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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 324-330
Mechanisms of Long-Term Donor-Specific Allograft Survival Induced
by Pretransplant Infusion of Lymphocytes
By
Liming Yang,
Barb Du Temple,
Qasim Khan, and
Li Zhang
From the Department of Cellular and Molecular Pathology, Multi Organ
Transplantation Program, The Toronto Hospital Research Institute,
University of Toronto, Toronto, Canada.
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ABSTRACT |
Pretransplantation donor-specific transfusion (DST) can enhance
allograft survival in man and animals. However, due to the lack of a
specific marker to identify donor-reactive cells in vivo in man and
normal (nontransgenic) animals, the underlying mechanism remains
unknown. In this study, we use 2CF1 transgenic mice
expressing a transgenic T-cell receptor (TCR) specifically recognizing
Ld, a major histocompatibility complex (MHC) class I
molecule, to delineate the role of DST in long-term skin allograft
survival and its underlying mechanisms. Our main findings include: (1)
in the absence of any other immunosuppressive treatment, a single dose
pretransplantation infusion of viable splenocytes from an
Ld+ donor is sufficient to induce permanent
donor-specific skin allograft survival in 2CF1
anti-Ld TCR transgenic mice; (2) DST leads to a
deletion of the majority (>60%) of donor-reactive T cells in the
periphery of the recipient. However, deletion does not necessarily
result in tolerance; (3) remaining donor-reactive T cells from
DST-treated mice are fully responsive to Ld in vitro, and
can suppress the antidonor response of naive T cells in vitro only when
exogenous interleukin (IL)-4 is provided; and (4) the sera level of
IL-4 in DST-treated tolerant mice is significantly increased. These
results suggest that the generation of a subset of T cells with the
potential to specifically inhibit antidonor responses, together with
promotion of IL-4 production in recipients, may be important mechanisms
for the induction and maintenance of antigen-specific tolerance.
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INTRODUCTION |
THE ULTIMATE goal of transplantation is
to induce long-term tolerance to a specific allograft without
continuing need for expensive, toxic and nonspecific immunosuppressive
drugs. Numerous studies have shown that pretransplantation infusion of
allogeneic donor lymphocytes, especially when some major
histocompatibility complex (MHC) antigens are shared between donor and
recipient, has the potential to fulfill such a goal.1-10
The underlying mechanisms, as suggested by previous studies, might
include clonal deletion, induction of anergy, generation of regulatory
cells, regulation of cytokine production, promotion of microchimerism,
provision of soluble MHC antigen (Ag), or a combination of
these.7 Although experiments to address these issues have
provided useful information, no definitive conclusions have been
reached. The major obstacle to delineating the mechanisms involved in
donor-specific transfusion (DST)-induced tolerance is the lack of a
specific marker to directly follow the fate of donor-reactive cells in
the recipient after DST. Therefore, it is difficult to view the precise
mechanisms involved in DST-induced tolerance in vivo. With the
development of transgenic mice that carry transgenic T-cell receptor
(TCR) specifically recognizing transplantation Ags, it became possible
to decipher this long-standing enigma in transplantation.
In our previous studies, anti-HY TCR transgenic mice were infused with
male HY+ lymphocytes and the fate of male-reactive T cells
was followed in vivo. It was found that the majority of male-reactive T
cells were deleted from the periphery of the recipients after
encountering male Ag, and about 20% to 30% of donor-reactive T cells
persisted in the periphery for the period of our study (up to 4 months
after DST).11-13 Because for unknown reasons, the anti-HY
TCR transgenic mice can neither generate functional cytotoxic T cells
(CTL) in vivo nor reject a male graft, it is not possible to study the
effect of DST on graft survival in this particular system.
In the present study, we use 2C transgenic mice that carry a transgenic
TCR that specifically recognizes an Ld (MHC class I
molecule) as a model to delineate the mechanisms involved in
DST-induced long-term allograft survival. We found that, in the absence
of any other immunosuppression, a single dose infusion of viable
lymphocytes from the graft donor before transplantation was sufficient
to induce long-term donor-specific skin allograft survival in 2C
transgenic mice. Because the tolerance was induced in transgenic mice,
we were able to directly monitor the fate of donor-reactive cells in
vivo and to explore the mechanisms of DST-induced long-term Ag-specific
tolerance.
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MATERIALS AND METHODS |
Mice.
C57BL/6 (B6, H-2b), SJL(H-2s),
C3H(H-2k), (B6×BALB/c)F1- (BYJ,
H-2b/d Ld+) and BALB/c H-2-dm2 (dm2, a BALB/c
Ld loss mutant, H-2 Dd+, Kd+,
Ld-) mice were purchased from Jackson Laboratories (Bar
Harbor, ME). Breeding stock of 2C transgenic mice (on B6 background)
were kindly provided by Dr Dennis Y. Loh (Nippon Roche Research Center,
Kamakura-shi, Japan).14 2C (H-2b/b) transgenic
mice carry functionally rearranged TCR  -(one copy) and  -chain
(eight copies) transgenes from a cytotoxic T-cell clone 2C, which is
specific for Ld MHC class I Ag. The specificity for
Ld requires both transgenic and
chains.14,15. The 2C clonotypic TCR is recognized by
monoclonal antibody (MoAb) 1B2.16 B62C mice
(H-2b/b) were bred with dm2 mice. The subsequent
1B2+ 2CF1 mice (H-2b/d,
Ld-, anti-Ld TCR+) were maintained
in the animal colony at the Ontario Cancer Institute and used as
recipients. The BYJ mice were used as lymphocytes and skin graft
donors.
