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* Harrison Department of Surgical Research, Department of Surgery, Hospital of the University of Pennsylvania, and
Wistar Institute, Philadelphia, PA 19104
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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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Our laboratory has previously reported that inoculation of donor Ags into the recipient thymus can promote donor-specific tolerance (2, 3, 4, 5). Although the mechanism underlying this finding remains unknown, several hypotheses have been put forward to explain the phenomenon. We and others have previously proposed that clonal deletion of Ag-reactive T cells may be predominantly responsible (6, 7, 8, 9, 10). However, this mechanism cannot account for the observation that depletion of already mature peripheral T cells is not invariably required by these protocols (11, 12, 13, 14). That the introduction of Ag into the thymus may exert a tolerogenic effect on an already established peripheral immune system requires further explanation.
The recent functional and phenotypic characterization of immunoregulatory CD4+CD25+ T cells invites the hypothesis that these cells may be responsible for the translation of tolerance from an Ag-inoculated thymus to a mature but naive peripheral immune system (15, 16, 17, 18, 19, 20). These cells which are characterized by expression of high levels of CD25 and intermediate levels of CD45RB are known to be weakly reactive to antigenic stimulation and able to mediate suppression of CD25- naive T cells. Until now, determination of the relative contribution of clonal deletion and active immunoregulation in intrathymic tolerance has been problematic given the inability to follow the fate of Ag-reactive T cells. However, the availability of TCR transgenic mouse lines in which the transgenic T cell is specific for transplantation Ags affords such investigation.
In the present work, we used the TS1 line of TCR transgenic mice to
probe the mechanisms responsible for the tolerant state that results
from intrathymic Ag inoculation (21). The TCR 
- and
-chain genes cloned to generate TS1 mice were derived from a
T cell line specific for the immunodominant epitope of influenza A
virus PR8 hemagglutinin
(HA)3 (22, 23). As a result, TS1 mice possess a high percentage of T cells
specific for the hemagglutinin molecule S1 determinant (aa 110–119)
presented in the context of MHC class II IEd.
We have found the TS1 line to be especially well-suited for studies on induced transplantation tolerance for the following reasons: 1) TS1 mice have been backcrossed extensively to the BALB/c parental line and are nearly genetically uniform; 2) a clonotypic Ab (6.5) is available to detect S1-reactive T cells; 3) HA transgenic BALB/c mice are available as a source of HA-expressing tissues for transplantation (20); and 4) a portion of both CD4 and CD8 T cell subsets are specific for S1. In addition, thymic self-tolerance in TS1 mice expressing HA as a "neo-self Ag" has been extensively studied (20, 24, 25) and has revealed the participation of multiple pathways of self-tolerance, including the development of CD4+CD25+ T cells that demonstrate potent immunoregulatory activity. We hypothesized that such a population of immunoregulatory cells might also contribute to the tolerant state induced by central Ag exposure via intrathymic injection of Ag and may provide a valuable component of protocols designed to achieve transplantation tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TS1 transgenic mice were created by von Boehmer and colleagues (21), and have been described in detail previously. HA28 and HA104 mice were created by Caton and colleagues (20) and have also been previously described. TS1, HA28, and HA104 transgenic lines are maintained as hemizygotes and repeatedly backcrossed with BALB/c mice (Taconic Farms, Germantown, NY). The progeny of these matings are screened by PCR for the presence of the relevant transgene. All animals are maintained in a sterile pathogen-free environment in the University of Pennsylvania animal facility (Philadelphia, PA).
Fluorescent labeling and in vitro culture conditions
Fluorescent labeling of lymphocytes was achieved as previously described (26). Briefly, lymph nodes or thymus was harvested and single-cell suspensions were prepared by passage of tissue through a cell strainer (70 µm; Falcon, Franklin Lakes, NJ). Cells were resuspended at a density of 1 x 107 cells/ml in IMDM. An equal volume of 5 mM CFSE (Molecular Probes, Eugene, OR) in IMDM was added and cells were cultured at 37°C for 5 min. The reaction was quenched through the addition of an equal volume of heat-inactivated FCS (Life Technologies, Rockville, MD). Labeled cells were washed two times with cold IMDM containing 10% heat-inactivated FCS. Stimulation of labeled cells in culture was achieved by the addition of stimulator splenocytes at a concentration of 3.5 x 106 cells/ml with varying concentrations of S1 peptide. In addition, parallel cultures were established to which IL-2 (10 ng/ml) was added (BioSource International, Camarillo, CA).
