to
alloantigen by the oral route (271.3 ± 77.4% for spleen and
302.7 ± 56.2% for MLN, taking the control mean as 100%). In
contrast, effector cells from animals orally exposed to syngeneic
spleen cells or to third-party (PVG) spleen cells did not show
increased MLR after kidney transplantation compared with control
animals without prior alloexposure before kidney transplantation
(syngeneic, 137.2 ± 41.4% of control; third-party exposure,
113.3 ± 28.6% of control). These results show that the feeding
of allogeneic cells to induce Ag-specific immune regulation does not
prolong allograft survival by decreasing allorecognition. On the
contrary, the treatment regimen appears to increase alloreactivity.
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The data reported above show that increased survival of kidney
allografts by prior alloexposure through the oral route was not due to
decreased recognition of the allochallenge. Therefore, we hypothesized
that the increased survival would be due to a modulation in the
generation or effector activity of CD8+ CTL in
the graft. To examine T cell responses in the graft, we isolated GIC on
day 5 post-transplant. Using flow cytometry, we found that the GIC
contained predominately T cells (
65%; labeled with anti-TCR
mAb; data not shown) and macrophages (35%; labeled with
anti-Mac-1 mAb; data not shown). We have previously observed that
on day 5 post-transplant, the total number of graft-infiltrating T
cells was decreased by 30% compared with that in controls
(8). The data in Fig. 2 show
that this decreased number of T cells is due to a decrease in the
number of CD4+ cells. In this figure data from a
representative experiment (Fig. 2A) and the mean of five
separate experiments (Fig. 2B) are shown. In fed animals
CD4+ GIC are present at only 60% of control
levels, whereas there is no significant difference in the
CD8+ GIC levels between fed and control animals.
Thus, there appears to be no defect in the ability of
CD8+ T cells to transit to the kidney allograft.
This suggests either that this CD8+ T cell
population does not contain CTL or that the CTL activity is somehow
modulated.
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To examine whether the CD8+ T cell
population in the GIC of the animals orally exposed to alloantigen
contained alloresponsive CTL, we assessed the expression of cytotoxic
mediators in the CD8+ T cell population of the
GIC, and we also conducted a direct CTL assay. The mediators we
assessed (by RT-PCR) were those well known to be associated with CTL
activity, granzyme B and perforin. In addition, we assessed the
presence of mRNA for FasL, which mediates apoptosis in target cells by
ligation of Fas. The results, shown in Fig. 3, clearly demonstrate the presence of
abundant mRNA for the CTL mediators granzyme B and perforin in the
CD8+ GIC T cell population from both control and
fed animals. There is no obvious diminution in the expression of the
message for these important cytotoxic mediators, suggesting that CTL
are present in the CD8+ T cell compartment of the
GIC. Furthermore, FasL expression appears to be increased, rather than
decreased, in the CD8+ T cell compartment of the
GIC from the transplanted kidneys of animals previously exposed to
alloantigen. These data indicate that CTL are present in the
CD8+ T cell compartment of these GIC.
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, clearly demonstrate that the
CD8+ T cell population in the GIC of both fed and
unfed animals contains mature alloresponsive CTL. Indeed, the CTL
activity in the animals that had been orally exposed to alloantigen
before kidney transplant is consistently higher than the CTL activity
in the animals without prior exposure to alloantigen by the oral route
(p < 0.04).
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Cytokine transcription of CD8+ GIC
It has been suggested that oral tolerance can generate
CD8+ regulatory cells (14, 20)
and/or switch immune responses toward type 2 responses (7, 12, 17). Because in our experiments the number of
CD8+ GIC T cells remains the same in fed and
unfed animals, but grafts survived much longer in the fed animals, we
postulated that the phenotype of the CD8+ GIC T
cells may have shifted to a type 2 CD8+ T cell
phenotype (Tc2). Therefore, we investigated cytokine mRNA from these
cells. RT-PCR was used to reveal the relative amount of cytokine mRNA
in these cells. As shown in Fig. 5, substantial amounts of IFN-
and TGF- mRNA were detected in
CD8+ GIC T cells from both fed and unfed animals.
