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Department of Medicine, Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215
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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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Although depletion of alloreactive clones may be critical to the induction of the tolerant state, the long-term maintenance of peripheral tolerance is believed to be dependent on self-perpetuating immunoregulatory mechanisms that actively constrain alloreaggressive T cell-mediated immune responses (7, 8, 9). The presence of suppressor or regulatory CD4+ T cells (T reg)4 in tolerant hosts has been described by several laboratories, including our own (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18). Regulatory CD4+CD25+ T cells, first identified as suppressor cells in 1990 (18, 19), have emerged as critical effectors in both the control of autoimmunity (20, 21, 22, 23) and the maintenance of peripheral allograft tolerance (24, 25).
The finding in hosts mounting graft rejection of lymphocytes capable of prolonging graft survival upon adoptive transfer into naive graft recipients (15) suggests that activation of T reg is an integral component of the allograft response. In the absence of ongoing drug therapy, however, we believe that the more rapid expansion of alloaggressive T cells overcomes the suppressor effect of T reg. We hypothesize, therefore, that the capacity of T reg to restrain naive lymphocytes from rejecting an allograft is not absolute and that it will fade as the numbers of potentially allodestructive T cells rise.
In this study we sought to characterize in detail, both in vivo and in vitro, the cells responsible for these immunoregulatory effects, focusing on their mechanism of action and costimulation requirements, and addressing how their impact on naive CD4+ and CD8+ T cells undergoing alloactivation fits into the predictions inherent in the pool size model of the allograft response (2).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 10-wk old DBA2 (H-2d) and B6AF1 (H-2b/k.d) mice were used as donor and recipient for islet transplantation, respectively. Male DBA1 (H-2q) were used as a third-party strain donor. Selected experiments were repeated using C57BL/6 (H-2b) and/or C57BL/6 congenic for CD45.1 as recipients. All animals were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed under standard conditions. All animal studies were approved by our institutional review board.
Islet transplantation
Islet transplantation was performed as previously described
(26). Briefly, DBA2 donor pancreata were perfused with 3.5
ml type IV collagenase (1.5 mg/dl; Worthington Biochemical, Freehold,
NJ) through the common bile duct, and incubated at 37°C for 40 min.
Islets were released from the pancreata and purified in discontinuous
Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) gradients.
The harvested islets were washed in HBSS, and 
600 islets were
transplanted under the renal capsule of each
B6AF1 recipient rendered diabetic by a single
i.p. injection of streptozotocin (225 mg/kg; Sigma-Aldrich, St. Louis,
MO). Allograft function was monitored by serial blood glucose
measurements. Primary graft function was defined as a blood glucose
level <200 mg/dl on day 3 post-transplantation, and graft rejection
was defined as an elevation in blood glucose >300 mg/dl following a
period of primary graft function.
Reagents and tolerizing protocol
B cell hybridomas were purchased from American Type Culture Collection (Manassas, VA) producing 1) a hamster mAb against mouse CD154 (MR1, IgG2a, ATCC HB11048) and 2) a rat anti-mouse CD25 (PC61 5.3, IgG1, ATCC TB222). The hybridoma cells were grown in Ultraculture medium (BioWhittaker, Walkersville, MD), and the mAbs were affinity-purified using protein G columns. Anti-CTLA4 mAb (UC10-4F10-11) was provided by Dr. M. Sayegh (Brigham and Women’s Hospital, Boston, MA). rIL-2 was obtained from BD PharMingen (San Diego, CA), and a nonlytic IL-2/Fc fusion protein that acts as a long-lived IL-2 molecule in vivo was produced in our laboratory as previously described (27).