DST.
Spleen and lymph nodes were harvested from BYJ mice that mismatch only
for Ld with the recipients. Single cell suspension was
prepared by gently pressing the spleen and lymph nodes against a fine
metal screen. The red blood cells were lyzed by lysing solution. Cells
were then washed three times with 1% bovine serum albumin
(BSA)/phosphate-buffered saline (PBS). The viability was
examined by eosin exclusion and was greater than 95%. One group of
recipients was injected intravenously (IV) with 3 ×
107/mouse sex-matched viable cells either on the day of
skin grafting or 18 days before transplantation. Another group of
recipients was injected with same number of syngeneic cells from
(B6×dm2)F1 mice at the same time points as controls.
Cell surface marker staining.
To follow the fate of donor-reactive T cells after encountering donor
Ag in vivo, lymphocytes from lymph nodes and spleen of recipient mice
were collected and analyzed at various time points after DST.
Lymphocytes were stained with fluorescein isothiocyanate (FITC)-labeled
clonotypic MoAb 1B2 (which recognizes the transgenic TCR, this
hybridoma was kindly provided by Dr Herman Eisen, Massachusetts
Institute of Technology, Cambridge, MA) and phycoerythrin
(PE)-conjugated anti-CD8 MoAb (Pharmingen, San Diego, CA). Data were
acquired and analyzed on an EPICS XL-MCL flow cytometry machine
(Coulter Corp, Miami, FL).
Tail skin grafting.
Skin grafting was performed as described previously.17
Briefly, a piece of donor tail skin about 1 × 0.5 cm2
and a thickness including the epidermis and most of the dermis was
removed with a sharp scalpel and transferred to the sides on the
recipient tail from which an equivalent amount of skin had been
removed. The grafts were covered with a clear spray bandage (NEW-Skin,
Dedtech Labs, Jackson, WY) and further protected with a light, loosely
fitting transparent glass tube. Grafts were visually monitored daily
and scored as rejected when greater than 90% dead.
Mixed lymphocyte responses (MLR).
At different time points after DST, splenocytes were collected from
recipient mice and used as responder cells in an MLR. Varying numbers
of responder cells (from 1 × 103 to 1
× 104 cells/well) were cocultured in 96-well plates
with irradiated (20 Gy) sex-matched splenocytes (3 ×
105 cells/well) from BYJ mice in -minimal essential
medium (MEM) supplemented with 10% fetal calf serum
(FCS) and 30 U/mL of recombinant
interleukin-2 (rIL-2) as a source of growth factor. After
3 days of incubation, 1 µCi of [3H] thymidine
deoxy-ribonucleic (TdR) was added to each well. Fifteen
hours later, cultures were procured and counted in a beta counter.
Cytotoxicity assay.
Varying numbers of donor-reactive T cells
(1B2+CD8+), from 30 to 3,000 cells/well, were
obtained from DST-treated and naive 2CF1 mice and
cocultured with irradiated (20 Gy) sex-matched splenocytes (3 ×
105 cells/well) from BYJ mice in -MEM supplemented with
10% FCS and 30 U/mL of rIL-2 as a source of growth
factor. After 5 days of incubation, percentage lysis of specific target
cells (Ld+ P815 cells, 3,000 cells/well) was measured by a
4-hour 51Cr release assay and specific killing was
calculated by the following formula: % Specific Lysis = (cpm
Experimental  cpm Background 51Cr release)/(cpm
Total 51Cr release in 2% Acetic Acid cpm
Background 51Cr release). All samples were performed in
five replicates.
Detection of interleukin-4 (IL-4) by enzyme-linked immunosorbent
assay (ELISA).
Serum samples were collected from recipient mice before, 4, and 18 days
after DST, as well as from the mice with donor skin allografts
surviving for 120 days. The concentration of IL-4 was measured using an
IL-4 ELISA kit (Genzyme Inc, Cambridge, MA). Samples were tested in
duplicates and the amounts of IL-4 were calculated from a standard
curve and expressed in pg/mL. The sensitivity of the assay is 4 to 6
pg/mL according to the manufacturer.
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RESULTS |
Induction of long-term donor-specific skin allograft survival in 2C
transgenic mice by pretransplantation DST.
To establish a system in which the response of recipient to a defined
donor MHC allo Ag can be monitored in vivo, B62C mice were bred with
dm2 mice. The subsequent 2CF1 mice were screened by
staining the blood samples using the clonotypic MoAb 1B2 and the
1B2+ 2CF1 mice were used as recipients. Because
sharing MHC class II Ag between donor and recipient has been suggested
to be important for the induction of donor-specific
tolerance,2-4 we chose sex-matched BYJ mice that are
mismatched only for Ld Ag with the 2CF1 mice as
lymphocyte and skin graft donors. As a result, the immune response that
took place in this system would be 1B2+CD8+
cells from the recipient reacting to Ld Ag expressed on the
donor.