However, compared with these cytokines, and to -actin, the amount of
IL-4 mRNA detected was markedly greater in the fed animals than in the
controls. Indeed, in multiple repeats, IL-4 amplicons were not detected
after RT-PCR of mRNA from CD8+ GIC from control
animals, but were always detectable in fed animals. In contrast,
IFN- was always detectable in both groups in comparable amounts
relative to the -actin control and other cytokines. In fact, IL-4
mRNA was only detectable in the CD8+ GIC T cells
from fed animals, not in the CD8+ GIC T cells
from unfed controls. These data suggest that in fed animals at least
some of the CD8+ GIC are Tc2 cells and
potentially regulatory cells.
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The data presented above suggest that the CD8+ GIC may be involved in intragraft modulation as a regulatory cell in animals that have been exposed to alloantigen by the oral route. To investigate the existence of regulatory T cells in the kidney, we transferred allograft GIC from fed or unfed animals into animals that had received a kidney allograft 1 day previously. GIC for transfer were taken on day 5 post-transplant, because our previous experimentation had shown significant damage to kidney grafts at this time point in control animals, but preservation of kidney architecture in animals orally exposed to alloantigen (thus indicating the presence of active regulatory events at this time point).
The results, shown in Fig. 6, confirm the
presence of a regulatory cell in the kidney allograft after oral
exposure to alloantigen. Survival of the kidney grafts in the animals
receiving GIC from allogeneic kidney grafts of animals orally exposed
to alloantigen was significantly increased compared with the survival
of grafts in animals receiving GIC from control grafts. The mean
survival in the control group was 9 days, with the longest survival
being 12 days. The mean survival of the kidneys in animals receiving
GIC transferred from orally treated animals was 22 days, with the
longest survival being 46 days (p < 0.03).
These data suggest that regulatory cells are present in the GIC of the
allograft kidney of orally treated animals.
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, transfer of CD8+
allograft GIC from orally pretreated rats into naive rats significantly
prolonged kidney allograft survival in those rats (mean survival, 89
days; longest survival, 215 days; p < 0.01).
CD4+ GIC, in contrast, did not confer increased
survival (mean survival, 10 days; longest survival, 13 days;
p > 0.05). These data indicate that a population of
CD8+, but not CD4+,
intragraft regulatory T cells is present in the kidney allografts of
animals orally exposed to alloantigen. Note that purified
CD8+ regulatory cells showed much more modulatory
activity than whole GIC (compare Figs. 6
and 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secreting T cell as a mediator in this complex interaction
(17). Others (11, 21, 22) have demonstrated
that oral tolerance can be induced in CD8 knockout mice and in mice
depleted of CD8+ T cells by anti-CD8 Ab
treatment. Thus, it appears that the generation of
CD8+ regulatory cells may be sufficient for, but
not essential to, the development of oral tolerance. In the experiments described here we have examined whether the generation of such regulatory cells plays a role in the significant prolongation of primary kidney allograft survival that we have observed following oral exposure to alloantigen (7, 8). We first assessed allorecognition by MLR in fed vs control animals. The MLR showed an increase in reactivity in the cells from the animals that had been previously exposed to alloantigen by the oral route before transplantation. From this we conclude that there is no defect in allorecognition produced by induction of immune modulation by pre-exposure to alloantigen by the oral route. These data confirm that prolongation of kidney allograft survival is not due to masking of allorecognition, but to an immunomodulatory effect on the immune response, presumably the development of an immunomodulatory cell.
Past research in oral tolerance has primarily concentrated on events in
the draining node (MLN) or spleen. However, the response to allogeneic
transplants is defined at the level of the organ transplant. We were
the first to show that oral tolerance could prolong primary solid organ
transplants (7, 8), and we chose to use the kidney as our
model so that cells infiltrating the graft would be readily available.