B6AF1 transplant recipients were tolerized by a single i.v. donor-specific transfusion (0.25 ml DBA2 blood) administered 28 days before transplantation together with a single 0.5 mg i.v. dose of anti-CD154 (28). To determine whether donor-specific tolerance had been achieved, euglycemic long-term survivors (>120 days) underwent left nephrectomy to remove the islet graft, and their blood glucose levels were allowed to rise before being retransplanted with either donor-type (DBA2) or third-party strain islets (DBA1) in the absence of further treatment. These mice accepted donor-type islets but rejected third-party strain grafts. In this model the administration of rIL-2 blocked the induction of tolerance but failed to provoke allograft rejection in fully tolerant hosts (29). Similar effects were noted when using a long-lived nonlytic IL-2/Fc fusion protein (our unpublished observations). Mononuclear leukocytes (MNLs) were harvested from tolerant mice at times ranging from 90 to 120 days after transplantation.
Flow cytometric analysis
Single-cell suspensions were stained with fluorochrome-conjugated mAbs, including anti-CD4, anti-CD8, anti-CD25, anti-CD69, and anti-CD45.1, or Ig isotype controls (all from BD PharMingen) and analyzed using CellQuest software on a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ).
Magnetic cell separation
Single-cell suspensions of B6AF1 spleens and lymph nodes were prepared, and RBC were removed by hypotonic shock. Magnetic beads coated with mAbs (Dynal Biotech, Oslo, Norway) were used to separate cells into CD25+ and CD25- subsets. Briefly, MNLs were incubated with anti-CD25 mAb-coated magnetic beads at a 10:1 bead to cell ratio for 30 min at 4°C with gentle rotation. Selected CD25+ T cells were isolated from the bead/cell mixture by exposure to a magnetic field using a magnetic particle concentrator (Dynal Biotech) according to the manufacturer’s instructions. The negatively selected CD25- cells were collected, and their purity, determined by FACS analysis, was >95% CD25-. Viability, determined by trypan blue staining, exceeded 95% in all cases.
Labeling of MNLs with CFSE
Single-cell suspensions of splenic and lymph node MNLs from either naive or tolerant B6AF1 mice were resuspended in HBSS at a concentration of 1 x 107 cells/ml and labeled with the tracking fluorochrome CFSE (Molecular Probes, Eugene, OR). MNLs were incubated with CFSE at a final concentration of 5 µM in HBSS for 6 min, and labeling was terminated by the addition of FCS (10% of the total volume). MNLs were washed twice in complete RPMI 1640 medium and resuspended for cell culture or i.v. injection.
In vitro MLR
Single-cell suspensions of splenic and lymph node MNLs were prepared as previously described. Next, 7.5 x 105 irradiated (3000 rad) DBA2 or DBA1 stimulator splenic MNLs were cultured in round-bottom 96-well microtiter plates with 7.5 x 105 responder cells obtained from naive and/or tolerant B6AF1 or C57BL/6 mice. In some cultures negatively selected CD25- MNLs from tolerant or naive mice were used as responding cells. Anti-CTLA4 mAb (or a hamster Ig isotype control) and rIL-2 were added to some wells at 25 µg/ml and 100 U/ml, respectively. Cell cultures were pulsed with 1 µCi/well [3H]TdR for 12 h and harvested on day 5. In some experiments 3.75 or 5 x 105 responder cells were used. Moreover, some cultures were harvested on day 4 to ensure that differences between tolerant and naive MNLs were not due to early proliferation peaking. Thymidine incorporation was measured using a liquid scintillation counter.
To analyze the interactions between naive and tolerant MNLs comixed and cultured in vitro, in some MLRs cells harvested from naive CD45.1 congenic C57BL/6 mice were stained with CFSE and used as responders alone or in combination with unlabeled MNLs obtained from tolerant C57BL/6 (CD45.2) hosts. MNLs were harvested on day 5 and stained with FITC-conjugated anti-CD45.1 mAb, and their proliferative profile was assessed by flow cytometry by gating on the CD45.1- and CFSE-positive populations.
In vivo quantitation of proliferating T cells
Approximately 7 x 107 CFSE-labeled MNLs from either naive or tolerant animals were adoptively transferred to B6AF1 irradiated transplant recipients, which were then transplanted with DBA2 (donor strain) or DBA1 (third-party strain) islet allografts. Additionally, 3.5 x 107 MNLs from naive or tolerant mice were CFSE-labeled and mixed together in equal proportions with unstained MNLs from tolerant or naive hosts, respectively. These cells were cotransferred into irradiated transplant recipients. Because cotransferred MNLs from either naive or tolerant mice could be labeled separately, this system enabled us to bidirectionally track the interactions between the lymphocytes from naive and tolerant hosts at the single-cell level.