Based on our studies on anti-HY TCR transgenic mice,11-13
2CF1 mice were injected IV with 3 × 107
viable lymphocytes from either sex-matched syngeneic 2CF1
transgene negative littermates (control group, Fig
1, top panel) or Ld+ cells from
BYJ mice (experimental group, Fig 1, bottom panel). Eighteen days after
injection, each recipient 2CF1 transgenic mouse from both
groups received three sex-matched skin grafts from each of BYJ
(donor-specific), (B6×dm2)F1 (syngeneic control) and
SJL or C3H (H-2S and H-2k, respectively, as
third party controls) mice. As shown in Fig 1, all syngeneic skin
grafts were accepted permanently and third party allogeneic ones were
rejected between 11 to 13 days by both groups of mice, indicating that
2CF1 transgenic mice are just as able to mediate allogeneic
skin graft rejection as nontransgenic mice. Interestingly, the BYJ skin
grafts survived permanently (over 120 days) in 13 of 15 mice that
received DST, whereas they were rejected within 2 weeks by all mice in
the control group. These results clearly indicate that rejection of a
single class I Ag mismatched skin allografts mounted by
2CF1 transgenic mice can be abolished by a single dose
pretransplant DST in the absence of any other immunosuppressive agent.
Moreover, because the fate of donor-reactive T cells can be directly
monitored in vivo after DST and transplantation, it provides a good
model for delineating the cellular and molecular mechanisms involved in
the induction and maintenance of Ag-specific tolerance in vivo.
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| Fig 1.
Induction of long-term donor-specific skin graft survival
by pretransplantation infusion of donor splenocytes. 2CF1
transgenic mice were injected IV with splenocytes from Ld-
syngeneic mice (top, n = 14) or Ld+ mice (bottom, n =
15). Eighteen days after injection, each mouse was given skin grafts
from SJL ( ), BYJ ( ) and (B6×dm2)F1 ( ). The
grafts were monitored and scored after skin transplantation for more
than 120 days.
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Reduction of the number of donor-reactive T cells in the periphery of
DST-treated recipient mice.
Clonal deletion has been considered as an important mechanism for both
negative selection in the thymus and induction of peripheral tolerance
to super Ags.18-20 Our previous studies have demonstrated
that encountering allo Ag in vivo results in a deletion of specific
allogeneic T cells in the periphery via activation-induced cell
death.11,12,21,22 However, it was not known whether
deletion of donor-reactive T cells, either centrally and/or
peripherally, was involved in DST-induced long-term allograft
acceptance. Theoretically, it is possible that the infused donor cells
or peptides derived from them might enter the thymus and induce a
native selection of 1B2+CD8+ cells in the
thymus. To see whether this was the case in our system, thymocytes were
collected from 2CF1 mice that were treated with DST and
accepted the donor-skin graft. Cells were triple stained with MoAbs
that specifically recognize 1B2, CD8, and CD4. The data were analyzed
using three-color flow cytometry. The thymocytes obtained from naive
2CF1 mice were stained in the same way and used as
controls. When the percentages of 1B2+CD8+,
1B2+CD4+, CD8+CD4+,
1B2+, CD8+ and CD4+ cells in the
thymus of DST-treated mice were compared with those in the naive
2CF1 mice, no significant difference was found (data not
shown), which is consistent with our previous
findings.11,12 These experiments suggest that no thymic
deletion of donor-reactive cells was involved in DST-induced long-term
allo skin graft survival.
Next, we followed the physiologic fate of anti-Ld T cells
in the periphery after encountering donor lymphocytes. As shown in Fig
2, 4 days after DST, the number of
1B2+CD8+ cells remained similar to that in
uninjected mice. By 18 days, however, the number of donor-reactive T
cells was significantly reduced in the periphery of the recipients.
This low number of 1B2+CD8+ cells remained for
at least 35 days after a single DST treatment. In contrast, no
significant changes were seen in the number of
1B2+CD8 cells after DST. The same
results were obtained in the lymph nodes (data not shown). Together,
these expriments show that DST can reduce the number of donor-reactive
T cells in the periphery of recipients.
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| Fig 2.
Decrease of 1B2+CD8+ cells in
2CF1 transgenic mice after DST. 2CF1 mice were
injected IV with splenocytes from BYJ mice. The percentages of
1B2+CD8+ cells (A) and
1B2+CD8- cells (B) in spleen are shown from
untreated 2CF1 mice (day 0,  ), day 4 (), day 18 ( ),
and day 35 ( ) after DST. *P < .0001.
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Decrease in the number of donor-reactive T cells did not correlate
with enhancement of donor-specific skin allograft survival.
To investigate further if the reduction of donor reactive T cells
before transplantation was essential for donor-specific skin graft
acceptance, 2CF1 mice were given DST and skin grafts on the
same day when no reduction of donor reactive T cells could be detected
in the recipient. As shown in Fig 3, skin
grafts from the lymphocyte donor strain survived over 120 days in five
of six mice, whereas the third party allografts were rejected within 2
weeks by all of the 2CF1 mice that received DST. These
findings were similar to those observed in mice that received skin
grafts 18 days after DST, at which time about 60% of donor-reactive T
cells had disappeared from the periphery of the recipients. Therefore,
reduction of donor-reactive T cells before transplantation seems not to
be essential for the induction of donor-specific skin graft survival.
