| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Pediatrics, Division of Bone Marrow Transplantation, University of Minnesota Cancer Center, Minneapolis, MN 55455;
SAIC-Frederick and the Laboratory of Leukocyte Biology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702;
Schering-Plough Research Institute, Kenilworth, NJ 07033; and
§
Telethon Institute of gene therapy-H. San Raffaele, Milan, Italy
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The anti-inflammatory members belonging to the TGF-ß family inhibit macrophage activation and Th1 inflammatory responses that occur during experimental allergic encephalomyelitis (10). The in vivo injection of TGF-ß protein or DNA plasmid delays cardiac allograft rejection in rodents (11). TGF-ß-deficient mice develop progressive inflammation with manifestations of autoimmunity that is dependent upon the regulation of CD4+ T cells by MHC class II/peptide complexes (12). Ag-specific T cell clones generated by exposure of CD4+ T cells to Ag in the presence of IL-10 have been shown to suppress the proliferation of naive CD4+ T cells to Ag partially by the secretion of TGF-ß and IL-10 (13). In the present study, we show that the anti-inflammatory cytokines IL-10 + TGF-ß have an additive effect on inducing CD4+ T cell Ag-specific hyporesponsiveness. Ag hyporesponsiveness was observed in vivo by the adoptive transfer of tolerized CD4+ T cells. Collectively, these data indicate that IL-10 and TGF-ß may play an important role in tolerance induction and suggest a new strategy for inducing Ag-specific tolerance via the regulation of IL-10 and TGF-ß function.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C.H2bm12 (termed bm12) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 (H-2b) (termed B6) and BALB/c (H-2d) mice were purchased from the National Institutes of Health (Bethesda, MD). D011.10 transgenic mice backcrossed >10 generations onto a BALB/c background were generated as described (14) and provided by Dr. Marc Jenkins (University of Minnesota, Minneapolis, MN). Donors and recipients were 8–10 wk of age at the time of bone marrow transplantation. All mice were housed in a specific pathogen-free facility in microisolator cages.
Cytokines
Human rIL-10 (Schering-Plough Research Institute, Kenilworth, NJ) (sp. act., 3.1 x 107 U/mg), active in mice because of species cross-reactivity, was added at initiation of MLR culture at a concentration of 100 and 1000 U/ml. IL-10 produced in Escherichia coli, isolated from inclusion bodies, was greater than 95% pure (as determined by gel electrophoresis), and contained less than 0.5 endotoxin U/mg of protein. Human rTGF-ß2 (Genzyme, Framingham, MA) (sp. act., 5 x 107 U/mg), active in mice because of species cross-reactivity, was added at the initiation of MLR culture at concentrations ranging from 0.1 to 1 ng/ml. TGF-ß2 was produced in E. coli.
In vitro MLR cultures
To purify CD4+ T cells, axillary,
mesenteric, and inguinal lymph nodes were mashed, and single cell
suspensions were passed through a wire mesh and collected into PBS
containing 2% FBS. Cell preparations were depleted of NK cells
(hybridoma PK136, rat IgG2a, provided by Dr. Gloria Koo, Rahway, NJ)
and CD8+ T cells (hybridoma 2.43, rat IgG2b,
provided by Dr. David Sachs, Charlestown, MA) by coating with mAb,
followed by passage through a goat anti-mouse and goat anti-rat
Ig-coated column (Biotex, Edmonton, Canada). The final composition of T
cells was determined by flow-cytometric analysis to be 
94%
CD4+ T cells. Responder
CD4+ T cells were mixed with irradiated (30
Gray), anti-Thy-1.2 mAb (hybridoma 30H-12, rat IgG2b, provided by
Dr. David Sachs), and anti-NK1.1 plus baby rabbit complement
(Nieffenegger, Woodland, CA)-depleted splenic stimulators. Three types
of cultures were established. In the first, bm12
CD4+ T cells were mixed with B6 splenic
stimulators. In the second, B6 CD4+ T cells were
mixed with bm12 splenic stimulators. In the third, nontransgenic BALB/c
CD4+ T cells were mixed with D011.10 BALB/c TCR
transgenic CD4+ T cells at a ratio of 3:1 and
then mixed with B6 splenic stimulator cells. Responder and stimulator
cells were suspended at a final concentration of 0.5 x
106/ml in 24-well plates (Costar, Acton, MA)
containing DMEM (BioWhittaker, Walkersville, MD) with 10% FBS
(HyClone, Logan, UT), 50 mM 2-ME (Sigma, St. Louis, MO), 10 mM HEPES
buffer, 1 mM sodium pyruvate (Life Technologies, Grand Island, NY), and
amino acid supplements (1.5 mM L-glutamine,
L-arginine, L-asparagine) (Sigma) and
antibiotics (penicillin, 100 U/ml; streptomycin, 100 mg/ml) (Sigma).
Plates were incubated at 37°C and 10% CO2 for
8 or 10 days. On day 5, the culture was split 1:1 with fresh media, and
human rIL-7 (R&D Systems, Minneapolis, MN) was added at a concentration
of 0.1 ng/ml. to increase cell recovery at the end of culture. To
monitor primary proliferation, 96-well round-bottom plates (Costar)
were set up to contain 105 responders and
105 stimulator cells per well in the presence or
absence of exogenous rhIL-2 (50 IU/ml) (Amgen, Thousand Oaks,
CA).