Mice were sacrificed 8 days following transplantation, and single-cell
suspensions were prepared from harvested spleens and lymph nodes as
described above. MNLs were stained for 20 min on ice with a
biotinylated Ab against mouse CD4 or CD8
(GK1.5 and 53-6.7,
respectively; BD PharMingen), and then stained with
streptavidin-CyChrome and PE-conjugated annexin V, anti-mouse CD69
(H1.2F3), or anti-mouse CD25 (7D4) (all from BD PharMingen), while
the cell populations were placed on ice for an additional 20 min.
Proliferation, apoptotic cell death, and activation markers of
CFSE-labeled CD4+ or CD8+ T
cells in each distinct generation of dividing cells were analyzed by
flow cytometry.
The responding frequency of T cells (percentage of dividing precursors among the recovered CFSE-positive MNLs harvested 8 days after adoptive transfer) was calculated by summing the extrapolated number of precursors for each division cycle and dividing this number by the total number of CFSE-labeled T cells (divided and undivided) recovered from lymph nodes and spleen. As recently reported (6), this calculation may overestimate the actual size of the alloreactive T cell pool, because only the portion of the donor cells that is recovered from the recipient’s lymph nodes and spleen is analyzed, and this portion very likely over-represents proliferating cells. Its value as a basis for comparisons of the proliferative profile of cell populations, however, remains valid and unquestioned.
Adoptive transfer of naive and tolerant MNLs into irradiated transplant recipients
B6AF1 mice were irradiated with 1000 rad
(GammaCell irradiator; Nordion, Kanata, Ontario, Canada) to
effectively ablate their immune system, and various mixtures of MNLs
from naive and/or tolerant hosts were injected through the tail vein.
Mice were then transplanted with DBA2 or third-party strain (DBA1)
islets. In the absence of lymphocyte transfer, irradiated recipients do
not reject allografts and die at 20 days of transplantation.
Statistical analyses
The nonparametric Mann-Whitney U test was performed to compare the responding frequencies of MNLs harvested from naive and tolerant hosts and transferred into irradiated transplant recipients. Graft survival was analyzed using Kaplan-Meier cumulative plots, and comparisons between groups were performed using a log-rank test. A value of p < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used a MLR system to compare the responses of MNLs from
tolerant and naive hosts to donor (DBA2) or third-party (DBA1)
alloantigen. The response of lymphocytes harvested from tolerant
B6AF1 hosts to donor stimulator cells was
dramatically less than that of lymphocytes obtained from naive
B6AF1 mice (Fig. 1
). Very similar results were obtained
when cultures were harvested on day 4 or when fewer responding cells
were used (data not shown). Moreover, the proliferation of lymphocytes
from naive and tolerant mice, mixed together in equal proportions, was
essentially equivalent to that of lymphocytes from tolerant hosts
alone, suggesting that tolerant MNLs suppress the ability of naive T
cells to proliferate in response to donor MNLs in the MLR. Identical
results were obtained when the responding cells were harvested from
naive and tolerant C57BL/6 mice (data not shown). Interestingly, the
responses of naive and tolerant lymphocytes were equivalent when
third-party strain stimulator cells were used (Fig. 1). Hence, T cells
from tolerant recipients are anergic to donor alloantigen but mount a
normal proliferative response when confronted with other
alloantigens.