Furthermore, the number of 1B2+CD8+ cells in
recipient mice treated with or without DST, 120 days after
transplantation, was compared with those in naive mice. Interestingly,
although a significantly (P < .01) lower number of
1B2+CD8+ cells was observed in both lymph nodes
and spleen of mice that had DST and accepted donor skin grafts for more
than 120 days compared with those in naive mice, the number of
1B2+CD8+ cells in the 2CF1 mice
that did not receive DST and rejected the Ld+ skin grafts
was also significantly reduced (Fig 4).
This finding suggests that deletion of donor-reactive T cells may be a
consequence of encountering allo Ag in vivo, either through infused
donor lymphocytes or donor skin graft. However, reduction in the number
of donor-reactive T cells in recipients may not necessarily lead to
allograft acceptance. Together, our data indicate that although DST can
induce deletion of donor-reactive T cells, this deletion appears not to
be essential for the induction of skin allograft survival.
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| Fig 3.
Survival of donor-specific skin grafts in
2CF1 transgenic mice given DST on the day of
transplantation. 2CF1 transgenic mice (n = 6)
were injected IV with splenocytes from BYJ (Ld+). On the
same day of the injection, each mouse was given skin grafts from BYJ
() and SJL (). The grafts were monitored and scored after skin
transplantation for more than 120 days.
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| Fig 4.
Deletion of 1B2+ CD8+ cells
after transplantation in both DST-treated and non-DST-treated mice.
The spleens (left panel) and lymph nodes (right panel) from naive
(), DST-treated (), and non-DST-treated () 2CF1
mice 120 days after transplantation were collected and the percentages
of 1B2+ CD8+ cells were compared. The
percentages of 1B2+ CD8+ cells in both
DST-treated and non-DST-treated mice were significantly reduced
compared with that in the naive mice (*P < .001).
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1B2+CD8+ cells remaining in DST-treated mice
are fully responsive.
Although DST can induce deletion of the majority of donor-reactive T
cells in the recipients, it was interesting to note that the deletion
was never complete. About 30% to 40% of donor-reactive
1B2+CD8+ cells were always found to persist in
the periphery of the recipients. To study if DST has any influence on
the function of those remaining donor-reactive T cells, the remaining
1B2+CD8+ cells from 2CF1 mice 18
days after DST were isolated and stimulated by irradiated
Ld+ lymphocytes from BYJ mice in vitro. Cell proliferation
was measured by 3H-thymidine incorporation and compared
with that of naive 2CF1 mice. As shown in the top panel of
Fig 5, when the same numbers of
1B2+CD8+ cells were seeded in the culture, the
remaining 1B2+CD8+ cells proliferated in the
same way as those from naive 2CF1 mice. The same results
were obtained from another four independent experiments.
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| Fig 5.
Full responsiveness of remaining donor-reactive T cells
to donor Ag 18 days after DST. Splenocytes from 2CF1 mice
either untreated ( ) or 18 days after DST () were stimulated by
irradiated (20 Gy) splenocytes from BYJ mice in -MEM
supplemented with 10% FCS and 30 U/mL rIL-2. In the top
panel, proliferation ([3H] TdR incorporation) was
measured after a 3.5-day culture. Data are plotted as cpm versus number
of 1B2+CD8+/well. Each data point
represents five replicates. In the bottom panel, percentage lysis of
specific Ld target cells (open symbols) was measured by
51Cr release assay after a 5-day culture. Data are plotted
as percentage specific killing versus number of 1B2+
CD8+ cells/well. These results represent four independent
experiments. Killing of third-party control (H-2K) target
cells by the highest number of effector cells is shown in solid
symbols. Each data represents five replicates. Similar results were
obtained at 35 days after DST.
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To further study if the remaining donor-reactive T cells can kill
Ld+ target cells, 1B2+CD8+ cells
were isolated from 2CF1 mice 35 days after DST. Their
capacity of lysing donor-specific, as well as third-party
(H-2K) target cells, was compared with
1B2+CD8+ cells from naive mice. The bottom
panel of Fig 5 shows identical killing of Ld+ target cells
by remaining and naive 1B2+CD8+ cells. No
difference was seen in killing of third-party targets. These data
indicate that remaining 1B2+CD8+ cells in
DST-treated mice are fully reactive to the donor antigen in vitro.
Suppression of naive donor-reactive T cells by residual
1B2+CD8+ cells from tolerant mice in the
presence of IL-4.
