Vigorous Allograft Rejection in the Absence
of Danger
This information is current as
of June 17, 2017.
Adam W. Bingaman, Jongwon Ha, Seung-Yeun Waitze,
Megan M. Durham, Hong Rae Cho, Carol Tucker-Burden,
Rose Hendrix, Shannon R. Cowan, Thomas C. Pearson and
Christian P. Larsen
J Immunol 2000; 164:3065-3071; ;
doi: 10.4049/jimmunol.164.6.3065
http://www.jimmunol.org/content/164/6/3065
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References
Vigorous Allograft Rejection in the Absence of Danger1
Adam W. Bingaman,* Jongwon Ha,* Seung-Yeun Waitze,* Megan M. Durham,*
Hong Rae Cho,† Carol Tucker-Burden,* Rose Hendrix,* Shannon R. Cowan,*
Thomas C. Pearson,2* and Christian P. Larsen2*
T
he adaptive immune system has a remarkable capacity to
respond to and control a myriad of pathogens and at the
same time only rarely causes destructive immune responses directed against autologous tissues. Several conceptual
models have been proposed to explain how the immune system so
effectively prevents the development of pathologic autoimmune
responses. The classic model of self-tolerance holds that developing T cells undergo an education process resulting in a repertoire
of T cells that distinguish between self- and non-self-Ags (1–3). It
is thought that most autoreactive T cells are deleted in the thymus
during the process of negative selection (4 – 6). However, some
self-molecules appear to be expressed exclusively in peripheral
(nonthymic) tissues such as inducible proteins, hormones, or proteins with a developmentally restricted pattern of expression. It has
been proposed that self-reactive T cells that escape from the thymus are deleted or inactivated by additional, less understood mechanisms in the periphery (7–10).
More recently, an alternative model has been proposed to explain the ability of T cells to control pathogens while failing to
injure host tissues (11, 12). This model suggests that the immune
system does not primarily distinguish self from non-self but rather
recognizes the context in which Ags are presented. In the setting of
inflammation or tissue injury, “danger” signals induce the expression of critical costimulatory molecules on APC, thus permitting T
cells to fully respond to Ags presented in this context. Conversely,
it is proposed that under homeostatic conditions, in the absence of
inflammation, Ag presentation occurs without costimulation, leading to specific T cell unresponsiveness and ultimately to tolerance.
Previous studies addressing mechanisms of peripheral tolerance
have explored the immunogenicity or tolerogenicity of neo-selfAgs expressed in the periphery using transgenic murine models.
These studies have yielded inconsistent results. Some models have
demonstrated development of T cell tolerance to the transgenic Ag
(13–15), whereas other models have resulted in T cell reactivity
against immunogenic forms of the same Ag (16 –21). In this report,
we have used immunodeficient Rag 1⫺/⫺ mice to study the response of T cells to neo-self peripheral Ags in the form of wellhealed skin or vascularized cardiac allografts. This approach ensures no thymic expression of the alloantigens and enables us to
study whether quiescent allografts can tolerize circulating T cells.
Surprisingly, we show that well-healed allografts with no evidence
of inflammation are promptly rejected by adoptive transfer of T
cells and by newly emerging T cells in recipients reconstituted by
bone graft transplantation. Furthermore, skin grafts with isolated
multiple major or multiple minor histocompatibility differences are
also rejected by newly emerging T cells. These experiments demonstrate for the first time in a nontransgenic model that newly
emerging T cells are neither ignorant to nor tolerized by alloantigens expressed on well-healed transplanted organs.
Materials and Methods
*Carlos and Marguerite Mason Transplantation Research Center, Department of Surgery, Emory University School of Medicine, Atlanta, GA, 30322; and †Ulsan University Department of Surgery, Ulsan, Korea
Received for publication July 27, 1999. Accepted for publication January 3, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health Grants 1 R01
DK50762-01, 1 R01 AI40519-01, and AR42687 and by the Carlos and Marguerite
Mason Trust.
2
Address correspondence and reprint requests to Dr. Christian P. Larsen or Dr.