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To test the hypothesis that the decreased proliferation in
response to donor strain MNLs observed when naive and tolerant MNLs
were mixed was due at least in part to a suppressive effect of tolerant
MNLs on naive MNLs, we performed additional MLRs in which naive CD45.1
congenic C57BL/6 MNLs were stained with CFSE and exposed to donor
strain stimulator cells in the presence or the absence of C57BL/6
(CD45.2) tolerant MNLs. As illustrated in Fig. 2A, the response of naive T
cells to donor strain MNLs was markedly curtailed when mixed
with tolerant MNLs. This suppressive effect was directed toward both
naive CD4+ and CD8+ T cells
(data not shown). Moreover, when the ratio of tolerant to naive MNLs
was titrated, this effect was shown to be dose dependent (Fig. 2B). This suppressive effect could not be reproduced when
the supernatant obtained from tolerant MNL cultures was added to wells
containing MNLs from naive mice alone, indicating that cell-to-cell
contact is probably required to confer suppression (data not
shown).
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Similarly, in the in vivo CFSE adoptive transfer model the
profiles of the passively transferred MNL populations revealed that 1)
in control B6AF1 mice in which no grafts are
placed or that are transplanted with isogeneic
B6AF1 islets, lymphocytes from tolerant and naive
donors exhibit similar spontaneous homeostatic proliferation (Fig. 3A); 2) the proliferative
response to donor strain (DBA2) islets of CD4+
and CD8+ T cells harvested from tolerant
B6AF1 mice is significantly weaker than that of
CD4+ and CD8+ T cells from
naive B6AF1 mice (Fig. 3, B and
D; mean decrease in responding frequency, 33 and 24%,
respectively); 3) with cotransfer of MNLs from naive and tolerant
hosts, the ability of CD4+ and
CD8+ T cells from naive mice to proliferate in
response to DBA2 grafts is impaired (Fig. 3, B and
D; mean decrease in responding frequency, 30 and 22%,
respectively). In contrast, CD4+ and
CD8+ T cells from naive and tolerant hosts mount
similar proliferative responses to third-party strain (DBA1) islets
(Fig. 3C).
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In the MLR model removal of
CD25+CD4+ T cells restores
the response of MNLs harvested from B6AF1
tolerant hosts or the mixture of MNLs from naive and tolerant
B6AF1 mice to donor cells (Fig. 4A). Moreover, provision of
either anti-CTLA4 mAb or rIL-2 to cultures containing either
lymphocytes from tolerant B6AF1 hosts or a
mixture of lymphocytes from tolerant and naive
B6AF1 mice disrupts the hyporesponsive state and
greatly increases the proliferative response to donor strain splenic
MNLs (Fig. 4B). Interestingly, the ability of anti-CTLA4
mAb to enhance the proliferative response of T cells from tolerant and
naive mice was noted even in cultures in which
CD4+CD25+ T cells were
removed (Fig. 4C). Similar results were observed when
responding MNLs were harvested from naive and tolerant C57BL/6 mice
(data not shown). The addition of a hamster Ig isotype control did not
modify the proliferation of either naive or tolerant MLNs (data not
shown).
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In the absence of syngeneic MNL transfer, irradiated
B6AF1 transplant recipients do not reject DBA2
islet allografts and die 20 days post-transplantation. Adoptive
transfer of 10 x 106 pooled lymph node and
splenic MNLs from naive, but not tolerant, B6AF1
mice uniformly leads to graft rejection (mean survival time (MST), 31
days; Fig. 5A). With transfer
of 15–70 x 106 MNLs from naive
B6AF1 mice the rejection is accelerated (MST, 22
days; Fig. 5B). In contrast, adoptive transfer of 70 x
106 MNLs from tolerant
B6AF1 hosts does not lead to rejection in 30% of
syngeneic recipients and, while the majority of recipients experience
rejection, this process occurs at a slower pace than noted with
transfer of MNLs from naive mice (MST, 38; p < 0.05;
Fig. 5B). In contrast, transfer of 70 x
106 MNLs from C57BL/6 tolerant hosts does not
elicit rejection (data not shown). These experiments highlight that T
cells capable of mediating allograft rejection persist in some, but
perhaps not all, tolerant hosts. Finally, MNLs from both naive and
tolerant B6AF1 hosts reject third-party (DBA1)
islets at same tempo (Fig. 5B).