Because none of our recipient mice were thymectomized and no deletion
of 1B2+CD8+ cells was seen in the thymus after
DST, it was possible that new naive 1B2+CD8+
cells were constantly produced by the thymus. Given the fact that
remaining 1B2+CD8+ cells in the periphery of
the DST recipients were fully capable of killing donor cells, yet did
not reject donor skin grafts, we reasoned that there must be an active
mechanism by which the function of 1B2+CD8+
cells could be inactivated in the donor graft accepted mice. In our
previous studies in which 1B2+CD8+ cells were
adoptively transferred into Ld-expressing severe combined
immunodeficiency (Scid) mice, the residual
1B2+CD8+ cells in the recipients could inhibit
proliferation of newly injected naive 1B2+CD8+
cells.21 It is possible that the remaining
1B2+CD8+ cells might themselves function as
regulatory/suppressor cells in the maintenance of donor-specific graft
survival. To test this possibility, 120 days after transplantation,
splenocytes were collected from 2CF1 mice that had received
DST and accepted skin allografts, as well as non-DST-treated
2CF1 mice that rejected skin allografts. These cells were
used as putative suppressor cells. Naive 2CF1 cells were
stimulated by irradiated Ld+ cells in an MLR to which
different ratios of suppressor cells were added. When the proliferation
of 1B2+CD8+ cells from naive 2CF1
mice was measured, a dose-dependent inhibition of
3H-incorporation by the 1B2+CD8+
cells from mice that accepted Ld skin allograft was
observed only when exogenous IL-4 was provided (Fig
6, bottom right panel). No inhibition was
observed in cells from mice that have rejected skin allografts
regardless of the presence or absence of IL-4 (Fig 6, top
and bottom, left panels), or by cells from graft tolerant mice in the
absence of IL-4 (Fig 6, top right panel). These findings
suggest that DST could promote the induction of suppressor cells, which
have the potential to inhibit the activation of naive donor-reactive T
cells. However, this suppression seems to be dependent on the presence
of IL-4.
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| Fig 6.
Suppression of naive 1B2+CD8+
T cells response to Ld in vitro by cells from tolerant mice
in the presence of exogenous IL-4. Naive
1B2+CD8+ cells (1 × 103
cells/well) from 2CF1 mice were used as responder cells and
stimulated by 3 × 105 cells/well of irradiated (20 Gy)
splenocytes from BYJ mice. 1B2+CD8+ cells
from 2CF1 mice that either rejected (left panels) or
accepted (right panels) Ld+ skin allografts 120 days
after transplantation were added into the MLR cultures as putative
suppressor cells. The suppressor to responder cell ratios were 0:1,
10:1, 5:1, 2:1, and 1:1 as indicated. Cells were cultured in -MEM
supplemented with 10% FCS, 30 U/mL rIL-2 in the presence (top panels)
or absence (bottom panels) of 50 U/mL rIL-4. Cell proliferation was
measured by [3H] TdR incorporation after a 3.5-day
culture. (), S/R = 0:1; (), S/R = 10:1; ( ), S/R = 5:1;
( ), S/R = 2:1; (), S/R = 1:1.
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Increased IL-4 expression in sera of DST-treated skin allograft
tolerant mice.
Since Mosmann et al23 first proposed the Th1/Th2 paradigm,
numerous studies have been performed to determine if Th1 and Th2 type
cytokine profiles are associated with rejection and tolerance,
respectively.24,25 Although some studies suggested the
presence of Th2 and absence of Th1-associated cytokines in graft
tolerant animals,26-27 the overall results are
controversial. Our previous work showed a significant increase in the
expression of IL-4 and decrease in IL-2 mRNA in the remaining
1B2+CD8+ cells after encountering
Ld+ Ag in vivo.21 As shown in Fig 6, only when
exogenous IL-4 was provided could the non-DST-depleted cells in skin
graft tolerant mice inhibit anti-Ld responses of naive
1B2+CD8+ cells. Based on these findings, we
wondered if the serum IL-4 level was increased in vivo after DST. Serum
samples were collected from 2CF1 mice before, 4, and 18
days after DST, as well as 120 days after skin grafting, and the
concentration of IL-4 was measured by ELISA. As shown in Fig
7, the sera level of IL-4 was increased
with time after DST and most significantly increased in mice that
permanently accepted donor skin allografts. These results suggest that
DST can promote IL-4 production, which may assist regulatory T cells in
the induction and maintenance of Ag-specific tolerance.
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| Fig 7.
Increase of IL-4 in the sera of DST-treated mice. Serum
samples were collected from 2CF1 mice before (), 4
(), and 18 () days after DST, as well as more than 120 days ()
after skin grafting. The concentration of IL-4 was measured by ELISA.
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DISCUSSION |
We have shown in this study that long-term donor-specific skin
allograft survival can be induced in 2C anti-Ld TCR
transgenic mice by a single dose infusion of donor lymphocytes before
transplantation. This donor-specific tolerance is induced and
maintained in the absence of any other immunosuppressive agent.
Moreover, the fate of donor-reactive T cells in the recipients was
directly monitored in vivo after DST and transplantation to further
delineate the mechanisms involved in the induction and maintenance of
tolerance.