To monitor secondary MLR proliferation, 3 x 104 responders and 105 irradiated (30 Gray) non-T cell-depleted stimulators were plated in the presence or absence of IL-2 (50 IU/ml). Neither IL-10 nor TGF-ß was added to the secondary cultures. To assess chicken egg OVA responses in secondary MLR cultures, MLR-cultured cells adjusted to contain 2.5 x 104 KJ1-26+ CD4+ T cells per well were mixed with 105 BALB/c splenic feeder cells in 96-well microtiter plates. OVA peptide 323–339 (ISQAVHAAHAEINEAGR) was added at a final concentration of 5 µg/ml. Microtiter wells were pulsed with tritiated thymidine (1 µCi) for 16–18 h before harvesting and counted in the absence of scintillation fluid on a beta plate reader. Three to six wells were analyzed for each data point.
Flow cytometry
T cells from bulk MLR cultures were assessed for evidence of
activation via the coexpression of CD4 and activation Ags, including
IL-2R 
-chain (CD25). The anti-clonotypic mAb KJ1-26 was used to
distinguish D011.10 TCR transgenic from BALB/c nontransgenic
CD4+ T cells. KJ1-26 recognizes a
CD4+ T cell population specific for chicken OVA
peptide 323–339 bound to IAd. All studies were
performed with two- or three-color flow cytometry using FITC- or
PE-conjugated CD4, CD25, CD62L, and control mAbs of the appropriate
isotypes (PharMingen, San Diego, CA). Irrelevant mAb control values
were subtracted from values obtained with relevant mAbs. Cultured cells
were also analyzed for the incorporation of
7-AAD3 (7-amino
actinomycin D) (Sigma, St. Louis, MO), which binds to intracellular
DNA. Cells with intact membranes exclude 7-AAD
(7-AAD-), while those undergoing early apoptosis
bind 7-AAD (7-AAD+). All results were obtained
using a FACScalibur (Becton Dickinson). Forward and side scatter
settings were gated to exclude debris. A total of 10,000–20,000 cells
was analyzed for each determination.
Quantitation of cytokine levels by ELISA
Murine cytokine levels (IL-2, IL-4, IL-10, IL-12, IL-13,
IFN-
, and acid-free and total TGF-ß) in the supernatant of MLR
cultures were quantitated by ELISA (R&D Systems). Sensitivity of the
assays was between 1 and 10 pg/ml for each assay. A standard curve
using recombinant protein was generated with each assay.
GVH induction
B6 or bm12 recipients were sublethally irradiated by exposing mice to 6 Gray total body irradiation from a 137Cesium source at a dose rate of 85 cGy/min on day 0. Day 10 MLR-cultured cells were injected i.v. at the doses indicated. Five to eight mice per group per experiment were studied. Peripheral blood was obtained by retroorbital venipuncture for measurement of day 14 and 28 hematocrit values as an indicator of the bone marrow-destructive effects of infused T cells.
GVHD assessment by tissue scoring
Recipients of adoptively transferred CD4+ T cells were monitored for the occurrence of GVHD symptomatology, including ruffled fur, diarrhea, hunched posture, and lethargy (15), and by twice weekly quantitation of body weights. Liver, lung, colon, and spleen were removed, embedded in O.C.T. compound, and snap frozen in liquid nitrogen. Cryosections were then stained by hematoxylin and eosin and examined in coded fashion using our documented GVHD semiquantitative scoring system (0.5–4 grades), as described previously (16) with each grade followed by its histological features.
Statistical analyses
Survival data were analyzed by lifetable methods, and actuarial survival rates are shown. p values <0.05 were considered significant. Group comparisons were made by logrank test statistics. For all other data, group comparisons were made by Student’s t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To determine whether exogenous IL-10 addition at the initiation of
an MLR culture would induce alloantigen hyporesponsiveness, highly
purified CD4+ T cells were incubated for 10 days
with irradiated, T cell-depleted MHC class II disparate splenic
stimulators in bulk cultures in the presence or absence of IL-10. bm12
responders and B6 stimulators differ at three amino acids due to
mutations in the IA region. Because the addition of 100 U/ml IL-10
significantly suppressed the primary MLR in only four of seven
experiments and did not suppress the secondary MLR (three of three
experiments), experiments were performed in which 1000 U/ml of
exogenous IL-10 was added to primary MLR cultures. In one experiment
directly comparing the two concentrations of IL-10, the higher dose was
significantly more suppressive than the lower dose (6% vs 24% of
control response, respectively), and neither dose was significantly
suppressive in secondary MLR. Therefore, we chose the higher IL-10
concentration of 1000 U/ml for all future experiments. At a
concentration of 1000 U/ml IL-10, proliferation in primary MLR cultures
was consistently suppressed (Fig. 1
A). Mean cell recoveries of
control and IL-10-treated cultures were 103% (range: 60–155%) and
41% (range: 13–84%), respectively, in six experiments. After
addition of exogenous IL-2, proliferative responses of control and
IL-10-treated cells in primary MLR were comparable (Fig. 1B). In secondary MLR, proliferative hyporesponsiveness of
IL-10-treated cells to alloantigen-bearing stimulators was only
transiently observed (e.g., Fig. 1C).
|
To compare the immunosuppressive effects of IL-10 and TGF-ß on
allogeneic responses, studies were performed to determine whether
exogenous TGF-ß would inhibit primary MLR and induce
hyporesponsiveness in secondary MLR. TGF-ß added at a concentration
of 0.1 ng/ml inhibited primary MLR proliferative responses by >50% on
days 4 to 7 (data not shown). TGF-ß added at a concentration of 0.3
ng/ml provided a greater inhibitory effect than TGF-ß at 0.1 ng/ml
(not shown). The greatest inhibition, which was comparable with that
observed with IL-10, was obtained in cultures with 1 ng/ml of TGF-ß,
in which the suppression of the proliferation was reproducibly >80%
by day 5 of primary MLR culture (Fig. 1
A). Secondary MLR
hyporesponsiveness was profound and consistently observed when TGF-ß
was added to the primary MLR culture at 1 ng/ml (Fig. 1C).