Flow cytometric analysis
Cells were either harvested directly from in vitro culture
flasks or were obtained from in vivo experiments by sacrificing animals
and harvesting thymi and lymph nodes followed by preparation of
single-cell suspensions. Cells were washed in biotin-free RPMI (Irvine
Scientific, Santa Ana, CA), and 1 x 106
cells were stained per sample. The following Abs were used for
analysis: anti-CD4 PE, anti-CD4 APC, anti-CD8 FITC,
anti-CD8 PE, anti-CD25 FITC, anti-CD25-PE, anti-CD45RB
PE (BD PharMingen, San Diego, CA), and anti-v8 FITC (Caltag
Laboratories, Burlingame, CA). In addition, 6.5 biotin
(21) and Strepavidin-Red670 (Life Technologies) were used
to detect the transgenic TCR. Flow cytometric analysis was performed on
a FACScan cytometer (BD Immunocytometry Systems, San Jose, CA). Data
acquisition and analysis were accomplished with BD Biosciences
CellQuest software (BD Biosciences, Mountain View, CA).
Peptide synthesis
All peptides were synthesized by the protein chemistry
laboratory of the University of Pennsylvania. All peptide preparations
are HPLC purified for 
90% purity. The sequences of the S1 and SCR
peptides are respectively: H-SFERFEIFPK-COOH and H-FIERKFPFES-COOH.
FACS purification of cell populations
Cells were sorted on a BD FACSVantage SE (BD Biosciences)
high-speed cell sorter. The dual laser Vantage is equipped with 5W
argon (Coherent Innova 305; Santa Clara, CA) and mixed gas
argon-krypton (Coherent Spectrum) lasers. The primary laser was tuned
to 488 nm excitation at 250 mW, and the secondary laser was tuned to
647 nm excitation at 150 mW. Our standard high-speed sort setup was
used: 45 psi, 70 µ nozzle, 80,000 kHz drop drive, Normal-R mode, 1.4
drop envelope. This configuration permitted trigger rates of 
15,000
cells/second with abort rates of <20%.
Cells were stained with CD25 or CD4 FITC collected through a 530/30 bandpass filter, CD45RB PE collected through a 575/26 filter, and CD4 or CD25 APC collected through a 675/20 bandpass filter. Sorted populations were gated on CD4 positive, CD25 negative, and CD45RB bright, or CD4 positive, CD25 positive, and CD45RB intermediate. Forward scatter pulse width was used as an additional gated parameter to exclude cell aggregates. Purity checks on the sorted populations ranged from 97–99%.
Stimulation of sorted T lymphocytes
The proliferation of CD25+ and CD25- T lymphocytes was assessed by tritiated thymidine uptake. A total of 10,000 FACS-purified responder cells were cultured in 96-well plates in the presence of 100,000 irradiated (2200 rad) BALB/c splenocytes as stimulator cells and 0.1 µM S1 peptide. [3H]Thymidine was added 18 h before harvesting. Some cultures were supplemented with 5 ng/ml rIL-2 (BioSource International).
Skin grafting
Skin grafts were transplanted to mice according to the technique of Billingham and Medawar (27). Mice were anesthetized, and skin grafts were placed in a lateral thoracic position and stabilized with vaseline gauze and band-aids. The bandages were removed on the tenth day. Grafts were scored as rejected when >75% of the grafted tissue area had been lost. In adoptive transfer experiments to assess the graft-reactive potential of sorted cells, animals received 500,000 donor cells in all cases.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our first studies, we evaluated the rejection response of TS1
recipients to HA-expressing skin grafts using two distinct HA
transgenic lines as skin graft donors (20, 24). The HA104
and HA28 mice carry the HA transgene controlled by the SV40 promoter
that results in diffuse expression that is not tissue-specific. Skin
grafts from each of the transgenic lines were rejected by TS1
recipients (Table I
). The mean survival
times of skin grafts from the two HA lines did not differ
significantly. All grafts from each group were rejected by 40 days
posttransplant.