Thomas C. Pearson, Emory University Transplantation Immunology Laboratory,
Suite 5105, WMB, 1639 Pierce Drive, Atlanta, GA 30322. E-mail addresses:
[email protected] or [email protected]
Copyright © 2000 by The American Association of Immunologists
Mice
Adult male 6- to 8-wk-old C57BL/6, BALB/c, C3H/HeJ, B6CBy F1,
B6.CH-2d, C.B10H-2b, C57BL/6 nude, and B6CBy F1 nude mice were
obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 Rag
1⫺/⫺ mice were obtained from The Jackson Laboratory and bred as homozygotes under sterile conditions at Emory University (Atlanta, GA).
Skin grafting
Full-thickness ear skin grafts (⬃1 cm2) were transplanted onto the thorax
of recipient mice and secured with a Band-Aid (Johnson & Johnson, Arlington, TX) for 7 days. Mice were housed individually under sterile conditions with sterile food and water for the duration of all experiments.
Rejection was defined as the complete loss of viable epidermal graft tissue.
0022-1767/00/$02.00
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Tolerance to self is a necessary attribute of the immune system. It is thought that most autoreactive T cells are deleted in the
thymus during the process of negative selection. However, peripheral tolerance mechanisms also exist to prevent development of
autoimmune diseases against peripheral self-Ags. It has been proposed that T cells develop tolerance to peripheral self-Ags
encountered in the absence of inflammation or “danger” signals. We have used immunodeficient Rag 1ⴚ/ⴚ mice to study the
response of T cells to neo-self peripheral Ags in the form of well-healed skin and vascularized cardiac allografts. In this paper we
report that skin and cardiac allografts without evidence of inflammation are vigorously rejected by transferred T cells or when
recipients are reconstituted with T cells at a physiologic rate by nude bone graft transplantation. These results provide new
insights into the role of inflammation or “danger” in the initiation of T cell-dependent immune responses. These findings also have
profound implications in organ transplantation and suggest that in the absence of central deletional tolerance, peripheral tolerance
mechanisms are not sufficient to inhibit alloimmune responses even in the absence of inflammation or danger. The Journal of
Immunology, 2000, 164: 3065–3071.
3066
VIGOROUS ALLOGRAFT REJECTION IN THE ABSENCE OF DANGER
Bone grafting
Femurs were harvested from donor mice and cleaned of connective tissue
under sterile conditions. Femurs were cut into 8 –10 small (1- to 2-mm)
fragments, and fragments of one femur were transplanted under the kidney
capsule of recipient mice.
Heart transplantation
Vascularized heterotopic heart transplantation was performed using microsurgical techniques essentially as described (22). Graft survival was followed by daily palpation. Rejection was defined by the loss of palpable
cardiac contractions, which was confirmed with direct visualization at
laparotomy.
Cell preparation for adoptive transfer
Flow cytometric analysis
Analysis of peripheral blood was conducted using fluorochrome-conjugated Abs (anti-CD4 and anti-CD8, PharMingen) or Ig isotype control (Rt
IgG2a, PharMingen) before lysis of red blood cells and washing with a
whole blood lysis kit (R&D Systems, Minneapolis, MN). Stained cells
were analyzed using Cellquest software on a FACScan flow cytometer
(Becton Dickinson).
Histology and immunocytochemistry
Fresh tissues were fixed in molecular biology fixative (Streck Laboratories,
Omaha, NE) for 2 h and then in 70% alcohol until ready for use. When
ready, tissues were processed and embedded in paraffin (Fisher Scientific,
Pittsburgh, PA). Five-micron thick tissue sections were cut on a microtome
and stained with hematoxylin and eosin according to standard procedures.
For immunohistochemical staining, sections were deparaffinized and then
rehydrated. Sections were then incubated with an avidin/biotin block kit
(Vector Laboratories, Burlingame, CA) according to the manufacturer’s
instructions before quenching with 3% H202 for 5 min. Sections were then
stained with biotinylated anti-Ly6-G or Rt IgG2b (PharMingen) before
incubation with ABC complex (Vector Laboratories). Peroxidase activity
was visualized using diaminobenzidine substrate (Pierce, Rockford, IL).