Exogenous IL-2 only partially restored the proliferation in primary MLR
(Fig. 1B), whereas addition of IL-2 to secondary MLR of
TGF-ß-treated cells resulted in proliferative responses comparable
with those of control (data not shown).
The effects of combined IL-10 + TGF-ß in inducing CD4+ T cell hyporesponsiveness in primary and secondary MLR cultures
CD4+ T cells from IL-10-treated human
primary MLR cultures have a consistent and profound degree of
proliferative hyporesponsiveness when restimulated with alloantigen in
the absence of exogenous IL-10 (13). In these cultures,
TGF-ß has been shown to be secreted (13). In contrast,
the analysis of supernatants from IL-10 (1000 U/ml)-treated cultures
revealed that biologically active TGF-ß was nondetectable (data not
shown). Therefore, a series of experiments was performed to determine
whether TGF-ß enhanced the suppressive effects of IL-10. IL-10 +
TGF-ß treatment resulted in significant suppression of MLR responses
in each of 18 experiments (range = 93–99% suppression) when
compared with control cultures (e.g., Fig. 1A). Mean cell
recoveries of IL-10 + TGF-ß-treated cultures in these experiments
were reduced by 84% as compared with control cultures. The addition of
exogenous IL-2 partially prevented (17 experiments) or fully prevented
(1 experiment) IL-10 + TGF-ß-mediated suppression of allogeneic
responses in primary MLR cultures (mean = 47% of control
responses) (e.g., Fig. 1B). Because this treatment would
affect the alloantigen-bearing stimulators in the primary MLR culture,
it is possible that the addition of exogenous IL-2 would have been more
potent in restoring proliferation of T cell responses in cultures
devoid of stimulator cells. In secondary MLR, cells treated with IL-10
+ TGF-ß showed profound hyporesponsiveness in each experiment
analyzed (mean = 84% suppressed vs control) (e.g., Fig. 1C). IL-2 added to the IL-10 + TGF-ß-treated cells
resulted in a significant restoration (mean = 155% restoration vs
controls) of the proliferative capacity in secondary MLRs.
In experiments directly comparing TGF-ß to IL-10 + TGF-ß treatment,
the addition of both cytokines resulted in a significantly greater
degree of suppression of primary MLR responses (mean values = 86%
vs 97%, respectively) (Fig. 1A), and 46% lower mean cell
recoveries were observed in IL-10 + TGF-ß-treated cultures compared
with TGF-ß only. In secondary MLR, cells treated with IL-10 + TGF-ß
had a comparable (six experiments) or significantly lower (two
experiments) proliferation than with TGF-ß treatment alone.
We have been able to determine that the optimal degree of inhibition of
proliferation in both primary and secondary MLR cultures required the
presence of IL-10 + TGF-ß (Fig. 1). In these experiments, flow
cytometry was performed at the end of culture to determine how
treatment affected the expression of activation Ags (Fig. 2). All four groups had high levels of
CD25 expression (mean values of 51%, 38%, 61%, and 40%,
respectively). Down-regulation of L-selectin in control (mean of 54%)
was higher than in the IL-10-, TGF-ß-, and IL-10 + TGF-ß-treated
groups (mean of 75%, 77%, and 80%, respectively), while
up-regulation of CD44 expression was higher in control (mean of 82%)
than in the IL-10-, TGF-ß-, and IL-10 + TGF-ß-treated groups (mean
of 66%, 68%, and 66%, respectively) (data not shown), suggesting
that each of the treatments inhibited the extent of T cell activation.
The frequency of 7-AAD+ T cells, an indicator of
early apoptosis, was similar in all groups with mean values of
4%.
|
). The Th1
cytokine, IFN-, was present at low levels in MLR with IL-10, but it
was undetectable in TGF-ß-containing cultures and in cultures with
both cytokines. Evaluation of murine Th2 cytokines (IL-4, IL-10, IL-13)
revealed that only IL-13 was produced in significant concentrations in
the control culture. A reproducible inhibition of IL-13 production was
observed in cultures treated with both cytokines (e.g., Table I). The
reduction in IL-12 production in cultures containing IL-10 is
consistent with the data of Murphy, O’Garra, and coworkers (17, 18) and the known inhibitory effects of IL-10 on monocytes and
macrophages (19), which are a source of IL-12 (reviewed in
Ref. 20). Because IL-12 is critical to the development of
Th1 cells (17, 18, 19, 20, 21), the inhibitory effects of IL-10 on
IL-12 production may be responsible for the observed inhibition of Th1
cell generation in IL-10-containing MLR cultures. Thus, both Th1 and
Th2 cytokine production is inhibited by IL-10 + TGF-ß treatment.