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). As expected, we found that TS1 skin grafts
transplanted to TS1 recipients are consistently accepted indefinitely,
documenting that the TS1 line is adequately inbred. BALB/c grafts
placed on TS1 recipients and reciprocal grafts from TS1 to BALB/c are
also accepted thus confirming that the TS1 line carries
histocompatibility Ags of the BALB/c background. Because the TS1 line
is maintained by backcross mating of TS1 transgene hemizygotes with
normal BALB/c mice, the transgene negative littermate siblings of the
TS1 transgene positive progeny provide ideal controls. Thus, we
transplanted HA104 skin grafts to TS1-negative BALB/c littermates
(TS1-) to assess the need for 6.5 transgenic T
cells in HA graft rejection. In no case was rejection observed in this
group that has now been followed for >200 days.
The rejection of HA104 skin by TS1 recipients provides a unique and
reliable model of rejection for use in transplantation tolerance
experiments. Rejection is reproducible and consistent in its evolution.
However, the fact that complete rejection of HA104 skin grafts required
almost 1 mo on average was somewhat surprising given the high frequency
of HA-reactive cells in TS1 hosts. One possible explanation is that
transgenic TCR expression leads to such severe perturbations in the T
cell repertoire that immune system function is globally compromised
(28, 29). To test this possibility, we examined the
response of TS1 mice to allogeneic skin grafts that were MHC
incompatible (B6 H-2b and C3H
H-2k). We found that TS1 mice reject allografts
promptly with a tempo similar to that of TS1-negative littermates,
indicating the presence of a competent immune system (Table I).
Collectively, data from these grafting experiments support the
conclusion that the rejection of HA104 skin by TS1 recipients is
dependent on both the transgenic T cells of the TS1 recipient and
expression of the HA transgene by the donor.
Induction of transplantation tolerance in TS1 mice by intrathymic inoculation with S1 peptide
Having established that HA104 skin graft rejection by TS1
recipients is dependent on HA-specific transgenic T cells, we next
determined whether these cells could be tolerized by intrathymic
exposure to the immunodominant epitope of HA (S1). TS1 mice were
inoculated intrathymically with 250 µg of S1 peptide (125 µg/lobe)
and grafted with skin from HA104 donors the next day. In control
experiments, recipient mice were untreated, treated with S1 peptide
i.v., or injected intrathymically with a control peptide (SCR)
comprised of the same amino acids present in S1 but scrambled in their
order. As detailed in Table I, intrathymic inoculation with S1 peptide
led to indefinite survival of HA104 skin grafts. In contrast,
prolongation of HA104 skin survival was not observed in untreated
controls, TS1 mice injected with S1 i.v., or TS1 mice inoculated
intrathymically with SCR. These results indicate a stable state of
specific transplantation tolerance to HA is induced by intrathymic S1
injection.
Phenotypic alteration of thymocytes following intrathymic inoculation with S1 peptide
To examine the effect of S1 peptide Ag exposure on developing
6.5+ thymocytes, we inoculated 6- to 8-wk-old TS1
mice with varying doses of S1 peptide directly into both thymic lobes.
Thymocytes were isolated 1 wk later and analyzed by three-color flow
cytometry. A representative pattern of 6.5 TCR expression in
CD4+CD8- thymocytes in S1
peptide-injected and saline-injected control TS1 mice is show in Fig. 1 with a quantitative summary in Table II. In general, the injection of
increasing doses of Ag produced a graded reduction in the intensity of
staining with anti-6.5 Ab. The greatest reduction we have detected
has been at a dose of 250 µg. Further increases in injected levels
did not result in increased reduction in staining intensity. A
combination of a decrease in receptor levels and deletion of transgenic
T cells may account for the disappearance of
6.5high cells. As there was no statistically
significant change in the proportion of 6.5+ T
cells even with high dose peptide administration, we favor receptor
down-regulation to explain these changes (Table II). To maintain high
levels of intrathymic peptide, we have selected a dose of 250 µg for
all additional experiments.
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). Reduction in
expression of high levels of the transgenic TCR on CD4 thymocytes was
most pronounced at 1 and 2 wk (p 
0.05 and
p = 0.06, respectively, vs control mice), and then
progressively returned to baseline at 3–4 wk. TS1 mice treated i.v.
with peptide did not reveal statistically significant reductions in
6.5+CD4+ thymocytes (see
below). We also observed that CD8 SP thymocytes that have high levels
of 6.5 (and are thought to be class II-restricted and S1-specific) are
efficiently eliminated (data not shown) (21).