Clonal deletion has been shown to be an important mechanism for the
induction of tolerance to self Ags and bacteria super
Ags.18-20 Its role in the induction of transplantation
tolerance, however, was not known. In this study, we found that after
DST, no significant change in the percentage of
1B2+CD8+ cells in the thymus of the recipients,
implying that the thymus may not be involved in DST-induced long-term
donor-skin graft survival. In contrast, there was a profound reduction
of donor-reactive T cells both in the lymph nodes and spleen (Fig 2),
suggesting that DST can induce deletion of donor-reactive T cells in
the periphery of the recipients. However, several lines of evidence
from our further studies suggested that clonal deletion may be an
epiphenomenon rather than a major mechanism involved in DST-induced
tolerance to allo Ags. First, long-term graft survival could also be
induced when DST was given on the day of transplantation, at which time
a reduction of donor reactive T cells had not occurred. Second,
significant deletion of donor-reactive T cells was also observed in
non-DST-treated mice that rejected their allografts. Third, deletion
of donor reactive T cells is never complete. In the lymph nodes and
spleen of all 2CF1 mice 18 or 35 days after DST, as well as
those with long-term surviving donor-specific skin grafts, about 30%
to 40% of the initial number of donor-reactive T cells was detected
(Figs 2 and 4).
There are three possible sources for the donor-reactive T cells seen in
the periphery after DST. (1) These cells have not encountered donor Ag;
(2) they are new emigrants from the thymus because the recipients were
not thymectomized; and (3) these cells are resistant to clonal deletion
and remain to serve as regulatory cells in the maintenance of
tolerance. Generally, a high dose of Ag and repeated stimulation are in
favor of deletion of Ag specific T cells. In this study, we gave only
one dose infusion of 3 × 107 donor lymphocytes and
the infused donor cells could be removed from within a few days to a
few weeks by CD8+ T cells in the recipient28
(and our unpublished data). It is thus possible that not every
potential donor-reactive T cell has encountered Ag. However, in our
previous studies in which 2CF1 cells were adoptively
transferred into scid mice that express Ld+,
otherwise completely matched for major and minor histocompatibility Ags
with the donor, about 20% of the initial
1B2+CD8+ cells were also found to persist in
the periphery of the recipients.22 It seems unlikely that
some anti-Ld cells persisted in the periphery due to
failure to encounter Ag in the Scid system because the Ag was
constitutively expressed on all of the nucleated cells of the
recipients. This finding also argues against the possibility that the
residual 1B2+CD8+ cells are new thymic
emigrants because the thymus of scid mice cannot produce
1B2+CD8+ cells.
In fact, the persistence of a small population of antigen-reactive T
cells in the periphery after encounter with Ags in vivo has been
observed in most of the systems where peripheral tolerance has been
induced by administration of Ags.11-13,20-22 Therefore, it
is unlikely to be a coincidence. Rather, it is possible that the
persistence of antigen-reactive T cells may represent an intrinsic
feature of the mechanism of peripheral tolerance, eg, some
antigen-reactive T cells may persist to function as
regulatory/suppressor cells in the maintenance of tolerance. This
possibility might prove to be true based on the following observations:
(1) mice made tolerant to allografts by injection of nondepleting MoAbs
to transiently block CD4 and CD8 molecules or anti-CD4 Ab plus
DST-generated lymphocytes, which could suppress naive syngeneic T cells
and also guide them to tolerance to the same Ags29,30; (2)
the T-cell clones generated from the remaining cells in mice that have
been made orally tolerant to myelin basic protein were able to protect
naive mice from developing encephalomyelitis induced by the same
protein31,32; (3) some T-cell clones from long-term
donor-specific skin graft tolerant 2CF1 mice could inhibit
the function of naive 1B2+CD8+ cells in an
Ag-specific, dose-dependent manner (Yang, et al, manuscript in
preparation). Together, these findings strongly suggest
that the generation of a population of T cells, which can specifically
suppress the function of naive syngeneic T cells in responding to the
same Ag, is one of the mechanisms of peripheral tolerance.
Our observation that the concentration of IL-4 in the serum of tolerant
mice was significantly increased supports the view that DST may promote
IL-4 production. It is not clear what the source of IL-4 is. We have
shown in the previous studies using an adoptive transfer system in
Scid mice that after encountering Ld Ag in vivo,
the persisting 1B2+CD8+ cells expressed
increased mRNA levels of IL-4 and IL-10 and decreased levels of
IL-2.21 In this study, when the mRNA level of IL-4 was
measured using reverse transcriptase-polymerase chain reaction
(RT-PCR), an increased expression of IL-4 mRNA was also observed in T
cells obtained from tolerant mice (data not shown). It is possible that
a high level of IL-4 was secreted by the non-DST-depleted T cells.
However, when cultured in vitro, these T cells seemed unable to produce
sufficient IL-4 and their suppressive function was dependent on
exogenous IL-4. These findings suggest that non-DST-depleted
donor-reactive T cells have the potential to produce IL-4, but the
production may be regulated by some other cells in vivo, such as mast
cells,33   T cells34 or a subset of
+ T cells expressing natural killer (NK) cell
markers.35
It is not known how IL-4 exerts its role in the induction
and/or maintenance of donor-specific allograft survival. It has
been suggested that IL-4 could induce immune deviation and tolerance to
autoimmune disease.36 In our system, when IL-4 was added to
an MLR without putative suppressor T cells, no inhibition of
proliferation of naive 1B2+CD8+ cells on
Ld+ cell stimulation was detected, suggesting that IL-4
alone is not able to produce tolerance. Furthermore, we found that only
when both IL-4 and regulatory cells were present at the same time could
a suppression of naive 1B2+CD8+ cells be
observed. This suggests that IL-4 may exert its effect indirectly
through regulatory T cells. These findings open a new window for our
understanding of the Th1/Th2 paradigm, as well as Ag-specific
suppression in peripheral tolerance.
| |
FOOTNOTES |
Submitted May 27, 1997;
accepted August 15, 1997.