|
A culture system was established in which T cell responses to an
Ag not present during the tolerization process could be accurately
quantified. To optimize quantification, the responses of D011.10 TCR
transgenic CD4+ T cells were physically tracked
via flow-cytometric analysis using an anti-clonotypic mAb. These T
cells express the appropriate TCR required for a response to the
nominal Ag OVA. CD4+ T cell cultures contained
25% D011.10 TCR transgenic T cells and 75% nontransgenic
CD4+ T cells of the same genetic (BALB/c)
background. As anticipated, the addition of TGF-ß and IL-10 + TGF-ß
to the primary MLR culture inhibited alloresponses (Fig. 3A). To determine whether
inhibition of alloantigen responses was specific, secondary cultures
were established to monitor alloantigen and nominal Ag responses. The
culture was adjusted such that the control and cytokine-treated
cultures contained the same number of nontransgenic
CD4+ T cells per well for measuring alloresponses
and the same number of transgenic CD4+ T cells
for measuring OVA responses. At the end of culture, cell recovery was
123% in the control group, 31% in the TGF-ß-treated group, and 20%
in the IL-10 + TGF-ß-treated group. TGF-ß and IL-10 + TGF-ß
treatment led to secondary alloantigen hyporesponsiveness (Fig. 3B), but did not impair responses to optimal concentrations
of OVA peptide in secondary culture (Fig. 3C).
|
To determine whether ex vivo tolerized T cells were impaired in
inducing GVHD, experiments were performed in which
105 control or an equal number of IL-10-,
TGF-ß-, or IL-10 + TGF-ß-treated CD4+ T cells
were infused into sublethally irradiated recipients bearing the same
alloantigen used as stimulator cells in MLR cultures. At this cell
dose, pooled data from three replicate experiments (n =
15 mice per group) show that the infusion of control cultured, IL-10-,
and TGF-ß-treated T cells was lethal to 100%, 100%, and 85% of
recipients, respectively (Fig. 4). In
contrast, when IL-10 + TGF-ß-treated T cells were injected, 75% of
the recipient mice survived. Mean weight values in the group that
received IL-10 + TGF-ß-treated cells remained constant over the
duration of the evaluation period, whereas mean weight values in the
group that received TGF-ß-treated cells began to decline at day 15
posttransfer, although not as rapidly as the groups that received
either control or IL-10-treated cells (Fig. 5). Long-term (2 mo posttransfer)
survivors (n = 5) that received IL-10 + TGF-ß-treated
T cells were not entirely GVH free by histological criteria. Using a
GVHD scoring system ranging from 0.5 (mildest) to 4 (most severe), we
observed that these survivors had mean GVH scores of the liver (mean
0.4), spleen (mean 1.0), lung (mean 1.3), and colon (mean 1.8). Day 14
mean hematocrit values, an indicator of GVHD-induced bone marrow
destruction, were not significantly different between the control,
IL-10-, and TGF-ß only-treated groups (18%, 16%, and 21%,
respectively) (Table II). In contrast,
mice in the IL-10 + TGF-ß-treated group had mean day 14 hematocrit
values of 28%, which were significantly higher
(p 0.01) than the other three groups. These
results conclusively demonstrate that both IL-10 and TGF-ß are at
least additive in inhibiting GVH lethality in vivo.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-10 has been shown to possess immunoregulatory properties, both stimulatory and suppressive, in human and murine models of autoimmune disease. Bacchetta et al. have shown that CD4+ host-reactive T cell clones isolated from a SCID patient transplanted with HLA-mismatched stem cells are hyporesponsive in vitro and do not cause GVHD in vivo (7). Ag-specific stimulation of these clones in vitro resulted in the production of unusually high levels of IL-10 and very low amounts of IL-2. The proliferative responses of these clones were restored in the presence of neutralizing concentrations of an anti-IL-10 mAb, suggesting that high levels of endogenous IL-10 suppress the activity of these cells. Buer et al. (22) have demonstrated that MHC class II-restricted T cells specific for influenza hemagglutinin became anergic in mice that expressed hemagglutinin, and that anergy was associated with IL-10 production. These findings provided indirect evidence that T cells anergized in vivo become regulatory T cells that may influence neighboring immune responses through the localized release of IL-10. IL-10 has previously been shown to affect many aspects of the immune response, including inhibiting Ag-specific activation and proliferation of human CD4+ T cells resulting in a long-term Ag-specific anergic state (23). IL-10 treatment of human CD4+ MLR cultures results in the generation of a subset of CD4+ T cells termed T-regulator type 1 that inhibit Ag-specific immune responses in part via TGF-ß and IL-10 production. In human culture systems, TGF-ß has been shown to be secreted by IL-10-treated CD4+ T cells (13). Our laboratory has previously shown that the exogenous administration of high doses of IL-10 to murine recipients of MHC-disparate grafts accelerates GHVD lethality (9). Paradoxically, low doses of IL-10 protected mice against GVHD lethality. In a mouse model of acute pancreatitis and diabetes, Pakala et al. (24) have demonstrated that Th2 cells mediate islet destruction by local IL-10 production, but not IL-4. These findings indicated that under certain conditions, Th2 T cells producing locally high levels of IL-10 may cause acute pathology and disease rather than a protective condition. Consistent with in vitro studies indicating that IL-10 can augment CD8+ CTL activity (25), Strom and coworkers (26) have shown that the in vivo administration of an IL-10 fusion protein enhanced CD8+ T cell infiltration and granzyme B gene expression in the pancreas of recipients of allogeneic islet grafts, suggesting that IL-10 can augment CTL alloreactivity. These findings indicated that under certain conditions, Th2 T cells producing locally high levels of IL-10 may cause acute pathology and disease rather than a protective condition. Therefore, in vivo regulation of immune responses by IL-10 may be concentration dependent or may be determined by the subset of T cells that produce IL-10.