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C). The ability of MNLs from tolerant hosts to suppress
allograft rejection was donor strain specific, because this suppression
was not observed when a mixture of MNLs from naive and tolerant mice
was passively transferred into syngeneic recipients of third-party
strain (DBA1) allografts (Fig. 5C).
In accordance with our pool size hypothesis, we found that the ability
of MNLs from tolerant B6AF1 hosts to constrain
rejection was dose limited. For example, at a mixture of 55 x
106 MNLs from tolerant hosts with 15 x
106 MNLs from naive mice, rejection was delayed
(Fig. 5C). When the number of MNLs from naive mice increased
while the number of MNLs from tolerant hosts decreased (mixture of
35 x 106 tolerant with 35 x
106 naive MNLs), tolerant MNLs were unable to
inhibit allograft rejection (Fig. 5C).
Insofar as the MLR experiments (Fig. 4A) indicated that
CD4+CD25+ T cells are
potent immunoregulatory cells, we conducted parallel experiments in the
passive transfer allograft response model to determine whether the
effect of MNLs from tolerant hosts upon MNLs from naive mice was
dependent on the presence of
CD4+CD25+ T cells among the
MNL populations harvested from B6AF1 tolerant
hosts. As shown in Fig. 5D, removal of this subpopulation
abolished the capacity of MNLs from tolerant hosts to delay graft
rejection in comixing experiments.
Again based on a precedent in the MLR model (Fig. 4B),
similar experiments were performed in which B6AF1
transplant recipients, receiving a mixed population of MNLs from naive
and tolerant B6AF1 hosts, were treated with
anti-CTLA4 mAb. Treatment with anti-CTLA4 mAb negated the
graft-protecting effect of tolerant MNLs and actually accelerated the
rate of graft rejection (Fig. 5D). By contrast, the
administration of anti-CTLA4 mAb and anti-CD25 mAb (1 mg/wk for
1 mo) to B6AF1 tolerant hosts >120 days after
transplantation failed to create allograft rejection or to prevent the
engraftment of a second graft from the same donor (n =
2; data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We now provide evidence that the immunoregulatory consequences of the
interaction between alloaggressive and T reg can be studied in vitro
and in vivo at the single-cell level. Observations made in an MLR
system indicate that 1) lymphocytes obtained from tolerant allograft
hosts proliferate weakly in response to donor cells, but vigorously in
response to third-party cells (Fig. 1); 2) these lymphocytes are not
only hyporesponsive, but, as our experiments using the CFSE dye clearly
reveal, they are also capable of suppressing the proliferation of both
CD4+ and CD8+ T cells
harvested from naive mice in response to donor cells (Fig. 2); 3) the
anergic/suppressive effect of MNLs harvested from tolerant hosts is
dependent on the presence of
CD4+CD25+ T cells (Fig. 4A); and 4) the immunoregulatory effect can be ablated
through provision of exogenous IL-2 or anti-CTLA4 mAb (Fig. 4B). Using the CFSE tracking dye method we were able to
confirm that several of the observations made in the MLR model can be
replicated in vivo. In these experiments we transferred CFSE-labeled
MNLs from naive or tolerant hosts into syngeneic irradiated mice
transplanted with allogeneic islets. In this model we determined that
CD4+ and CD8+ T cells
harvested from tolerant hosts proliferated weakly upon encounter with
donor, but not third-party, alloantigen. Moreover, MNLs from tolerant
hosts restrain the proliferation of both CD4+ and
CD8+ T cells from naive syngeneic mice in an
Ag-specific fashion (Fig. 3, B and
C).
In addition, the present study clearly shows, using an in vivo adoptive
transfer model of the allograft response, that the graft-protective
effect of regulatory lymphocytes is far from infinite. The clinical
outcome, rejection or tolerance, resides in a fragile balance between
the contingent immunoregulatory and alloaggressive T cells. A certain
ratio of regulatory to alloaggressive lymphocytes is required to
produce effective donor-specific suppression of allograft rejection
(Fig. 5C). These findings are in agreement with our pool
size model (2), which hypothesizes that depletion or
inactivation of alloaggressive T cells during the period of tolerance
induction is required to permit the pool size of T reg to reach the
critical levels that enable them to dominate the allograft response. In
accordance with results in both allograft (24, 25, 30) and
autoimmune models (20, 22, 31),
CD4+CD25+ T cells are
required for effective immunoregulation (Fig. 5D).