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We next characterized the effect of intrathymic Ag injection on
peripheral T cells. To evaluate the effect of peptide that might escape
to the periphery during or immediately after thymic injection, we
performed parallel experiments in mice receiving an i.v. injection of
the S1 peptide. Thus, TS1 mice were inoculated either intrathymically
(Fig. 3, left panels)
or i.v. (Fig. 3, right panels) with 250 µg of S1 peptide
and sacrificed at day 7 for analysis of the thymic (top) and
lymph node cells (bottom) for CD4, CD8, and 6.5 expression.
We observed a marked reduction in CD4 SP thymocytes that expressed high
levels of 6.5 TCR following intrathymic but not i.v. delivery of Ag
(compared with naive TS1, solid black line in each). In addition, we
observed little change in the pattern of peripheral 6.5 expression from
either intrathymic (Fig. 3, lower left panel) or i.v. (Fig. 3, lower right panel) treatment. The lack of peripheral
alterations following intrathymic injection of 6.5 suggested
that alterations at the thymic level must be exported to the periphery
to promote the observed tolerogenic effect. We hypothesized that this
conversion of the periphery might be accomplished by intrathymic
generation of CD25+ regulatory T cells. Our
phenotypic analysis of the thymocyte population also suggests this
possibility in that TCR down-regulation is characteristic of
CD25+ T cell generation.
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To assess this hypothesis, we undertook a detailed analysis of the
response of peripheral T cells to stimulation with the target Ag S1.
TS1 lymph node cells were labeled with CFSE and cultured with 5 µM S1
peptide and irradiated syngeneic BALB/c spleen cells as a source of
APCs. After 3 days of culture, cells were labeled with anti-CD4 and
anti-6.5, and mitotic activity was measured by flow cytometry. The
responder precursor frequency was calculated as described previously
(26). To assess the response of tolerant mice, TS1 mice
were injected intrathymically at 6–8 wk of age with either saline, 50
µg, or 250 µg of SI peptide and analyzed 1 wk later. Mice injected
intrathymically with a high dose of peptide demonstrated an abortive
proliferative response in vitro (Fig. 4)
as evidenced by a marked reduction in the precursor frequency of cells
responding and lack of progression of cells to late division peaks.
This pattern, characterized by a limited number of mitotic events and
an absence of cells accumulating in later peaks, has been noted
previously in the response of T cells which are cocultured with
CD25+ regulatory cells (20). Thus,
despite the absence of gross phenotypic alterations in the peripheral T
cells, the response of 6.5 T cells from tolerant mice to S1 peptide in
vitro is markedly abnormal.
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To determine whether intrathymic peptide inoculation generates
cells with a regulatory phenotype similar to that reported by Jordan et
al. (20), we studied thymocytes of naive TS1 control mice
and TS1 mice that had been rendered tolerant by intrathymic inoculation
with S1 peptide 1 wk earlier. When CD4 SP 6.5+
thymocytes were examined selectively, we observed a dramatic increase
in the percentage of CD25+ cells comparing
tolerant with untreated control mice (Fig. 5). This increase was evident as early as
1 wk after injection and CD25 levels returned to normal at 3 wk.
Moreover, in TS1 mice injected with S1 peptide intrathymically, the
increase in CD25+ expression was confined to the
6.5+ population of CD4 SP thymocytes, indicating
that it was the result of an Ag-specific interaction. Analysis of CD4
SP subpopulations found CD25+ cells to be first
evident at the early SP stage. No change in the percentage of
CD25+ T cells was evident in the
6.5- CD4 SP population.
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). Again the effect was not observed in
the population of peripheral T cells that do not express the transgenic
6.5 TCR. Simultaneous analysis for CD25 and CD45RB found the majority
of CD25+ T cells to have intermediate staining
for CD45RB (data not shown). As was the case for thymocytes, peripheral
levels of CD25+ cells also returned to normal
levels after 1 mo. However, this finding does not preclude their
continued involvement in the protection of the grafted tissue. Recent
analyses have suggested that these cells may continue to reside and
function within the graft (30); determination of the
distribution of these cells throughout the animal following this
therapy will require further investigation.
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, the
uptake of tritiated thymidine by these cells is greatly diminished in
comparison to the CD25- population. However, the
addition of IL-2 to the culture medium enhanced proliferation to
supranormal levels. The sorted CD25+ cell
fraction is likely to be enriched in 6.5-expressing T cells; this
increase in responder cell number may account for the augmented
response engendered by the addition of IL-2.