TGF-ß has been shown to exhibit dose-dependent effects, with low
concentrations and high concentrations exerting distinct physiologic
effects (27, 28, 29). Mice deficient in TGF-ß develop a
lethal multiorgan inflammatory immune infiltrate at 3 wk of age
(12, 30, 31) with increased expression of the inflammatory
cytokines IFN- and TNF- (12) and the inflammatory
mediator nitric oxide (32). Conversely, high levels of
TGF-ß expression in recipients of transplanted organ allografts have
been associated with the development of chronic rejection
(33, 34, 35). In vivo neutralization of TGF-ß in
Leishmania amazonensis-infected BALB/c mice resulted in
susceptibility to the organism, demonstrating that endogenous TGF-ß
suppressed Th1 cellular immune responses (36). Letterio et
al. (37) have shown that the progressive inflammatory
process found in TGF-ß-deficient mice is associated with several
autoimmune conditions, including circulating Abs to nuclear Ags, immune
complex deposition, and increased expression of both MHC class I and
class II. Fukaura et al. (38) found that oral
administration of myelin basic protein and proteolipid protein resulted
in a marked increase in the relative frequencies of both myelin basic
protein and proteolipid protein-specific TGF-ß1-secreting T cells.
Their results demonstrate the possibility of inducing a distinct
lineage of TGF-ß1-secreting Th3 T cells that migrates to target
organs and suppresses inflammation in the local microenvironment.
Powrie et al. (39) have identified a subpopulation of
peripheral CD4+ T cells that secreted TGF-ß,
thus contributing to a natural immune regulatory mechanism that
prevented the development of pathogenic Th1 responses in the gut of
mice. This immunoregulatory T cell population appeared to be distinct
from Th2 cells. By treating infected mice with rTGF-ß, Omer and Riley
(40) described a direct role for TGF-ß in inducing
protective immune responses in the resolution of malaria in BALB/c
mice. TGF-ß induced protective immune responses that led to slower
parasite growth early in infection, which down-regulated pathogenic
responses late in infection.
In our study, IL-10 and TGF-ß were necessary, but alone not sufficient to induce Ag-specific T cell hyporesponsiveness and to prevent GHVD in vivo. In an attempt to unify T cell anergy induced by TCR occupancy in the absence of costimulation with anergy induced by IL-10, Schwartz (41) has proposed a model in which IL-10 facilitates anergy by blocking the delivery of B7/CD28 costimulation. The small amount of proliferation observed in our IL-10 + TGF-ß-treated T cell cultures suggests that there is a minor threshold level of costimulation for IL-2 production. Therefore, as noted by Schwartz for IL-10 treatment of human T cells, anergy in our IL-10 + TGF-ß-treated cultures may result from the augmentation in the production of Nil-2a or the inhibitor of p21ras activation in T cells. Alternatively, IL-10 + TGF-ß treatment could block the down-regulation of these components by signal transduction through the IL-2R. Whether the effects of IL-10 + TGF-ß are due to the more potent inhibition of a common downstream signal transduction pathway by each protein or to the combined inhibition of two distinct signaling pathways that lead to a more profound inhibition is not known.
Interestingly, we have shown that alloantigen-specific hyporesponsiveness can be achieved without losing the capacity of the cells to respond to other Ags. Using OVA-responsive TCR transgenic T cells in an MLR culture containing wild-type CD4+ T cells and IL-10 + TGF-ß, but not OVA Ag, we quantified the proliferation of OVA-responsive TCR transgenic cells to an optimal concentration of OVA peptide in secondary culture and demonstrated that it was not inhibited. Prevention of CD4+ T cell responses to alloantigens by ex vivo IL-10 + TGF-ß treatment, therefore, was Ag specific and not due to the global immune suppression of both Ag-specific and bystander cells. However, we cannot exclude, from these studies, the possibility that IL-10 + TGF-ß treatment may be detrimental to either responses to other types of Ags not present during the initial culture or responses to lower concentrations of OVA that provide less vigorous TCR signal transduction.
Our data are the first to demonstrate direct evidence that IL-10 and TGF-ß work in concert to regulate Ag-specific immune responses that are biologically relevant in vivo. Whatever the molecular mechanism, an ex vivo Ag-specific tolerization strategy would have an advantage over ex vivo tolerization approaches that rigorously deplete T cells or in vivo approaches that globally immunosuppress the recipient (42). Indeed, these latter currently available approaches are associated with extended periods of immune suppression and consequent high mortality in an allogeneic bone marrow transplantation setting. In addition, the finding that nominal Ag responses remain intact after ex vivo tolerization induced by IL-10 + TGF-ß treatment suggests that the antiviral or antileukemia responses of alloantigen nonresponsive cells may be preserved. Because CD8+ T cells can play an important role in antiviral or antileukemia responses, future studies should be undertaken to determine whether IL-10 + TGF-ß treatment would be similarly beneficial on selectively inhibiting CD8+ T cell responses to alloantigen of sufficient magnitude to inhibit GVHD lethality. The possible clinical translation of these basic observations in patients undergoing allogeneic bone marrow transplantation warrants further investigation.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Bruce R. Blazar, University of Minnesota Hospital, Box 109 Mayo Building, 420 S.E. Delaware Street, Minneapolis, MN 55455. E-mail address:
3 Abbreviations used in this paper: 7-AAD, 7-amino actinomycin D; GVH, graft-vs-host; GVHD, GVH disease.
Received for publication May 18, 1999. Accepted for publication July 19, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Transplant. Proc. 25:2937.[Medline]
production by mouse T helper clones that are unresponsive to B7 costimulation. J. Exp. Med. 180:223.This article has been cited by other articles:
|
|
 |
|
  A. S. Almeida, P. M. Lago, N. Boechat, R. C. Huard, L. C. O. Lazzarini, A. R. Santos, M. Nociari, H. Zhu, B. M. Perez-Sweeney, H. Bang, et al. Tuberculosis Is Associated with a Down-Modulatory Lung Immune Response That Impairs Th1-Type Immunity J. Immunol., July 1, 2009; 183(1): 718 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Solvason, Y.-P. Lou, W. Peters, E. Evans, J. Martinez, U. Ramirez, A. Ocampo, R. Yun, S. Ahmad, E. Liu, et al. Improved Efficacy of a Tolerizing DNA Vaccine for Reversal of Hyperglycemia through Enhancement of Gene Expression and Localization to Intracellular Sites J. Immunol., December 15, 2008; 181(12): 8298 - 8307. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Chen, X. Liang, A. J. Peterson, D. H. Munn, and B. R. Blazar The Indoleamine 2,3-Dioxygenase Pathway Is Essential for Human Plasmacytoid Dendritic Cell-Induced Adaptive T Regulatory Cell Generation J. Immunol., October 15, 2008; 181(8): 5396 - 5404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. V. Poncini, C. D. A. Soto, E. Batalla, M. E. Solana, and S. M. Gonzalez Cappa Trypanosoma cruzi Induces Regulatory Dendritic Cells In Vitro Infect. Immun., June 1, 2008; 76(6): 2633 - 2641. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Dittrich, A. Erbacher, S. Specht, F. Diesner, M. Krokowski, A. Avagyan, P. Stock, B. Ahrens, W. H. Hoffmann, A. Hoerauf, et al. Helminth Infection with Litomosoides sigmodontis Induces Regulatory T Cells and Inhibits Allergic Sensitization, Airway Inflammation, and Hyperreactivity in a Murine Asthma Model J. Immunol., February 1, 2008; 180(3): 1792 - 1799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. P. Beiting, L. F. Gagliardo, M. Hesse, S. K. Bliss, D. Meskill, and J. A. Appleton Coordinated Control of Immunity to Muscle Stage Trichinella spiralis by IL-10, Regulatory T Cells, and TGF-beta J. Immunol., January 15, 2007; 178(2): 1039 - 1047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Y. Lan, Z. Wang, G. Raimondi, W. Wu, B. L. Colvin, A. De Creus, and A. W. Thomson "Alternatively Activated" Dendritic Cells Preferentially Secrete IL-10, Expand Foxp3+CD4+ T Cells, and Induce Long-Term Organ Allograft Survival in Combination with CTLA4-Ig J. Immunol., November 1, 2006; 177(9): 5868 - 5877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Sinuani, Z. Averbukh, I. Gitelman, M. J. Rapoport, J. Sandbank, M. Albeck, B. Sredni, and J. Weissgarten Mesangial cells initiate compensatory renal tubular hypertrophy via IL-10-induced TGF-beta secretion: effect of the immunomodulator AS101 on this process Am J Physiol Renal Physiol, August 1, 2006; 291(2): F384 - F394. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M C Fantini, C Becker, I Tubbe, A Nikolaev, H A Lehr, P Galle, and M F Neurath Transforming growth factor {beta} induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis Gut, May 1, 2006; 55(5): 671 - 680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Keino, M. Takeuchi, T. Kezuka, T. Hattori, M. Usui, O. Taguchi, J. W. Streilein, and J. Stein-Streilein Induction of Eye-Derived Tolerance Does Not Depend on Naturally Occurring CD4+CD25+ T Regulatory Cells. Invest. Ophthalmol. Vis. Sci., March 1, 2006; 47(3): 1047 - 1055. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-T. Lin, B. Storer, P. J. Martin, L.-H. Tseng, B. Grogan, P.-J. Chen, L. P. Zhao, and J. A. Hansen Genetic variation in the IL-10 pathway modulates severity of acute graft-versus-host disease following hematopoietic cell transplantation: synergism between IL-10 genotype of patient and IL-10 receptor {beta} genotype of donor Blood, December 1, 2005; 106(12): 3995 - 4001. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mocellin, F. M. Marincola, and H. A. Young Interleukin-10 and the immune response against cancer: a counterpoint J. Leukoc. Biol., November 1, 2005; 78(5): 1043 - 1051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. M. A. de Graaff, E. C. de Jong, T. M. van Capel, M. E. A. van Dijk, P. J. M. Roholl, J. Boes, W. Luytjes, J. L. L. Kimpen, and G. M. van Bleek Respiratory Syncytial Virus Infection of Monocyte-Derived Dendritic Cells Decreases Their Capacity to Activate CD4 T Cells J. Immunol., November 1, 2005; 175(9): 5904 - 5911. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R Blazar and W. J Murphy Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) Phil Trans R Soc B, September 29, 2005; 360(1461): 1747 - 1767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Banovic, K. P. A. MacDonald, E. S. Morris, V. Rowe, R. Kuns, A. Don, J. Kelly, S. Ledbetter, A. D. Clouston, and G. R. Hill TGF-{beta} in allogeneic stem cell transplantation: friend or foe? Blood, September 15, 2005; 106(6): 2206 - 2214. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Kang and P. M. Allen Priming in the Presence of IL-10 Results in Direct Enhancement of CD8+ T Cell Primary Responses and Inhibition of Secondary Responses J. Immunol., May 1, 2005; 174(9): 5382 - 5389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Waiczies, T. Prozorovski, C. Infante-Duarte, A. Hahner, O. Aktas, O. Ullrich, and F. Zipp Atorvastatin Induces T Cell Anergy via Phosphorylation of ERK1 J. Immunol., May 1, 2005; 174(9): 5630 - 5635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hanada, K. Tanaka, Y. Matsumura, M. Yamauchi, H. Nishinakamura, H. Aburatani, R. Mashima, M. Kubo, T. Kobayashi, and A. Yoshimura Induction of Hyper Th1 Cell-Type Immune Responses by Dendritic Cells Lacking the Suppressor of Cytokine Signaling-1 Gene J. Immunol., April 1, 2005; 174(7): 4325 - 4332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. W. Blaser, S. Roychowdhury, D. J. Kim, N. R. Schwind, D. Bhatt, W. Yuan, D. F. Kusewitt, A. K. Ferketich, M. A. Caligiuri, and M. Guimond Donor-derived IL-15 is critical for acute allogeneic graft-versus-host disease Blood, January 15, 2005; 105(2): 894 - 901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kranzer, A. Eckhardt, M. Aigner, G. Knoll, L. Deml, C. Speth, N. Lehn, M. Rehli, and W. Schneider-Brachert Induction of Maturation and Cytokine Release of Human Dendritic Cells by Helicobacter pylori Infect. Immun., August 1, 2004; 72(8): 4416 - 4423. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. G. Bonecini-Almeida, J. L. Ho, N. Boechat, R. C. Huard, S. Chitale, H. Doo, J. Geng, L. Rego, L. C. O. Lazzarini, A. L. Kritski, et al. Down-Modulation of Lung Immune Responses by Interleukin-10 and Transforming Growth Factor {beta} (TGF-{beta}) and Analysis of TGF-{beta} Receptors I and II in Active Tuberculosis Infect. Immun., May 1, 2004; 72(5): 2628 - 2634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Chen, X. Cui, G. D. Sempowski, C. Liu, and N. J. Chao Transfer of allogeneic CD62L- memory T cells without graft-versus-host disease Blood, February 15, 2004; 103(4): 1534 - 1541. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-T. Lin, B. Storer, P. J. Martin, L.-H. Tseng, T. Gooley, P.-J. Chen, and J. A. Hansen Relation of an Interleukin-10 Promoter Polymorphism to Graft-versus-Host Disease and Survival after Hematopoietic-Cell Transplantation N. Engl. J. Med., December 4, 2003; 349(23): 2201 - 2210. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Foussat, F. Cottrez, V. Brun, N. Fournier, J.-P. Breittmayer, and H. Groux A Comparative Study between T Regulatory Type 1 and CD4+CD25+ T Cells in the Control of Inflammation J. Immunol., November 15, 2003; 171(10): 5018 - 5026. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Horwitz, S. G. Zheng, and J. D. Gray The role of the combination of IL-2 and TGF-{beta} or IL-10 in the generation and function of CD4+ CD25+ and CD8+regulatory T cell subsets J. Leukoc. Biol., October 1, 2003; 74(4): 471 - 478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. von Boehmer Dynamics of Suppressor T Cells: In Vivo Veritas J. Exp. Med., September 15, 2003; 198(6): 845 - 849. [Full Text] [PDF]
|
|
|
|
|
|
M. D. Elhalel, J.-H. Huang, W. Schmidt, J. Rachmilewitz, and M. L. Tykocinski CTLA-4 {middle dot} FasL Induces Alloantigen-Specific Hyporesponsiveness J. Immunol., June 15, 2003; 170(12): 5842 - 5850. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. V. Parekh, D. V. R. Prasad, P. P. Banerjee, B. N. Joshi, A. Kumar, and G. C. Mishra B Cells Activated by Lipopolysaccharide, But Not By Anti-Ig and Anti-CD40 Antibody, Induce Anergy in CD8+ T Cells: Role of TGF-{beta}1 J. Immunol., June 15, 2003; 170(12): 5897 - 5911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Thomas, R. Kumar, A. Preda-Pais, S. Casares, and T.-D. Brumeanu A Model for Antigen-Specific T-Cell Anergy: Displacement of CD4-p56lck Signalosome from the Lipid Rafts by a Soluble, Dimeric Peptide-MHC Class II Chimera J. Immunol., June 15, 2003; 170(12): 5981 - 5992. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-m. Chen, M. J. O'Shaughnessy, I. Gramaglia, A. Panoskaltsis-Mortari, W. J. Murphy, S. Narula, M. G. Roncarolo, and B. R. Blazar IL-10 and TGF-{beta} induce alloreactive CD4+CD25- T cells to acquire regulatory cell function Blood, June 15, 2003; 101(12): 5076 - 5083. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Asadullah, W. Sterry, and H. D. Volk Interleukin-10 Therapy--Review of a New Approach Pharmacol. Rev., June 1, 2003; 55(2): 241 - 269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. P. A. MacDonald, V. Rowe, C. Filippich, R. Thomas, A. D. Clouston, J. K. Welply, D. N. J. Hart, J. L. M. Ferrara, and G. R. Hill Donor pretreatment with progenipoietin-1 is superior to granulocyte colony-stimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation Blood, March 1, 2003; 101(5): 2033 - 2042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Weigel, N. Nath, P. A. Taylor, A. Panoskaltsis-Mortari, W. Chen, A. M. Krieg, K. Brasel, and B. R. Blazar Comparative analysis of murine marrow-derived dendritic cells generated by Flt3L or GM-CSF/IL-4 and matured with immune stimulatory agents on the in vivo induction of antileukemia responses Blood, December 1, 2002; 100(12): 4169 - 4176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