Supported by Medical Research Council of Canada Grant No. MT
12639 (to L.Z.).
Address reprint requests to Li Zhang, MD, PhD, Department
of Cellular and Molecular Pathology, CCRW 2-852, 101 College St,
Toronto, Canada, M5G 2C4.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Drs D.Y. Loh and H.M. Eisen for kindly providing breeding
stock of 2C transgenic mouse and hybridoma 1B2, respectively. The
authors also thank Drs D.H. Sachs, R.G. Miller, R. Gorczynski, and S.
Albert for critically reading the manuscript and giving useful
comments.
| |
REFERENCES |
1.
Sharabi Y,
Sachs DH:
Mixed chimerism and permanent specific transpalntation tolerance induced by a nonlethal preparative regimem.
J Exp Med
169:493,
1989[Abstract/Free Full Text]
2.
Lagaaij EL,
Henneman IPH,
Ruigrok M,
De Haan MW,
Persijn GG,
Termijtelen A,
Hendriks GFJ,
Weimar W,
Claas FHJ,
Van Rood JJ:
Effect of one HLA-DR antigen-matched and completely HLA-DR-mismatched blood transfusion on survival of heart and kidney allografts.
N Engl J Med
321:701,
1989[Abstract]
3.
Van Rood JJ,
Class FHJ:
The influence of allogeneic cells on the human T and B cell repertoire.
Science
248:1388,
1990[Abstract/Free Full Text]
4.
Middleton D,
Martin J,
Douglas J,
McClelland M:
Transfusion of one HLA-DR antigen-matched blood to potential recipients of a renal allograft.
Transplantation
58:845,
1994[Medline]
[Order article via Infotrieve]
5.
Barber WH,
Mankin JA,
Laskow DA,
Deierhoi MH,
Julian BA,
Diethelm AG:
Long-term results of a controlled prospective study with transfusion of donor specific bone marrow in 57 cadaver renal allograft recipients.
Transplantation
51:70,
1991[Medline]
[Order article via Infotrieve]
6.
van Twuyver E,
Mooijaart RJD,
ten Berge JM,
van der Horst AR,
Wilmink JM,
Kast WM,
Melief CJM,
de Waal LP:
Pretransplant blood transfusion revisited.
N Engl J Med
325:1210,
1991[Abstract]
7.
Brennan DC,
Flye MW:
Donor-specific transfusion and donor bone marrow infusion in renal transplantation tolerance: A review of efficacy and mechanisms.
Am J Kidney Dis
26:701,
1995[Medline]
[Order article via Infotrieve]
8.
Anderson CB,
Brennan D,
Keller C,
Goss J,
Shenoy S,
Burton K,
Sicard G,
Flye MW:
Beneficial effects of donor-specific transplantation on long-term renal allograft function.
Transplant Proc
271:991,
1995
9.
Wong W,
Morris PJ,
Wood KJ:
Syngeneic bone marrow expressing a single donor class I MHC molecule permits acceptance of a fully allogeneic cardiac allograft.
Transplantation
62:1462,
1996[Medline]
[Order article via Infotrieve]
10.
der Zwan AV,
Besseling AC,
de Waal LP,
Boog CJP:
Specific tolerance induction and transplantation: A single-day protocol.
Blood
89:2596,
1997[Abstract/Free Full Text]
11.
Zhang L,
Martin DR,
Fung-Leung WP,
Teh HS,
Miller RG:
Peripheral deletion of mature CD8+antigen-specific T cells after in vivo exposure to male antigen.
J Immunol
148:3740,
1992[Abstract]
12.
Zhang L,
Shannon J,
Sheldon J,
Teh HS,
Mak TW,
Miller RG:
Role of infused CD8+ cells in the induction of peripheral tolerance.
J Immunol
152:2222,
1994[Abstract]
13.
Zhang L,
Fung-Leung W,
Miller RG:
Down-regulation of CD8 on mature antigen-reactive T cells as a mechanisms of peripheral tolerance.
J Immunol
155:3464,
1995[Abstract]
14.
Sha WC,
Nelson CA,
Newberry RD,
Kranz DM,
Russell JH,
Loh DY:
Selective expression of an antigen receptor on CD8-bearing T lymphocytes in transgenic mice.
Nature
335:271,
1988[Medline]
[Order article via Infotrieve]
15.
Sha WC,
Nelson CA,
Newberry RD,
Kranz DM,
Russell JH,
Loh DY:
Positive and negative selection of an antigen receptor on T cells in transgenic mice.
Nature
336:73,
1988[Medline]
[Order article via Infotrieve]
16.
Kranz DM,
Tonegawa S,
Eisen HN:
Attachment of an anti-receptor antibody to non-target cells renders them susceptible to lysis by a clone of cytotoxic T lymphocytes.