In concert with the implications inherent in the model of
infectious tolerance (7, 8, 9), MNLs harvested from tolerant
hosts inhibit the ability of naive T cells to respond to donor
alloantigen in vitro (Fig. 2A) and in vivo (Fig. 3B). Indeed, tolerant MNLs appear to render naive T cells
anergic to donor alloantigen, and this process is specific for donor
cells. Further exploration of these experimental systems should enable
a thorough dissection of the molecular basis for infectious
tolerance.
The role of CD4+CD25+ T lymphocytes as suppressor or regulatory cells is well established in several autoimmune models (20, 31). In a rat allograft model IL-2R+ T cells were identified as essential to the immunoregulatory cell process (18). In several more recent reports the CD25+ marker or the CD45RBlow phenotype have been used to identify T reg among the lymphocytes of tolerant allograft recipients (24, 30) or in a graft-vs-host disease model (25). The effector function of CD4+CD25+ T reg has been studied in vitro with respect to the sensitivity to anti-CTLA mAb. The results have not been uniform; in some settings anti-CTLA4 mAb blocks T reg effects (20), while the action of T reg is insensitive to anti-CTLA mAb in other models (32). Because of uncertainties regarding the consequences of anti-CTLA4 mAb for T reg function, we undertook parallel experiments in the MLR and adoptive transfer models of transplant biology.
Our data indicate that an intact B7/CTLA4 pathway is required to
maintain the anergic/suppressor phenotype of tolerant lymphocytes in
the MLR (Fig. 4B). In this system anti-CTLA4 mAb acts at
least in part by increasing the proliferation of conventional
CD25- T cell subsets in response to donor Ag
even in the absence of
CD4+CD25+ T reg (Fig. 4C). The validity of these observations made in an in vitro
model are strongly supported by the experiments summarized in Fig. 5D. While adoptive transfer of MNLs from tolerant hosts
comixed with MNLs from naive hosts constrains the ability of
alloaggressive T cells to mediate rejection, concomitant treatment with
anti-CTLA4 mAb destroys this effect (Fig. 5D). This
result is parallel to that of experiments conducted in a murine model
of autoimmune colitis (31), in which T reg function was
blocked by administration of anti-CTLA4 mAb.
In our islet transplantation model, blockade of the CTLA4-mediated negative signal with either CTLA4Ig or anti-CTLA4 mAb completely prevents tolerance induction (33). Whether the tolerance-blocking effects of anti-CTLA4 mAb occur through the direct targeting of T reg or by increasing the activity of cytopathic T cells has not yet been completely elucidated. Again, our results suggest that blockade of CTLA4 signaling acts at least in part through an effect that is directed at the cellular targets of T reg action.
In light of our findings, the failure of systemically administered rIL-2 or high-dose anti-CTLA4 mAb (alone or together with anti-CD25 mAb) to break tolerance in graft-bearing recipients may seem paradoxical. Indeed, under the cover of anti-CTLA4 mAb plus anti-CD25 mAb, these recipients accept a second donor strain graft in the absence of further immunosuppression. We believe that our present results, together with our previous findings (33), indicate that tolerance induction and the metastable phase of immune ignorance period that precedes the development of full tolerance are the states that can be broken by anti-CTLA4 mAb or exogenous rIL-2 (29, 33). In contrast, established tolerance is resistant to these treatments. Hence, CTLA4-dependent mechanisms are crucial to the induction, but not to the maintenance, of the tolerant state.
In short, our data support the pool size model and the critical role of CTLA4-triggered negative signal in the induction of tolerance. A link between loss of donor-specific proliferation and donor-specific tolerance is evident, but it is not at all certain whether this is sufficient to create and maintain immunological tolerance.