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To demonstrate that the CD25+,
CD4+, 6.5+ T cells we
identified in peripheral lymph nodes were generated intrathymically
rather than as a result of encountering Ag that escaped to the
periphery, we tracked the phenotype of recent thymic emigrants
following thymic Ag inoculation. To accomplish this analysis, we
labeled thymocytes in vivo with CFSE, as described by Graziano et al.
(33). With this technique, CFSE-positive T cells could be
identified subsequently in peripheral lymphoid organs with 2% of
peripheral T cells CFSE+ at 4 and 7
days.
To examine the fate of CD25+ thymocytes generated
by intrathymic S1 peptide inoculation, TS1 mice were injected
intrathymically either with CFSE alone, or with a mixture of CFSE and
the S1 peptide to provide a peptide dose of 250 µg in 20 µl. Mice
were sacrificed either 4 h or 4 days later and thymic and lymph
node tissue suspensions prepared and labeled for four-color flow
cytometry with CFSE and Abs for CD4, CD8, and 6.5. In lymph node cells,
we found that CFSE-positive cells were not detectable at 4 h
postinjection but comprised 2% of CD4 T cells at 4 days. By
analyzing CFSE-positive cells, we were able to characterize the
phenotype of T cells that had migrated to the periphery during the
prior 4 days. We found that recent thymic emigrants in peptide injected
tolerant mice revealed both a relative reduction in
6.5high T cells (Fig. 8A) and an increase in the
proportion that were CD25+ (Fig. 8, B
and C).
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To investigate the function of tolerant T cells in vivo, we
developed an adoptive transfer model of skin graft rejection by
transferring TS1 T cells to naive BALB/c hosts grafted with HA104 skin.
Lymph node cells or thymocytes from naive or tolerant TS1 mice were
transferred to BALB/c host that had been grafted with HA104 skin at
least 4 wk earlier. The use of established grafts avoids the
nonspecific inflammation accompanying the skin grafting procedure that
could override the immunoregulatory effects of
CD25+ cells. As shown in Table III, TS1 lymph node cells (5 x
105) transferred to BALB/c hosts with established
HA104 skin resulted in the prompt destruction of the skin. Similarly,
transfer of thymocytes from TS1 hosts that had received an intrathymic
injection with SCR control peptide 1 wk earlier also efficiently
mediated rejection of established HA104 skin grafts. In contrast,
thymocytes harvested from mice rendered tolerant by intrathymic
inoculation with S1 peptide 1 wk earlier did not mediate graft
rejection upon transfer to BALB/c hosts. To determine whether the
presence of CD4 SP, CD25+ regulatory T cells was
responsible for the lack of rejection, tolerant thymocytes were
negatively sorted to remove CD25+ cells. The
resulting population of CD4 SP, CD25- thymocytes
from intrathymic S1-injected mice mediated efficient graft
rejection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of immunoregulatory T cells may result directly from Ag contact in the thymus at the appropriate stage of thymocyte development. Cells with an immunoregulatory phenotype (CD4+, CD25+, CD45RBint) migrate from the thymus to the peripheral lymphoid organs within the first 2 wk following tolerance induction and may be responsible for exporting tolerance from the thymus to the periphery. Using an adoptive transfer approach, we demonstrated that unlike T cells from naive TS1 mice, T cells from TS1 mice rendered tolerant by intrathymic S1 peptide inoculation do not mount a productive response to HA104 skin upon transfer to BALB/c hosts. Elimination of CD4+CD25+ T cells from the tolerant population restores a prompt rejection response, confirming a key regulatory role for this population in the tolerant state. These results suggest that, as with tolerance to self Ags, multiple mechanisms contribute to the development of acquired central tolerance following intrathymic inoculation with Ag, and that regulatory cells generated by the tolerant thymus may be primarily responsible for translation of tolerance to the peripheral immune compartment.
Additional insight into the generation of intrathymic tolerance is
afforded by a number of transgenic models in which self-tolerance has
been studied. A particularly incisive line of investigation resulted
from intercrossing TS1 mice with the HA-expressing transgenic lines
HA104 and HA28. These transgenic mice lines each express the HA Ag
under the direction of the SV40 promoter, but they are distinguished by
the amount of Ag produced from this construct. HA104 mice express high
levels of the HA Ag in all tissues whereas HA28 mice have been found to
express lower levels. When TS1 mice are crossed with HA104 mice, the
resulting double transgenic progeny delete the majority of all
6.5-expressing thymocytes (25). In contrast, (TS1 x
HA28)F1 mice delete few of these cells, and rather
have a peripheral repertoire characterized by large numbers of
6.5+ CD4 T cells with an immunoregulatory
phenotype (CD25+CD45RBint)
(20). Almost 50% of 6.5high lymph
node cells from (TS1 x HA28)F1 mice express CD25 and an
intermediate level of the CD45RB marker. Functional analysis of these
CD25+, CD4+,
6.5+ T cells reveals not only a lack of
responsiveness to the S1 peptide but also potent regulatory activity.
In cell mixing experiments, CD25+,
CD4+ T cells suppressed the response of naive
6.5+ CD4 TS1 T cells to the S1 peptide even under
conditions of more than a 10-fold excess of naive cells. The functional
activity and phenotypic characteristics of the
CD4+, CD25+ T Cells
described by Jordan et al. (20) are similar in many
ways to those we have identified in a model of acquired central
transplantation tolerance. Whether they constitutively express cell
surface CTLA4 or TGF- as has been described for other
immunoregulatory T cells is under study (31, 37).
These additional models of central tolerance support the hypothesis that avidity is a primary determinant of Ag-specific cell fate following intrathymic peptide inoculation. Our current work provides further understanding of earlier findings using the intrathymic tolerance model. In virtually all of the initial transplant investigations of intrathymic tolerance induction, treatment of the recipient with a peripheral T cell-depleting agent such as anti-lymphocyte serum or anti-CD4 was required to achieve tolerance to a subsequent tissue graft. However, more recently, a growing number of cases have been reported in which peripheral T cell depletion has been found to be unnecessary (12, 13, 14, 38, 39, 40, 41, 42). Interestingly, in each case, either purified protein preparations or synthetic peptide, but not donor cells, has been used as the inoculated Ag. Although the immunological mechanisms underlying these apparently contradictory findings are unclear, it is tempting to speculate that peptide preparations may promote lower avidity interactions that generate CD25+ regulatory T cells while inoculated cells which often repeat the same determinant may fall at a higher end of the avidity spectrum.
The TS1 TCR model provides a unique opportunity for dissection of the events promoting rejection and tolerance to allografts. However, a recurring concern in using a TCR transgenic host for such studies is whether in the setting of an overabundance of a single clone of T cells, the response under study still recapitulates a physiological response. In fact, our observation that TS1 hosts reject MHC disparate allografts in 10 days compared with an average of 28 days for HA104 graft rejection was unexpected in light of the fact that the precursor frequency of T cells reactive for the allogeneic donor is likely to be many fold lower than the frequency of HA-specific T cells in TS1 recipients. Although differences in Ag density (HA vs Allo-MHC) on the graft could contribute to this result, an alternate hypothesis is that rejection of HA grafts by TS1 hosts is impeded by too high a frequency of responding T cells (43). In support of the latter possibility is our finding that HA104 graft rejection is typically more rapid following transfer of a small number of TS1 T cells to BALB/c hosts than in naive TS1 recipients.
Intrathymic inoculation with Ag may provide a unique opportunity to study the generation of immunoregulatory T cells. As far as we are aware, this technique is the first reported to permit induction of such cells in the thymus of adult mice. This innovation should permit further manipulations that define the events leading to the pathway of clonal deletion vs anergic regulatory T cell induction. Generation of transplantation tolerance based on immunoregulatory T cells may provide an ideal means to maintain graft survival without the need for chronic immunosuppression.
In summary, the current results indicate that immunoregulatory T cells may play a critical role in the generation and maintenance of acquired central transplantation tolerance. Our results delineate a simple means both to generate immunoregulatory T cells in normal adult mice by central tolerance induction and provide a relevant assay for their function in vivo, in the form of regulation of a response to an allograft. Further study of their mechanism of action and the events that determine whether a developing T cell will undergo deletion or will assume an anergic regulatory cell role, has the potential to define new strategies to induce stable transplantation tolerance.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. James F. Markmann, Harrison Department of Surgical Research, Department of Surgery, Hospital of the University of Pennsylvania, 3400 Spruce Street, Philadelphia, PA 19104. E-mail address: James.Markmann{at}UPHS.upenn.edu