Proc Natl Acad Sci USA
81:7922,
1984[Abstract/Free Full Text]
17.
Zhang L,
Miller RG:
The correlation of prolonged survival of maternal skin grafts with the presence of naturally transferred maternal T cells.
Transplantation
56:918,
1993[Medline]
[Order article via Infotrieve]
18.
Kappler J,
Wade T,
White J,
Kushnir E,
Blackman M,
Bill J,
Roehm N,
Marrack P:
A T cell receptor V segment that imparts reactivity to a class-II major histocompatibility complex product.
Cell
49:272,
1987
19.
Kisielow P,
Bluthmann H,
Staerz UD,
Steinmetz M,
Von Boehmer H:
Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+CD8+ thymocytes.
Nature
333:742,
1988[Medline]
[Order article via Infotrieve]
20.
Webb S,
Morris C,
Sprent J:
Extrathymic tolerance of mature T cells: Clonal elimination as a consequence of immunity.
Cell
63:1249,
1990[Medline]
[Order article via Infotrieve]
21.
Zhang L,
Miller RG,
Zhang J:
Characterization of apoptosis-resistant antigen-specific T cells in vivo.
J Exp Med
183:2065,
1996[Abstract/Free Full Text]
22.
Zhang L:
The fate of adoptively transferred antigen-specific T cells in vivo.
Eur J Immunol
26:2208,
1996[Medline]
[Order article via Infotrieve]
23.
Mosmann TR,
Coffman RL:
Th1 and Th2 cells: Different patterns of lymphokine secretion lead to different functional properties.
Annu Rev Immunol
7:145,
1989[Medline]
[Order article via Infotrieve]
24.
Dallman M:
Cytokines and transplantation: Th1/Th2 regulation of the immune response to solid organ transplants in the adult.
Curr Opin Immunol
7:632,
1995[Medline]
[Order article via Infotrieve]
25.
Strom TB,
Roy-Chaudhury P,
Manfro R,
Zheng XX,
Nickerson PW,
Wood K,
Bushell A:
The Th1/Th2 paradigm and the allograft response.
Curr Opin Immunol
8:688,
1996[Medline]
[Order article via Infotrieve]
26.
Siegling A,
Lehmann M,
Riedel H,
Platzer C,
Brock J,
Emmrich F,
Volk H:
A nondepleting anti-rat CD4 monoclonal antibody that suppresses T helper 1-like but not T helper 2-like intragraft lymphokine secretion induces long-term survival of renal allografts.
Transplantation
57:464,
1994[Medline]
[Order article via Infotrieve]
27.
Levy AE,
Alexander JW:
Administration of intragraft interleukin-4 prolongs cardiac allograft survival in rats treated with donor-specific transfusion/cyclosporine.
Transplantation
60:405,
1995[Medline]
[Order article via Infotrieve]
28.
Fast LD:
Recipient CD8+ cells are responsible for the rapid elimination of allogeneic donor lymphoid cells.
J Immunol
157:4805,
1996[Abstract]
29.
Cobbold SP,
Adams E,
Marshall SE,
Davies JD,
Waldmann H:
Mechanisms of peripheral tolerance and suppression induced by monoclonal antibodies to CD4 and CD8.
Immunol Rev
149:5,
1996[Medline]
[Order article via Infotrieve]
30.
Chen ZK,
Cobbold SP,
Waldmann H,
Metcalfe S:
Amplification of natural regulatory immune mechanisms for transplantation tolerance.
Transplantation
9:1200,
1996
31.
Weiner HL,
Friedman A,
Miller A,
Khoury SJ,
Al-Sabbagh A,
Santos L,
Sayegh M,
Nussenblatt RB,
Trentham DE,
Hafler DA:
Oral tolerance: Immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens.
Annu Rev Immunol
12:809,
1994[Medline]
[Order article via Infotrieve]
32.
Chen Y,
Inobe J,
Marks R,
Gonnella P,
Kuchroo VK,
Weiner HL:
Peripheral deletion of antigen-reactive T cells in oral tolerance.
Nature
376:177,
1995[Medline]
[Order article via Infotrieve]
33.
Brown MA,
Pierce JH,
Watson CJ,
Falco J,
Ihle JN,
Paul WE:
B cell stimulatory factor-1/interleukin-4 mRNA is expressed by normal and transformed mast cells.
Cell
50:809,
1987[Medline]
[Order article via Infotrieve]
34.
Ferrick DA,
Schrenzel MD,
Mulvania T,
Hsieh B,
Ferlin WG,
Lepper H:
Differential production of interferon- and interleukin-4 in response to Th1- and Th2-stimulating pathogens by T cells in vivo.
Nature
373:255,
1995[Medline]
[Order article via Infotrieve]
35.
Arase H,
Arase N,
Nakagawa K,
Good RA,
Onoe K:
NK1.1+ CD4+CD8- thymocytes with specific lymphokine secretion.
Eur J Immunol
23:307,
1993[Medline]
[Order article via Infotrieve]
36.
Rocken M,
Racke M,
Shevach EM:
IL-4-induced immune deviation as antigen-specific therapy for inflammmatory autoimmune disease.
Immunol Today
17:225,
1996[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract