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+ Dendritic Cells Expressing Indoleamine 2,3-Dioxygenase1
*
Department of Experimental Medicine, University of Perugia, Perugia, Italy; and
Ludwig Institute for Cancer Research, Brussels, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-) and tolerogenic
(CD8+) DC. By virtue of their respective actions on
CD8- and CD8+ DC, IL-12 and IFN-
have
functionally opposing effects on peptide presentation by the
CD8- DC subset, and IFN--activated CD8+ DC
mediate tolerogenic effects that prevail over the adjuvant activity of
IL-12 on CD8- DC. We have previously shown that CD40
ligation abrogates the tolerogenic potential of CD8+ DC, an
effect associated with an impaired capacity of the CD40-modulated and
IFN--treated DC to degrade tryptophan and initiate T cell apoptosis
in vitro. We report here that IL-6 may both replace (upon
administration of the recombinant cytokine) and mediate (as assessed by
the use of neutralizing Abs) the effect of CD40 ligation in ablating
the tolerogenic activity of CD8+ DC. The activity of IL-6
includes down-regulation of IFN-R expression in the CD8+
DC subset and correlates to a reduced ability of these cells to
metabolize tryptophan and initiate T cell apoptosis in
vitro. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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enhances the
tolerogenic activity of CD8+ DC on Ag
presentation by the other subset, CD40 activation on the former cells
will abolish their tolerogenic capacity or even trigger the potential
for immunogenic presentation of P815AB (9, 10). This
effect is accompanied in vitro by an impaired ability of the
CD40-modulated and IFN--treated CD8+ DC to
produce indoleamine 2,3-dioxygenase (IDO) and to initiate apoptosis of
Ag-specific CD4+ T cells (10).
Much evidence indicates that DC can present Ag in an immunogenic or
tolerogenic fashion and that autoimmunity can be primed by DC
(11, 12, 13). Many autoimmune states involve an imbalance of
cytokines, and it has been proposed that DC may induce autoimmunity
because of intrinsic defects in genes controlling DC function or
following their differentiation by certain cytokines (14).
In several experimental models, IL-6 appears to be required for the
development of Ag- or collagen-induced arthritis (15, 16),
myelin oligodendrocyte protein-induced experimental autoimmune
encephalomyelitis (17, 18, 19, 20), and autoantibodies against DNA
in pristane-induced systemic lupus erythematosus (21).
However, how IL-6 acts to induce disease whereas TNF-
(22) and IFN- (23, 24, 25) can, in at least
some cases, protect against autoimmunity is not clear
(14). Regarding the possible role of IFN-, we have
previously proposed that the cytokine acting on
CD8+ DC might contribute to the maintenance of T
cell tolerance to self Ags via tryptophan degradation affecting T cell
responses (8, 10).
We have adopted an experimental design relying on the in vitro
treatment of DC with cytokines before their transfer into recipient
hosts to confine the analysis of cytokine effects to DC in the face of
the high pleiotropy of these molecules in vivo. Our earlier studies
were largely based on the use of P815AB, which was shown by us to
possess both class I- and class II-restricted epitopes
(3), such that transfer of peptide-pulsed DC would result
in an IL-12-dependent response in the host requiring the presence of
CD4+ T cells (1, 2). However,
because of the low intrinsic immunogenicity of the peptide, effective
priming in vivo called for the use of added adjuvanticity in vitro
(2, 6, 7) or ablation of the tolerogenic effect of a
specific subset of splenic DC (8, 10). In the present
study we have further explored the issue of cytokine regulation of Ag
presentation in an immunogenic vs tolerogenic fashion as resulting from
the combined actions of different DC subsets. We demonstrate that,
similar to CD40 activation, and largely mediating the effect of this
maneuver, IL-6 will abolish the susceptibility of
CD8+ DC to the tolerogenic activity of IFN- in
the induction of T cell reactivity to P815AB or NRP-A7. These data
strengthen the hypothesis that the DC system can present tumor/self Ags
in an immunogenic or tolerogenic fashion depending on the cytokine
balance and that IL-6 may be a major cytokine involved in the induction
of autoimmunity by DC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DBA/2J (H-2d) mice were obtained from
Charles River Laboratories (Calco, Milan, Italy). Male mice were used
at the age of 2–4 mo. The source and characteristics of murine rIL-12
(6), murine rIFN- (8), hamster
anti-murine CD40 (HM40-3) mAb used in combination with goat
anti-hamster IgG (9, 10), and biotinylated rat IgG to
murine IFN-R -chain (10) were previously described.