RT-PCR
At specified intervals after transplantation, the skin allografts were removed. Assessment of transcript expression was performed using RT-PCR
on a Perkin-Elmer 9600 Thermocycler (Norwalk, CT) as described (23).
The annealing temperature for IL-1␤ was 57°C; for macrophage-inflammatory protein-2 (MIP-2),3 56°C; for B7.1, 50°C; and for ␤-actin, 50°C.
The primers and cycle numbers used were as follows: for IL-1␤, forward
5⬘-GCAACTGTTCCTGAACTCA-3⬘ and reverse 5⬘-CTCGGAGCCTG
TAGTGCAG-3⬘, 22 cycles; for MIP-2, forward 5⬘-TGTCAATGCCT
GAAGACCCTGC-3⬘ and reverse 5⬘-CCGACTGCATCTATTTGTCCCC3⬘, 20 cycles; for B7.1, forward 5⬘-AGTTGTCCATCAAAGCTGAC-3⬘
and reverse 5⬘-CTAAAGGAAGACGGTCT-3⬘, 26 cycles; and for ␤-actin,
forward 5⬘-ATGGATGACGATATCGCT-3⬘ and reverse 5⬘-ATGAGG
TAGTCTGTCAGGT-3⬘, 17 cycles. PCR products were analyzed on
3
Abbreviation used in this paper: MIP, macrophage-inflammatory protein.
Results
Well-healed allogeneic skin grafts on Rag 1⫺/⫺ mice have no
evidence of inflammation or “danger”
To study the role of acute tissue injury (“danger”) in the initiation
of allograft rejection and the induction of T cell tolerance to transplanted tissues, we developed an experimental model using T celldeficient C57BL/6 (H-2b, B6) Rag 1⫺/⫺ mice, which are unable to
reject allografts. By reconstituting these mice with T cells at various times after transplantation of a BALB/c (H-2d) skin graft, we
were able to study responses to acutely inflamed, freshly transplanted allografts or to uninflamed, well-healed allografts (quiescent allografts). First, we compared the freshly transplanted allografts with allografts that had been allowed to heal for 50 days on
unreconstituted B6 Rag 1⫺/⫺ recipients. Freshly transplanted skin
grafts appeared thickened and edematous, with granulation tissue
around the edges of the graft. Histologically, these grafts showed
prominent neovascularization and diffuse infiltration with Ly6-G⫹
polymorphonuclear leukocytes (Fig. 1A). In contrast, 50 days after
transplantation, epithelialization of the surrounding graft bed was
complete and the BALB/c skin grafts appeared grossly wellhealed. Histologically, the quiescent allografts were indistinguishable from normal skin with complete resolution of the granulocytic
infiltrate (Fig. 1A). To determine whether the freshly transplanted
and quiescent allografts differed in expression of mediators of inflammation or costimulation, we analyzed transcript expression by
RT-PCR. Analysis of freshly transplanted skin harvested from Rag
1⫺/⫺ recipients demonstrated markedly increased expression of
transcripts for IL-1␤, MIP-2, and B7.1, which is indicative of a
local innate immune response consistent with acute inflammation
associated with tissue injury. By 50 days, the expression of these
transcripts had returned to basal levels observed in normal skin,
confirming the resolution of inflammation or “danger” within the
quiescent allografts (Fig. 1B).
Adoptively transferred T cells mediate prompt rejection of
well-healed skin allografts
Next, we used this model to test the hypothesis that alloantigens
presented in an inflammatory or dangerous environment would
elicit immunity and rejection, whereas the same alloantigens presented in the absence of inflammation would fail to generate a
rejection response and would instead induce tolerance. For this, we
adoptively transferred 1 ⫻ 107 B6 T cells into B6 Rag 1⫺/⫺ mice
that had freshly transplanted skin allografts or well-healed allografts that had been transplanted 50 days before reconstitution
(Fig. 2a). As expected, Rag 1⫺/⫺ mice that were not reconstituted
with T cells accepted allogeneic skin grafts indefinitely. Surprisingly, Rag 1⫺/⫺ mice that were reconstituted with B6 T cells
promptly rejected both well-healed and acutely transplanted skin
allografts with identical kinetics. Because there is evidence that the
costimulatory requirements of CD4 and CD8 T cells differ (24, 25)
and that high-affinity CD8 T cells may be activated in the absence
of any costimulation (26, 27), we performed similar experiments
using purified CD4 and CD8 T cells for the reconstitution. As for
the mixed T cell population, we observed that either CD4 or CD8
T cells could mediate prompt rejection of both acute and wellhealed BALB/c skin allografts (Figs. 2, b and c). These results
suggest that acute tissue injury is not required in order for transplanted allogeneic tissues to elicit prompt rejection responses.