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Acknowledgments |
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Footnotes |
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2 T.B.S. and X.X.Z. share senior authorship.
3 Address correspondence and reprint requests to Dr. Terry B. Strom, Department of Medicine, Division of Immunology, Beth Israel Deaconess Medical Center, Harvard Medical School, Room 380, Research North, 99 Brookline Avenue, Boston, MA 02215. E-mail address: tstrom{at}caregroup.harvard.edu
4 Abbreviations used in this paper: T reg, regulatory T cell; MNL, mononuclear leukocyte; MST, mean survival time.
Received for publication September 5, 2001. Accepted for publication January 2, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. Sho, K. Kishimoto, H. Harada, M. Livak, A. Sanchez-Fueyo, A. Yamada, X. X. Zheng, T. B. Strom, G. P. Basadonna, M. H. Sayegh, et al. Requirements for induction and maintenance of peripheral tolerance in stringent allograft models PNAS, September 13, 2005; 102(37): 13230 - 13235. [Abstract] [Full Text] [PDF]
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W. Chen, D. Zhou, J. R. Torrealba, T. K. Waddell, D. Grant, and L. Zhang Donor Lymphocyte Infusion Induces Long-Term Donor-Specific Cardiac Xenograft Survival through Activation of Recipient Double-Negative Regulatory T Cells J. Immunol., September 1, 2005; 175(5): 3409 - 3416. [Abstract] [Full Text] [PDF]
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C. Domenig, A. Sanchez-Fueyo, J. Kurtz, S. P. Alexopoulos, C. Mariat, M. Sykes, T. B. Strom, and X. X. Zheng Roles of Deletion and Regulation in Creating Mixed Chimerism and Allograft Tolerance Using a Nonlymphoablative Irradiation-Free Protocol J. Immunol., July 1, 2005; 175(1): 51 - 60. [Abstract] [Full Text] [PDF]
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M. Karim, G. Feng, K. J. Wood, and A. R. Bushell CD25+CD4+ regulatory T cells generated by exposure to a model protein antigen prevent allograft rejection: antigen-specific reactivation in vivo is critical for bystander regulation Blood, June 15, 2005; 105(12): 4871 - 4877. [Abstract] [Full Text] [PDF]
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K. Fischer, S. Voelkl, J. Heymann, G. K. Przybylski, K. Mondal, M. Laumer, L. Kunz-Schughart, C. A. Schmidt, R. Andreesen, and A. Mackensen Isolation and characterization of human antigen-specific TCR{alpha}{beta}+ CD4-CD8- double-negative regulatory T cells Blood, April 1, 2005; 105(7): 2828 - 2835. [Abstract] [Full Text] [PDF]
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S. E. Sandner, M. R. Clarkson, A. D. Salama, A. Sanchez-Fueyo, C. Domenig, A. Habicht, N. Najafian, H. Yagita, M. Azuma, L. A. Turka, et al. Role of the Programmed Death-1 Pathway in Regulation of Alloimmune Responses In Vivo J. Immunol., March 15, 2005; 174(6): 3408 - 3415. [Abstract] [Full Text] [PDF]
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S. Schenk, D. D. Kish, C. He, T. El-Sawy, E. Chiffoleau, C. Chen, Z. Wu, S. Sandner, A. V. Gorbachev, K. Fukamachi, et al. Alloreactive T Cell Responses and Acute Rejection of Single Class II MHC-Disparate Heart Allografts Are under Strict Regulation by CD4+CD25+ T Cells J. Immunol., March 15, 2005; 174(6): 3741 - 3748. [Abstract] [Full Text] [PDF]
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K. Oh, S. Kim, S.-H. Park, H. Gu, D. Roopenian, D. H. Chung, Y. S. Kim, and D.-S. Lee Direct Regulatory Role of NKT Cells in Allogeneic Graft Survival Is Dependent on the Quantitative Strength of Antigenicity J. Immunol., February 15, 2005; 174(4): 2030 - 2036. [Abstract] [Full Text] [PDF]