IL-12 was 98.8% pure, as assessed by SDS-PAGE, and endotoxin
contamination was <0.9 endotoxin U/mg on Limulus
amebocyte assay. Murine rIL-6 (109 U/mg) was
produced in the baculovirus system, as previously described (26, 27). Endotoxin was removed from all solutions containing rIL-12,
anti-CD40 Abs, or rIL-6 with Detoxi-gel (Pierce, Rockford, IL),
resulting in endotoxin contamination below the detection limit (0.05
endotoxin U/ml) of the assay (Coatest Endotoxin, Chromogenix AB,
Molndal, Sweden) (9). Rat monoclonal 6B4 (anti-mouse
IL-6) and 15A7 (anti-mouse IL-6R) Abs were previously described
(28, 29). The enzyme inhibitor
1-methyl-DL-tryptophan (1-MT) waspurchased
from Aldrich (Milan, Italy).
Peptides
P815AB (amino acid sequence LPYLGWLVF) and NRP-A7 (KYNKANAFL)
were synthesized on solid phase using F-moc for transient N-terminal
protection, purified by means of reverse phase HPLC, and characterized
by amino acid analysis. NRP-A7 is an alanine mutant analog, with
superior agonistic properties, of NRP, a synthetic peptide mimotope
recognized in the context of H-2Kd class I
molecules by a prevailing, pancreatic 
-cell-specific T lymphocyte
population in NOD mice (4, 5).
DC purification
DC were prepared and fractionated according to CD11c/CD8
expression using positive selection columns in combination with CD11c
and CD8 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany) as
previously described (6, 7, 8). Briefly, DC were obtained
from collagenase-treated spleens (collagenase type IV, Sigma, St.
Louis, MO). Total spleen cells were treated with EDTA to disrupt DC-T
cell complexes according to a previous described procedure
(30), and EDTA was also present in subsequent steps
involving the use of positive selection columns. Cells were resuspended
in a 1.080 g/cm3 isoosmotic Nycodenz
medium (Sigma, St. Louis, MO), and centrifuged at 3000 rpm for 15 min
at 4°C. The low density fraction at the interface was collected and
washed several times. The recovered cells were incubated with CD11c
microbeads and separated using a positive selection column. Cells were
then resuspended in RPMI medium supplemented with 10% FCS and allowed
to adhere for 2 h; this was followed by an additional 18-h
incubation to allow DC to detach. The recovered cells were routinely
96–98% CD11c+, >99%
I-A+, >98% B7-2+, and
<0.1% CD3+ and appeared to consist of 90–95%
CD8- and 5–10% CD8+
cells. For preparation of CD8+ and
CD8- fractions, the purified DC were
separated using a positive selection column and CD8 microbeads.
After cell fractionation the recovered CD8-
cells typically contained <0.5% contaminating
CD8+ DC, whereas the CD8+
fraction was made up of >90% CD8+ DC.
DC treatments and immunization
Cytokine treatments of DC were performed at 37°C by incubation
with 100 ng/ml rIL-12, 200 U/ml rIFN-, or 10 ng/ml rIL-6 for 18
h before peptide pulsing, unless otherwise stated. Control cultures
were incubated with medium alone. In all CD40 stimulations (9, 10), DC were incubated on ice for 10 min in PBS plus 10% mouse
serum, for 20 min with hamster anti-mouse CD40 mAb (5 µg/ml), and
then overnight at 37°C with goat anti-hamster Ab (5 µg/ml) in
Iscove’s medium plus 10% FCS. CD40 ligation on DC routinely involved
the use of the second cross-linking Ab, as the latter appears to be
necessary for effective DC activation. To check for possible
nonspecific effects of anti-CD40 ligation, appropriate controls
included incubation of the CD8+ DC in the
presence of the second Ab alone, which treatment appeared to be devoid
of any functional effect. As an additional control, isotype-matched
anti-mouse H-2Kd mAb 31-3-4S, capable of
binding to DC, was also used in place of the primary anti-CD40
reagent (9). For IL-6 neutralization, DC were subjected to
CD40 activation in vitro in the presence of 6B4 (anti-IL-6) and
15A7 (anti-IL-6R) mAbs, each at 10 µg/ml. In all experiments,
appropriate controls for anti-IL-6 treatments included the use of
isotype-matched mAbs in the place of 6B4 and 15A7 mAbs. In general,
preincubation of the DC with cytokines or anti-CD40 Abs
significantly reduced their viability, with the
CD8+ subset being apparently more affected than
the CD8- subset. For most experiments, the
viability of the CD8- DC that were used for in
vivo immunization would exceed 90% and that of
CD8+ DC would exceed 50%. For some experiments,
nonviable cells in the CD8+ DC fraction were
removed by selecting low density viable cells using a Ficoll
centrifugation procedure. Cells were washed between and after the
incubations before peptide loading (5 µM, 2 h at 37°C),
irradiation, and i.v. injection into recipient hosts of 3 x
105 CD8- DC, either alone
or in combination with 3% CD8+ DC. Appropriate
controls included the use of rTNF- (8) and
baculovirus-derived IL-9 (31) to check for possible
nonspecific effects of cytokine treatment, and none was found.
Skin test assay
A skin test assay was employed for measuring class I-restricted delayed-type hypersensitivity responses to P815AB and NRP-A7, as previously described (1, 2, 3). Measurements were made in a blind fashion, and results were expressed as the increase in footpad weight of peptide-injected footpads over that of vehicle-injected counterparts. Data are the mean ± SD for at least six mice per group. The statistical analysis was performed using Student’s paired t test by comparing the mean weight of experimental footpads with that of control counterparts (1, 2, 3). The data reported are from representative experiments, and experiments with similar results were performed three to six times.
Cytofluorometric analysis
Surface expression of the IFN-R -chain was performed using
biotinylated rat IgG to murine CD119 (clone GR20; PharMingen, San
Diego, CA) in conjunction with avidin-FITC. Biotinylated rat IgG2a
(PharMingen) was used as an isotype-matched control mAb.
Kynurenine assay
The kynurenine assay was performed as previously described (10). Briefly, CD8+ DC, either untreated or treated with different agents, were washed and resuspended in HBSS containing 100 µM tryptophan (Sigma, St. Louis, MO). Cells were incubated for an additional 4 h at 37°C, followed by harvest of supernatant that was stored at -80°C before quantitation of kynurenine by HPLC. IDO activity was expressed as the concentration (micromoles per liter kynurenine) in the sample.