However, it is possible that other danger signals provided by the
innate immune system play an important role in this process. Although Rag 1⫺/⫺ mice do not develop T or B cells, they have a
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Splenic and mesenteric lymph node cells were harvested from B6 mice.
After red blood cell lysis with Tris-buffered ammonium chloride (Sigma,
St. Louis, MO), T cell-enriched populations were prepared as nylon woolnonadherent cells. T cell subsets were prepared from nylon wool-nonadherent cells by incubation with rat anti-mouse CD11b (M1/70), anti-mouse
CD45R (B220), and either anti-mouse CD4 (GK1.5) or anti-mouse CD8
(TIB105) (all from American Type Culture Collection, Manassas, VA) for
20 min on ice. Cells were then washed and incubated with goat anti-rat IgG
biospheres (BioSource International, Camarillo, CA) (20:1 bead:target ratio) for 20 min on ice. Cell suspensions were then placed on a magnet for
15 min and collected, and viable cells were counted using trypan blue
exclusion. Adequacy of T cell subset depletion (⬍1% contaminating cells)
was confirmed on a FACScan flow cytometer (Becton Dickinson, Braintree, MA) using PE-conjugated Abs (anti-CD4 and anti-CD8, PharMingen,
San Diego, CA) or isotype control (Rt IgG2a, PharMingen). T cells (1 ⫻
107) were adoptively transferred into recipient mice via penile vein injection. For adoptive transfer of thymocytes, thymuses were harvested from
6-wk-old B6 mice, red blood cells were lysed in Tris-buffered ammonium
chloride, and viable cells were counted using trypan blue exclusion. For
adoptive transfer experiments, mice received 5 ⫻ 107 thymocytes i.v. by
penile vein injection.
ethidium bromide-stained, 1.5% Separide (Life Technologies, Gaithersburg, MD) gels.
The Journal of Immunology
3067
normal population of NK cells that may be activated by the absence of self-MHC class I molecules on the transplanted allograft
(28, 29). Therefore, it is possible that NK cells in the Rag 1⫺/⫺
recipients may have provided danger signals that were responsible
for the activation of the adoptively transferred T cells and rejection
of the well-healed allografts. To explore this possibility, we performed similar experiments in which semiallogeneic B6⫻BALB/c
F1 (H-2bxd) skin grafts were transplanted onto B6 Rag 1⫺/⫺ recipients. In this experiment the transplanted skin graft would express the same alloantigens as a fully allogeneic BALB/c skin
graft. However, because the semiallogeneic grafts also express recipient (H-2b) MHC class I genes, NK cell recognition of “missing
self” on the transplanted grafts will not occur, and therefore, NK
cells will not be activated against the graft. As with the previous
experiments, adoptive transfer of T cells resulted in prompt rejection of both the acutely transplanted and well-healed allografts
(Fig. 2d). Thus, adoptively transferred T cells do not require inflammation or NK cell activation via recognition of “missing self”
to initiate or mediate skin allograft rejection.
Our initial experiments were performed using splenic T cells
prepared from adult B6 mice. Such populations would be expected
to contain both naive and memory T cells. “Experienced” or memory T cells have a lower threshold for activation than naive (virgin)
T cells do (30 –32). Because T cells that recognize environmental
Ags may cross-react with alloantigens (33–35), memory T cells
within these preparations may have been responsible for the re-
jection of the well-healed allografts in the absence of “danger.” To
determine whether naive T cells are able to generate immune responses to well-healed allografts, we adoptively transferred thymocytes, which contain only T cell progenitors or naive T cells,
from B6 mice into B6 Rag 1⫺/⫺ mice with well-healed or freshly
transplanted skin allografts (Fig. 2e). As in the previous experiments with splenic T cells, Rag 1⫺/⫺ recipients reconstituted with
thymocytes promptly rejected both acute and well-healed skin allografts at the same rate. As an alternative approach to addressing
this issue, we used neonatal splenocytes to perform the reconstitution and again observed similar rejection of both acute and wellhealed allografts (data not shown). These data suggest that naive T
cells can mediate prompt rejection of fully allogeneic skin grafts in
the absence of any detectable inflammation.