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 A. L. Kinter, M. Hennessey, A. Bell, S. Kern, Y. Lin, M. Daucher, M. Planta, M. McGlaughlin, R. Jackson, S. F. Ziegler, et al. CD25+CD4+ Regulatory T Cells from the Peripheral Blood of Asymptomatic HIV-infected Individuals Regulate CD4+ and CD8+ HIV-specific T Cell Immune Responses In Vitro and Are Associated with Favorable Clinical Markers of Disease Status J. Exp. Med., August 2, 2004; 200(3): 331 - 343. [Abstract] [Full Text] [PDF]
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 T. L. Sumpter and D. S. Wilkes Role of autoimmunity in organ allograft rejection: a focus on immunity to type V collagen in the pathogenesis of lung transplant rejection Am J Physiol Lung Cell Mol Physiol, June 1, 2004; 286(6): L1129 - L1139. [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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 E. M. Aandahl, J. Michaelsson, W. J. Moretto, F. M. Hecht, and D. F. Nixon Human CD4+ CD25+ Regulatory T Cells Control T-Cell Responses to Human Immunodeficiency Virus and Cytomegalovirus Antigens J. Virol., March 1, 2004; 78(5): 2454 - 2459. [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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M. Kataoka, J. A. Margenthaler, G. Ku, and M. W. Flye Development of Infectious Tolerance After Donor-Specific Transfusion and Rat Heart Transplantation J. Immunol., July 1, 2003; 171(1): 204 - 211. [Abstract] [Full Text] [PDF]
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D. S. Game, M. P. Hernandez-Fuentes, A. N. Chaudhry, and R. I. Lechler CD4+CD25+ Regulatory T Cells Do Not Significantly Contribute to Direct Pathway Hyporesponsiveness in Stable Renal Transplant Patients J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1652 - 1661. [Abstract] [Full Text] [PDF]
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Y. Zhai and J. W. Kupiec-Weglinski Regulatory T Cells in Kidney Transplant Recipients: Active Players but to What Extent? J. Am. Soc. Nephrol., June 1, 2003; 14(6): 1706 - 1708. [Full Text] [PDF]
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N. E. Phillips, T. G. Markees, J. P. Mordes, D. L. Greiner, and A. A. Rossini Blockade of CD40-Mediated Signaling Is Sufficient for Inducing Islet But Not Skin Transplantation Tolerance J. Immunol., March 15, 2003; 170(6): 3015 - 3023. [Abstract] [Full Text] [PDF]
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W. Chen, M. S. Ford, K. J. Young, M. I. Cybulsky, and L. Zhang Role of Double-Negative Regulatory T Cells in Long-Term Cardiac Xenograft Survival J. Immunol., February 15, 2003; 170(4): 1846 - 1853. [Abstract] [Full Text] [PDF]
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W.-P. Min, D. Zhou, T. E. Ichim, G. H. Strejan, X. Xia, J. Yang, X. Huang, B. Garcia, D. White, P. Dutartre, et al. Inhibitory Feedback Loop Between Tolerogenic Dendritic Cells and Regulatory T Cells in Transplant Tolerance J. Immunol., February 1, 2003; 170(3): 1304 - 1312. [Abstract] [Full Text] [PDF]
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M. R. Nicolls, M. Coulombe, J. Beilke, H. C. Gelhaus, and R. G. Gill CD4-Dependent Generation of Dominant Transplantation Tolerance Induced by Simultaneous Perturbation of CD154 and LFA-1 Pathways J. Immunol., November 1, 2002; 169(9): 4831 - 4839. [Abstract] [Full Text] [PDF]
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S. Rutella, L. Pierelli, G. Bonanno, S. Sica, F. Ameglio, E. Capoluongo, A. Mariotti, G. Scambia, G. d'Onofrio, and G. Leone Role for granulocyte colony-stimulating factor in the generation of human T regulatory type 1 cells Blood, September 18, 2002; 100(7): 2562 - 2571. [Abstract] [Full Text] [PDF]
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