Induction and assay of apoptosis
DC (2.5 x 105) treated overnight
with IL-6 and/or IFN- were washed and cultured for 3 days with
5 x 105 F76 cells, a Th1-type
P815AB-specific CD4+ T cell clone
(10), in the presence of 5 µM P815AB peptide. At the end
of the coculture the CD11c+ cells were removed by
the use of CD11c microbeads (Miltenyi Biotec), and the remainder of the
population was surface stained with anti-CD4-PE and FITC-labeled
annexin V and propidium iodide (PI; PharMingen). For measurement of
apoptosis, a gate was set on CD4+ T cells, and
the percentage of cells in the very early stages of apoptosis was
determined by annexin V staining excluding PI+
cells.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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effects on CD8+ DC
expressing IDO activity
To confirm and expand our previous observations with P815AB and
NRP-A7 (10), we conducted parallel experiments aimed at
ascertaining any regulatory function of CD40 activation on
CD8+ DC treated with IFN- and assayed for
suppressive activity. The effect of CD40 activation on IDO induction by
IFN- was also investigated. Recipient mice were injected with
CD8- DC or a combination of
CD8- and 3% CD8+ DC
pulsed with P815AB or NRP-A7. Each DC fraction was used either as such
or after cytokine (IL-12 or IFN-) treatment in vitro. Groups of
CD8+ DC were exposed to CD40 activation as
described in Materials and Methods before IFN- treatment
(200 U/ml for 6 h). After 2 wk recipients of DC transfer were
challenged intrafootpad with the appropriate peptide. Fig. 1
A shows that CD40 activation of CD8+ DC
blocked the IFN--induced inhibition of CD8-
DC presentation of P815AB or NRP-A7. Thus, similar to the pattern of
P815AB, negative modulation of CD8- DC
presentation of NRP-A7 is induced by IFN- acting on
CD8+ DC, an effect that can be prevented by CD40
activation in the latter cells. In addition, Fig. 1B shows
that CD40 activation blocked IDO induction by IFN- in
CD8+ DC. The effect was quantitatively similar to
that observed with the use of the enzyme inhibitor 1-MT.
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on CD8+ DC
In several experimental models it has been shown that IL-6 acts to
induce autoimmune disease and that the cytokine may exert effects on DC
that could contribute to the priming of autoimmunity (14).
Therefore, we wanted to investigate whether exposure of
CD8+ DC to IL-6 might affect their negative
regulatory function in the priming to P815AB or NRP-A7. Recipient mice
were injected with a combination of peptide-pulsed
CD8- and 3% CD8+ DC. Each
DC fraction was used either as such or after cytokine treatment in
vitro. Fig. 2
shows that IL-6 treatment of CD8+ DC blocked
their inhibitory function on peptide presentation by the
CD8- subset. In addition, this maneuver
abolished the effect of IFN- on CD8+ DC when
these cells were exposed sequentially to IL-6 (18 h) and IFN- (6 h)
before peptide pulsing. Fig. 2 also shows that combined treatment of
mice with IL-12-treated CD8- DC and IL-6-treated
CD8+ DC would not result in increased reactivity
in vivo to P815AB or NRP-A7, according to a pattern previously observed
on testing the effect of combined treatment of DC with IL-12 and
anti-CD40 agonistic mAb (9). In experiments not
reported here we also found that the modulatory effect of IL-6 was lost
when the CD8+ DC were exposed concurrently,
rather than sequentially, to IL-6 and IFN- for 18 h.
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Because the effect of rIL-6 on the basal or IFN--induced
suppressive activity of CD8+ DC appeared to be
similar to that of CD40 activation, we examined whether induction of
IL-6 might be involved in the modulatory properties of CD40 activation.
In preliminary experiments we found that triggering of CD40 would
result in the production of high levels of IL-6 (up to 5 ng/ml) by both
CD8+ and CD8- DC. We thus
examined the effect of IL-6-neutralizing Abs added to cultures of
CD8+ DC treated with CD40-stimulating Abs.
Mixtures of CD8- and CD8+
DC pulsed with P815AB or NRP-A7, as illustrated above, were injected
into recipient mice. Each DC fraction was used either as such or after
cytokine (IL-12 or IFN-) treatment. Groups of
CD8+ DC were also exposed to CD40 agonistic mAb
either alone or in combination with anti-IL-6 mAbs. Fig. 3
shows that the presence of IL-6-neutralizing Abs would completely block
the effect of CD40 activation in triggering reactivity to P815AB or
NRP-A7. An absolute requirement for IL-6 induced by CD40 activation
appeared to be evident both in the modulation of the basal inhibitory
activity of CD8+ DC and in the reversal of the
effect mediated by IFN-. It is interesting that the ability of IL-6
neutralization to ablate the effect of CD40 activation argues against
the possibility that selective death or paralysis of
CD8+ DC in culture is a major mechanism through
which CD40 ligation exerts its effect. In addition, we have previously
demonstrated that CD40 ligation renders CD8+ DC
capable of presenting P815AB in an immunogenic fashion
(10). Furthermore, in experiments not reported here we
have obtained evidence that both CD40 ligation and IL-6 treatment
up-regulate the expression of B7-1 in CD8+ DC.