Developing T cells populate the periphery and mediate rejection
of well-healed skin allografts
These initial experiments indicated that acute tissue injury was not
required for the initiation of T cell responses to transplanted tissues. Furthermore, these results suggested that encounter with Ags
in the absence of costimulation failed to induce tolerance. It is
possible that some of the transferred cells became activated during
ex vivo preparation before adoptive transfer. Additionally, there is
evidence that peripheral tolerance mechanisms can be overcome
by a large dose of potentially autoreactive cells (36, 37). To address these possibilities, we studied whether alloreactive T cells
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FIGURE 1. Histology and transcript expression of freshly transplanted and quiescent
skin allografts. BALB/c skin allografts were
harvested from B6 Rag 1⫺/⫺ recipients on
days 2, 28, and 50 after transplantation. Normal BALB/c skin was harvested for comparison. A, Tissue sections were stained with a
standard histochemical procedure for hematoxylin and eosin before immunohistochemical
staining with anti-Ly6-G Ab. a, Normal
BALB/c skin showing typical skin architecture
with an absence of Ly6-G⫹ cells. b, Skin allograft harvested 2 days after transplantation
showing diffuse infiltration of Ly6-G⫹ polymorphonuclear leukocytes into the dermis. c
and d, Skin allografts harvested 28 and 50 days
after transplantation showing restoration of
normal skin architecture with complete resolution of Ly6-G⫹ polymorphonuclear infiltrate.
B, Total RNA from harvested tissues was analyzed by RT-PCR for IL-1␤, MIP-2, B7.1,
and ␤-actin. PCR products were analyzed on
ethidium bromide-stained agarose gels. IL-1␤,
MIP-2, and B7.1 transcripts were all induced
in day 2 skin allografts. Expression patterns
returned to basal levels in allografts harvested
on days 28 and 50.
3068
VIGOROUS ALLOGRAFT REJECTION IN THE ABSENCE OF DANGER
emerging from the thymus at a physiologic rate would be tolerized
or activated by encounter with alloantigens in the form of a wellhealed skin allograft. To investigate this possibility, we developed
a model to physiologically reconstitute Rag 1⫺/⫺ mice by transplanting bone marrow grafts from T cell-deficient B6 nude mice
under the kidney capsule of the Rag 1⫺/⫺ recipient. In this system,
progenitors from the nude bone graft traffic to the Rag 1⫺/⫺ thymus mature into CD4⫹ and CD8⫹ T cells and populate the pe-
4
A. W. Bingaman, S. Waitze, D. F. Alexander, H. R. Cho, A. Lin, C. Tucker-Burden,
S. R. Cowan, T. C., Pearson, and C. P. Larsen. Transplantion of the bone marrow
microenvironment leads to hematopoietic chimerism without cytoreductive conditioning. Submitted for publication.
FIGURE 3. Emerging T cells reject well-healed skin allografts. B6 Rag 1⫺/⫺ mice were transplanted with a B6 nude bone graft under the kidney capsule
(f) and skin grafts from BALB/c (a); C.B10.H-2b (b), and B6.C.H-2d (c) donors. On day 28 after transplantation, skin grafts were well-healed (Fig. 1) and
recipients had no detectable T cells by flow cytometry (data not shown). In nude bone graft recipients, skin grafts in each group were rejected with similar
kinetics. Rag 1⫺/⫺ recipients that did not receive B6 nude bone grafts (〫) did not reject allografts. Similar results were obtained in experiments in which
transplantation of the nude bone graft was delayed for 90 days.