On the premise that rejection is defined at the level of the organ, we
postulated that critical immunomodulatory events would also be defined
at the level of the transplanted organ, and examining the
graft-infiltrating lymphocytes would be of prime importance for
developing an understanding of oral transplantation tolerance. We
isolated GIC on day 5 post-transplant. This is the latest time point at
which we can obtain GIC from control animals. We have reported
previously that the total number of allograft-infiltrating T cells in
orally treated animals was decreased at this time by 30% compared
with that in normal untreated control allografts (8). In
the current study we have found that this decreased number of T cells
is due to a decrease in the number of CD4+ cells.
In contrast, we found that the CD8+ cells, a
subset of which is presumably allospecific CTL, remain intact.
CD8+ T cells are the major source of CTL and have
been suggested to destroy graft tissue predominantly through direct
cytotoxic effects against foreign class I MHC-expressing cells
(23). Many treatments that prolong allograft survival,
including anti-CD4 mAb (24), portal venous inoculation
with alloantigen (25), and nematode infection
(26), are associated with decreased numbers of
CD8+ T cells or decreased CTL activity. The
undiminished numbers of intragraft CD8+ T cells
in our study suggests that the animals that have been orally
pre-exposed to alloantigen do not have a defect in the generation or
transit of active CTL to the allograft. However, it was possible that
the CD8+ T cells in the graft did not contain
active CTL and that the modulation lay in a defect in
CD8+ CTL differentiation.
To address this we examined whether that the known characteristics of CTL, that is the expression of perforin, granzyme, and FasL (27), were present in a highly purified CD8+ T cell population from the GIC derived from allografts in orally treated animals. We found no substantial difference in the level of mRNA for the cytotoxic mediators perforin and granzyme in the GIC from orally treated animals vs controls. Furthermore, FasL expression appeared to be increased, rather than decreased, in these highly enriched CD8+ GIC T cell populations. This supports the hypothesis that similar numbers of potentially active mature CTL are present in the grafts of orally treated animals even in the face of prolonged graft survival. This hypothesis was confirmed by our experimentation showing increased, rather than decreased, cytotoxicity to allogeneic targets in a direct CTL assay, thus amply demonstrating the presence of mature CTL in the GIC population. This increase in cytotoxicity could be due to the increased levels of FasL on these cells.
Although many studies have demonstrated that CD8+ T cells are not absolutely essential for allograft rejection, including heart, kidney, and skin allografts (28, 29), CTL have been suggested to play an important role in normal MHC-mismatched allograft rejection (30). The potential mechanism of CTL involvement in acute renal allograft rejection has been suggested to be mediated by cytotoxic granule-based killing, but not FasL-induced killing. This is because granule proteins are expressed on biopsies of kidney undergoing acute rejection, whereas FasL is expressed mostly in the absence of acute rejection episodes (31, 32). In addition, there is a paucity of FasL-induced apoptosis seen in renal allograft rejection, which may be due to low levels of Fas expression on graft cells (31) or resistance of kidney tubule cells to FasL-mediated events (33). In our experiments the presence of these mature alloreactive CTL in the orally treated animals exhibiting prolonged graft survival was perplexing. Interestingly, similar observations of increased CTL activity exhibited by GIC from kidney allografts have also been reported in studies of blood transfusion transplantation tolerance, although further characterization of the cells was not performed (34). One potential explanation could be that the mature alloreactive CTL fall under the control of regulatory elements in situ, and that they are somehow freed from those regulatory elements in the artificial conditions of the in vitro assay. Another possibility is that the increased CTL activity observed in our in vitro experiments is mediated by increased FasL expression, which may be less significant in vivo, as described above. However, increased FasL expression by CD8+ intragraft regulatory cells may, in fact, represent another level of regulation. Some immune-privileged sites, such as the testes and the anterior chamber of the eye, are thought to express their privileged phenotype because of the high levels of FasL expression in these sites (35, 36, 37, 38). Indeed, Swenson and coworkers (39) have provided evidence that kidney allografts transfected with FasL cDNA were protected from rejection, presumably through interference with alloreactive T cells by a Fas/FasL interaction. In addition, high levels of FasL expression in renal biopsies from kidneys that are not undergoing acute rejection support the idea that some degree of FasL-dependent immune privilege occurs in renal graft protection (32). Therefore, it is possible that the observed CD8+ T cells may be involved in graft protection, rather than destruction, due to their level of FasL expression, which may interact with alloreactive Fas-bearing T cells.