P. Hoffmann, J. Ermann, M. Edinger, C. G. Fathman, and S. Strober Donor-type CD4+CD25+ Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation J. Exp. Med., August 5, 2002; 196(3): 389 - 399. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Daddi, T. Suda, F. D'Ovidio, S. A. Kanaan, T. Tagawa, K. Grapperhaus, B. D. Kozower, J. H. Ritter, N. S Yew, T. Mohanakumar, et al. Recipient intramuscular cotransfection of naked plasmid transforming growth factor {beta}1 and interleukin 10 ameliorates lung graft ischemia-reperfusion injury J. Thorac. Cardiovasc. Surg., August 1, 2002; 124(2): 259 - 269. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. M. Janssen, M. H. M. Wauben, F. P. Nijkamp, W. van Eden, and A. J. M. van Oosterhout Immunomodulatory Effects of Antigen-Pulsed Macrophages in a Murine Model of Allergic Asthma Am. J. Respir. Cell Mol. Biol., August 1, 2002; 27(2): 257 - 264. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Piccirillo, J. J. Letterio, A. M. Thornton, R. S. McHugh, M. Mamura, H. Mizuhara, and E. M. Shevach CD4+CD25+ Regulatory T Cells Can Mediate Suppressor Function in the Absence of Transforming Growth Factor {beta}1 Production and Responsiveness J. Exp. Med., July 15, 2002; 196(2): 237 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Taylor, T. M. Friedman, R. Korngold, R. J. Noelle, and B. R. Blazar Tolerance induction of alloreactive T cells via ex vivo blockade of the CD40:CD40L costimulatory pathway results in the generation of a potent immune regulatory cell Blood, May 29, 2002; 99(12): 4601 - 4609. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Chen, X. Cui, C. Liu, and N. J. Chao Prevention of graft-versus-host disease while preserving graft-versus-leukemia effect after selective depletion of host-reactive T cells by photodynamic cell purging process Blood, May 1, 2002; 99(9): 3083 - 3088. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-N. Avice, M. Rubio, M. Sergerie, G. Delespesse, and M. Sarfati Role of CD47 in the Induction of Human Naive T Cell Anergy J. Immunol., September 1, 2001; 167(5): 2459 - 2468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Cottrez and H. Groux Regulation of TGF-{{beta}} Response During T Cell Activation Is Modulated by IL-10 J. Immunol., July 15, 2001; 167(2): 773 - 778. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Taylor, R. J. Noelle, and B. R. Blazar Cd4+Cd25+ Immune Regulatory Cells Are Required for Induction of Tolerance to Alloantigen via Costimulatory Blockade J. Exp. Med., June 4, 2001; 193(11): 1311 - 1318. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. K. Levings, R. Sangregorio, F. Galbiati, S. Squadrone, R. de Waal Malefyt, and M.-G. Roncarolo IFN-{{alpha}} and IL-10 Induce the Differentiation of Human Type 1 T Regulatory Cells J. Immunol., May 1, 2001; 166(9): 5530 - 5539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Rossi, J. Sarkar, and D. Scandella Long-term induction of immune tolerance after blockade of CD40-CD40L interaction in a mouse model of hemophilia A Blood, May 1, 2001; 97(9): 2750 - 2757. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Weinberg, B. R. Blazar, J. E. Wagner, E. Agura, B. J. Hill, M. Smogorzewska, R. A. Koup, M. R. Betts, R. H. Collins, and D. C. Douek Factors affecting thymic function after allogeneic hematopoietic stem cell transplantation Blood, March 1, 2001; 97(5): 1458 - 1466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. A. Boussiotis, Z.-M. Chen, J. C. Zeller, W. J. Murphy, A. Berezovskaya, S. Narula, M. G. Roncarolo, and B. R. Blazar Altered T-cell receptor + CD28-mediated signaling and blocked cell cycle progression in interleukin 10 and transforming growth factor-{beta}-treated alloreactive T cells that do not induce graft-versus-host disease Blood, January 15, 2001; 97(2): 565 - 571. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-H. Sonoda, D. E. Faunce, M. Taniguchi, M. Exley, S. Balk, and J. Stein-Streilein NK T Cell-Derived IL-10 Is Essential for the Differentiation of Antigen-Specific T Regulatory Cells in Systemic Tolerance J. Immunol., January 1, 2001; 166(1): 42 - 50. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