3 Abbreviations used in this paper: HA, hemagglutinin; SP, single positive; int, intermediate.
Received for publication March 29, 2002. Accepted for publication October 30, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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8.1 transgenic mice peripherally tolerized to Mls-1. Eur. J. Immunol. 24:1710.[Medline]
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  S. S. Lee, W. Gao, S. Mazzola, M. N. Thomas, E. Csizmadia, L. E Otterbein, F. H. Bach, and H. Wang Heme oxygenase-1, carbon monoxide, and bilirubin induce tolerance in recipients toward islet allografts by modulating T regulatory cells FASEB J, November 1, 2007; 21(13): 3450 - 3457. [Abstract] [Full Text] [PDF]
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 S. Deng, D. J. Moore, X. Huang, M.-M. Lian, M. Mohiuddin, E. Velededeoglu, M. K. Lee IV, S. Sonawane, J. Kim, J. Wang, et al. Cutting Edge: Transplant Tolerance Induced by Anti-CD45RB Requires B Lymphocytes J. Immunol., May 15, 2007; 178(10): 6028 - 6032. [Abstract] [Full Text] [PDF]
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S. Deng, D. J. Moore, X. Huang, M. Mohiuddin, M. K. Lee IV, E. Velidedeoglu, M.-M. Lian, M. Chiaccio, S. Sonawane, A. Orlin, et al. Antibody-induced transplantation tolerance that is dependent on thymus-derived regulatory T cells. J. Immunol., March 1, 2006; 176(5): 2799 - 2807. [Abstract] [Full Text] [PDF]
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X. Huang, J. Zhu, and Y. Yang Protection against Autoimmunity in Nonlymphopenic Hosts by CD4+CD25+ Regulatory T Cells Is Antigen-Specific and Requires IL-10 and TGF-{beta} J. Immunol., October 1, 2005; 175(7): 4283 - 4291. [Abstract] [Full Text] [PDF]
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T. Higuchi, T. Maruyama, A. Jaramillo, and T. Mohanakumar Induction of Obliterative Airway Disease in Murine Tracheal Allografts by CD8+ CTLs Recognizing a Single Minor Histocompatibility Antigen J. Immunol., February 15, 2005; 174(4): 1871 - 1878. [Abstract] [Full Text] [PDF]
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M. K. Lee IV, D. J. Moore, B. P. Jarrett, M. M. Lian, S. Deng, X. Huang, J. W. Markmann, M. Chiaccio, C. F. Barker, A. J. Caton, et al. Promotion of Allograft Survival by CD4+CD25+ Regulatory T Cells: Evidence for In Vivo Inhibition of Effector Cell Proliferation J. Immunol., June 1, 2004; 172(11): 6539 - 6544. [Abstract] [Full Text] [PDF]
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O. Joffre, N. Gorsse, P. Romagnoli, D. Hudrisier, and J. P. M. van Meerwijk Induction of antigen-specific tolerance to bone marrow allografts with CD4+CD25+ T lymphocytes Blood, June 1, 2004; 103(11): 4216 - 4221. [Abstract] [Full Text] [PDF]
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Y. Hayashi, S.-i. Tsukumo, H. Shiota, K. Kishihara, and K. Yasutomo Antigen-Specific T Cell Repertoire Modification of CD4+CD25+ Regulatory T Cells J. Immunol., May 1, 2004; 172(9): 5240 - 5248. [Abstract] [Full Text] [PDF]
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M. Stanzani, S. L. R. Martins, R. M. Saliba, L. S. St. John, S. Bryan, D. Couriel, J. McMannis, R. E. Champlin, J. J. Molldrem, and K. V. Komanduri CD25 expression on donor CD4+ or CD8+ T cells is associated with an increased risk for graft-versus-host disease after HLA-identical stem cell transplantation in humans Blood, February 1, 2004; 103(3): 1140 - 1146. [Abstract] [Full Text] [PDF]
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M. Karim, C. I. Kingsley, A. R. Bushell, B. S. Sawitzki, and K. J. Wood Alloantigen-Induced CD25+CD4+ Regulatory T Cells Can Develop In Vivo from CD25-CD4+ Precursors in a Thymus-Independent Process J. Immunol., January 15, 2004; 172(2): 923 - 928. [Abstract] [Full Text] [PDF]
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