This study focused on CD8+ GIC T cells isolated from allografts on day 5 post-transplant, a time point when robust graft destruction occurs in control animals and the latest time point that we could obtain GIC from control transplants. We currently have no information on whether these CD8+ T cells persist throughout the entire period of graft prolongation.
It has been suggested that type 1 immunity, mediated mainly by CD4+ Th1 cells, is the major effector mechanism of allograft rejection, whereas type 2 immunity favors allograft survival (40, 41). A shift from type 1 T cell to type 2 T cell responsiveness has been reported to prolong allograft survival in several experimental models (6, 12, 26), and this shift has been associated with an increased presence of IL-4 (6, 9, 42). Because IL-4 is the only type 2 cytokine that can be reliably correlated with type 2 T cell activity, the presence of increased levels of IL-4 mRNA in the CD8+ GIC from allografts of fed animals indicates an increased intragraft type 2 response. IL-4 mRNA has often been found in whole graft extracts or the whole GIC population of control rejecting grafts and in biopsies of human grafts, but has not been previously demonstrated in a graft-infiltrating CD8+ T cell population. This finding, in the context of the tolerizing protocol, suggests that the regulatory cell is a CD8+ Tc2 cell (43, 44).
The presence of regulatory cells can be demonstrated by in vivo cell transfer (45, 46). Others have shown that oral tolerance can induce regulatory cells in the spleen and draining lymph nodes, using autoimmune disease models (14, 17). We also found that regulatory cells are present in the spleen (the reactive node for kidney transplants) and MLN (the draining node of the gastrointestinal tract) following oral exposure to alloantigens. We were able to transfer graft protection using cells from these sites from fed animals, but not from unfed control animals (mean survival of rats that received splenocytes or MLN cells from fed animals, 38 and 24 days, respectively; mean survival of rats that received splenocytes or MLN cells from unfed control rats, 9 day). The presence of regulatory cells in the reactive node may indicate an effect of oral tolerance and the inductive phase of the responses. Importantly, however, we demonstrated that regulatory cells were also present in the kidney allograft of fed animals. Transfer of kidney GIC from orally tolerized animals into naive animals prolonged graft survival in these animals. This confirms that oral transplantation tolerance mediates a change at the effector site (i.e., within the transplant). We chose to harvest cells from the kidney allografts of orally pre-exposed animals on day 5 post-transplant because 1) our preliminary histologic evidence has shown that immune modulation is ongoing at this point (8); 2) this is the point at which we know there are mature CTL in the GIC population of the graft; and 3) this is the latest time point that we could reliably obtain GIC from control transplants. We compared the effect of transfer of these cells harvested from orally treated animals with that in cells obtained from control non-pretreated animals on allograft survival in naive recipients. Our results confirmed that oral exposure to alloantigen generates intragraft regulatory cells that are present in the kidney allograft.
Once we had demonstrated that regulatory T cells exist in the kidney
allograft of animals orally exposed to alloantigen, it was important to
differentiate between CD4+ and
CD8+ effects. Because others have previously
shown the existence of a CD8+ regulatory cell in
the spleens and lymph nodes of orally tolerized animals in other models
(14), we suspected that regulation would occur in this
compartment. The data obtained from our subsequent transfer experiments
confirmed that CD8+, but not
CD4+, T cells in the GIC transfer tolerance.