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FIGURE 2. Freshly transplanted and quiescent skin allografts are rejected by adoptively transferred T cells with similar kinetics. B6 Rag 1⫺/⫺
recipients of freshly transplanted (day 0 with respect to adoptive transfer,
〫) or quiescent (day 50, f) BALB/c skin allografts were reconstituted
with 1 ⫻ 107 syngeneic (B6) unseparated T cells (a), CD4⫹ T cells (b), or
CD8⫹ T cells (c). Freshly transplanted and quiescent allografts were
promptly rejected in all groups with similar kinetics. d, B6 Rag 1⫺/⫺ recipients of freshly transplanted and quiescent B6C skin allografts were
reconstituted with 1 ⫻ 107 syngeneic (B6) unseparated T cells with similar
prompt rejection in both groups. e, Adoptive transfer of 5 ⫻ 107 B6 thymocytes resulted in prompt rejection of well-healed and freshly transplanted BALB/c skin allografts. a, Recipients with quiescent allografts
reconstituted with saline (E) did not reject allografts. Similar results were
obtained in experiments in which T cell reconstitution was delayed for
⬎100 days.
riphery between 4 and 8 wk after transplantation.4 Therefore, B6
Rag 1⫺/⫺ recipients of BALB/c skin grafts were reconstituted by
transplantation of B6 nude bone marrow grafts. Flow cytometry of
peripheral blood confirmed that recipient mice contained no detectable T cells 4 wk after transplantation (data not shown). Furthermore, skin allografts harvested from Rag 1⫺/⫺ recipient mice
4 wk after transplantation appeared well-healed and demonstrated
no evidence of inflammation or “danger” by histology or RT-PCR
(Fig. 1). Nonetheless, as T cells emerged over the ensuing several
weeks, recipients of B6 nude bone grafts uniformly rejected their
BALB skin allografts (Fig. 3a). To confirm that rejection was specific for the allogeneic skin grafts, we performed similar experiments in which B6⫻BALB/c F1 (B6C) nude bone grafts were
transplanted into B6 Rag 1⫺/⫺ recipients simultaneously with
BALB/c and C3H/HeJ (H-2k) skin grafts. After 6 –12 wk, recipients of B6C nude bone grafts rejected all well-healed C3H/HeJ
skin grafts, whereas BALB/c grafts remained well-healed indefinitely (data not shown). These data indicate that naive alloreactive
T cells emerging from the thymus at a physiologic rate are not
tolerized by encounter with alloantigens in the form of a quiescent
skin allograft. Rather, even uninflamed allogeneic tissues elicit a
vigorous rejection response. Thus, whereas T cell recognition of
Ag in the thymus confers robust tolerance, encounter with the
same Ag in the periphery elicits immunity. Therefore, in the setting of an allogeneic transplant, peripheral tolerance mechanisms
appear to be unable to fully compensate for failure to delete Agspecific T cells in the thymus.
Previous studies have demonstrated peripheral tolerance using
transgenic mice that express single allogeneic MHC class I or class
II genes on peripheral tissues (13–15). Because our studies examined allogeneic skin grafts with multiple major and minor histocompatibility differences, it is possible in the setting of these multiple antigenic differences that peripheral tolerance mechanisms
alone might not be sufficient to tolerize a strong polyclonal T cell
response with a wide range of affinities. Additionally, it is possible
that unidentified superantigens expressed by the BALB/c genome
could induce an immune response in the absence of costimulation.
To address these possibilities, we performed similar experiments
with simultaneous transplantation of B6 nude bone graft and
The Journal of Immunology
3069
isolated multiple major (B6.C.H-2d) or minor (C.B10.H-2b) histocompatibility disparate skin grafts from congenic mice onto B6
Rag 1⫺/⫺ mice. After 6 wk, all skin grafts were smooth and had
healthy hair growth. However, grafts slowly began to show evidence of rejection with small ulcerations, and all skin grafts from
both groups with multiple major and multiple minor histocompatibility differences were completely rejected (Fig. 3, b and c). Thus,
emerging T cells populate the periphery and reject well-healed
skin grafts with isolated multiple major or multiple minor histocompatibility differences.