Thus, animals that have been orally exposed to alloantigen develop
CD8+ regulatory cells that are present in the
kidney on day 5 post-transplant. This cell population will transfer
graft prolongation to a naive animal receiving a kidney transplant.
Interestingly, the transferred CD8+ GIC were much
better at transferring graft prolongation than transferred whole GIC,
even though the same number of CD8+ T cells was
transferred in each instance (compare Figs. 6 and 7). This suggests
that the whole GIC population contains both effector and regulatory
cells.
These data do not rule out a contribution of CD4+ T cells to oral transplantation tolerance in ways other than active regulation. However, these data confirm that generation of CD8+ regulatory cells, a response to oral pre-exposure to alloantigen, is sufficient to induce graft prolongation. These intragraft regulatory cells are capable of transferring prolongation of graft survival to naive animals and may mediate their effects by IL-4 secretion and/or Fas/FasL interaction with alloreactive T cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Timothy D. G. Lee, Department of Microbiology and Immunology, Transplantation and Immunology Research Laboratory, Tupper Medical Building, Halifax, Nova Scotia, Canada B3H 4H7. E-mail address: tim.lee{at}dal.ca
3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; GIC, graft-infiltrating cells; FasL, Fas ligand; MLN, mesenteric lymph nodes; BN, Brown Norway.
Received for publication September 13, 2000. Accepted for publication April 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T lymphocytes regulate the induction and maintenance of oral tolerance. J. Immunol. 158:3610.[Abstract]
+ T cells. Immunology 81:27.[Medline]
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 H. L. WEINER Current Issues in the Treatment of Human Diseases by Mucosal Tolerance Ann. N.Y. Acad. Sci., December 1, 2004; 1029(1): 211 - 224. [Abstract] [Full Text] [PDF]
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E. S. Morris, K. P. A. MacDonald, V. Rowe, D. H. Johnson, T. Banovic, A. D. Clouston, and G. R. Hill Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance Blood, May 1, 2004; 103(9): 3573 - 3581. [Abstract] [Full Text] [PDF]
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 J. C. Zimring, S. B. Levery, B. Kniep, L. M. Kapp, M. Fuller, and J. A. Kapp CD75s is a marker of murine CD8+ suppressor T cells Int. Immunol., November 1, 2003; 15(11): 1389 - 1399. [Abstract] [Full Text] [PDF]
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M. Kataoka, J. A. Margenthaler, G. Ku, and M. W. Flye Development of Infectious Tolerance After Donor-Specific Transfusion and Rat Heart Transplantation J. Immunol., July 1, 2003; 171(1): 204 - 211. [Abstract] [Full Text] [PDF]
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W. Chen, M. S. Ford, K. J. Young, M. I. Cybulsky, and L. Zhang Role of Double-Negative Regulatory T Cells in Long-Term Cardiac Xenograft Survival J. Immunol., February 15, 2003; 170(4): 1846 - 1853. [Abstract] [Full Text] [PDF]
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K. J. Young, L. Yang, M. J. Phillips, and L. Zhang Donor-lymphocyte infusion induces transplantation tolerance by activating systemic and graft-infiltrating double-negative regulatory T cells Blood, October 16, 2002; 100(9): 3408 - 3414. [Abstract] [Full Text] [PDF]
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E. Chiffoleau, G. Beriou, P. Dutartre, C. Usal, J.-P. Soulillou, and M. C. Cuturi Role for Thymic and Splenic Regulatory CD4+ T Cells Induced by Donor Dendritic Cells in Allograft Tolerance by LF15-0195 Treatment J. Immunol., May 15, 2002; 168(10): 5058 - 5069. [Abstract] [Full Text] [PDF]
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;
n = 10) to alloantigen, but not by transferring
total GIC from unfed control rats (
; n = 5).
Total GIC isolated from a single kidney allograft (
; n = 8;