T cells mediate rejection of vascularized cardiac allografts in
the absence of danger
Our data demonstrate that well-healed skin allografts can be rejected by circulating T cells and that they do not induce tolerance
of newly emerging T cells. Recent evidence in a transgenic model
suggests that adult mice fail to develop tolerance to allogeneic
MHC class I Ag in the skin due to low levels of intradermal T cell
traffic (38). Therefore, it is possible that circulating naive T cells
are never exposed to the well-healed skin allograft and thus are not
rendered tolerant to it. Furthermore, because the skin allograft is
exposed to the external environment, minor trauma could elicit a
cutaneous inflammatory response and rejection of the allograft.
Additionally, there is evidence that vascularized allografts can induce tolerance to themselves under some circumstances (39) and
therefore may influence the responses of peripheral alloreactive T
cells more effectively than nonvascularized grafts. Thus, it was
possible that well-healed vascularized allografts with higher levels
of T cell traffic through the donor-derived endothelium could induce peripheral tolerance in this model. To address this possibility,
we performed similar experiments to determine whether adoptively transferred T cells or newly emerging T cells could reject or
be tolerized by the presence of a well-healed heterotopic vascularized cardiac allograft (Fig. 4). Surprisingly, adoptive transfer of
T cells into Rag 1⫺/⫺ mice on the day of cardiac transplantation or
50 days after transplantation resulted in prompt rejection and extensive lymphocytic infiltration and myocardial damage of the vascularized allografts. Furthermore, simultaneous cardiac allograft
and nude bone graft transplantation resulted in rejection of cardiac
allografts between 8 and 12 wk, which was associated with a diffuse lymphocytic infiltrate and destruction of myocytes. Thus,
well-healed vascularized cardiac allografts are also promptly rejected by T cells and are unable to independently promote peripheral tolerance induction of naive thymic emigrants.
Discussion
The results presented here provide new insights into the role of
inflammation or “danger” in the initiation of T cell-dependent immune responses. In our model, T cell-deficient mice reject wellhealed skin and vascularized cardiac allografts upon adoptive
transfer of T cells or when they develop T cells from a transplanted
bone marrow graft. There are several possible explanations for the
immunogenicity of the well-healed allografts. It is well known that
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FIGURE 4. A, Transferred T cells
infiltrate well-healed cardiac allografts.
B6 Rag 1⫺/⫺ recipients of BALB/c cardiac allografts were reconstituted with
saline 50 days after heart transplantation (a), 1 ⫻ 107 B6 T cells on the day
of transplantation (b), and 1 ⫻ 107 B6
T cells 50 days after transplantation (c).
Cardiac allografts were harvested 7
days after reconstitution and stained
with hematoxylin and eosin. Allografts
from recipients reconstituted with saline
50 days after transplantation demonstrate
no evidence of rejection or infiltrate. Recipients of freshly transplanted allografts
(reconstituted with T cells on day 0) or
quiescent allografts (reconstituted with T
cells on day 50) demonstrated comparable marked lymphocytic infiltration of
the myocardium. B, Newly emerging T
cells reject well-healed cardiac allografts. B6 Rag 1⫺/⫺ recipients of
BALB/c vascularized cardiac allografts
and B6 nude bone grafts (f) rejected
well-healed allografts between 50 and
70 days. Rag 1⫺/⫺ recipients of
BALB/c cardiac allografts that did not
receive B6 nude bone grafts (〫open
diamonds) did not reject allografts.
3070
VIGOROUS ALLOGRAFT REJECTION IN THE ABSENCE OF DANGER
Finally, these results have profound implications in organ transplantation. These findings suggest that in the absence of central
deletional tolerance, peripheral tolerance mechanisms are not sufficient to inhibit alloimmune responses even in the absence of inflammation or danger. These data are consistent with reports of
patients with well-functioning allografts many years after transplantation who promptly reject their allografts upon cessation of
immunosuppressive drugs (47– 49). Because new T cells continue
to develop into late adult life (50), future tolerance-induction protocols cannot rely on the well-healed allograft to induce peripheral
tolerance of emerging T cells. Rather, strategies to effect thymic
selection, such as bone marrow transplantation to induce hematopoietic chimerism or strategies to induce active donor-specific regulatory T cells, may be required to maintain long-term allograft
acceptance in the absence of chronic immunosuppression.
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