Therefore, differential cell death in culture and the mere selection of
nonsuppressive cells are unlikely to underlie the apparent loss of
suppressive function resulting from CD40 ligation or treatment with
IL-6 in vitro.
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We have previously shown that the effect triggered by IFN- in
CD8+ DC involves interference with tryptophan
metabolism in vivo upon transfer of P815AB-pulsed DC (8).
Furthermore, CD40 activation prevents the effect of IFN- on IDO
expression by CD8+ DC (10).
Therefore, we sought to determine whether IL-6 might interfere with IDO
activity as induced by IFN- and whether the former cytokine might be
involved in the effect of CD40 activation. The functional activity of
IDO produced by activated CD8+ DC was measured in
terms of its ability to metabolize tryptophan to kynurenine.
CD8+ DC were treated with IFN- or exposed
sequentially to IL-6 (18 h) and IFN- (6 h). Alternatively, the
CD8+ DC were exposed sequentially to a
combination of anti-CD40 mAb plus anti-IL-6 for 18 h,
followed by IFN- treatment. Fig. 4
shows that IL-6 would greatly impair the effect of IFN- on IDO
production. Furthermore, IL-6 neutralization ablated the effect of CD40
modulation on IDO induction by IFN-.
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R expression by IL-6
We have previously shown that CD40 activation on
CD8+ DC negatively regulates the expression of
the IFN-R. We therefore examined any possible effect of IL-6 on this
expression. We assayed the surface expression of the IFN-R -chain
by flow cytometry using biotinylated rat IgG to murine CD119.
CD8- DC were included as a control because CD40
activation on these cells has previously been shown to lack any effect
on IFN-R expression. Fig. 5
shows that IL-6 resulted in a marked decrease in the -chain of the
IFN-R. This effect was in contrast with that on
CD8- DC, where no significant changes were
observed.
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on induction of apoptosis
by CD8+ DC
We have previously suggested that IDO might contribute to the
negative regulatory and tolerogenic properties of IFN- via induction
of T cell apoptosis in our model system with P815AB. Using a
P815AB-specific CD4+ T cell clone cultured with
DC treated with IFN-, we have found that CD40 activation may block
the induction of T cell apoptosis by CD8+ DC
(10). We therefore became interested in ascertaining any
possible regulatory activity of IL-6 on T cell apoptosis mediated by
IFN--activated CD8+ DC. The P815AB-specific
CD4+ T cell clone F76 was used for measurement of
apoptosis upon 24- or 72-h coculture of the latter cells with
CD8+ DC exposed to IFN- either alone or in
combination with IL-6. No significant apoptosis was observed in the
24-h cocultures (data not shown). In contrast, Fig. 6
shows that the coculture of clone F76 cells and
CD8+ DC for 72 h in the presence of P815AB
resulted in apoptosis of approximately 10% CD4+
cells. This effect could be explained at least in part by the
production of IFN- by the Th1 clone cells, and in fact, the
proportion of apoptotic T cells in the absence of DC would not exceed
5% (data not shown). Upon exposure of the CD8+
DC to rIFN- (6 h), the proportion of apoptotic cells rose to >30%.
However, sequential exposure of the CD8+ DC to
IL-6 (18 h) and IFN- (6 h) ablated the modulatory role of externally
added IFN-. The effect of IL-6 was quantitatively similar to that
observed upon addition of the competitive inhibitor of IDO, 1-MT, to
the cocultures of DC and CD4+ T cells. These data
suggest that IL-6 may act through modulation of IDO induction by
IFN- in CD8+ DC to regulate T cell apoptosis.
It is interesting to note that although annexin V staining measures the
very early stages of apoptosis, the generation of effective
proapoptotic signals in the cocultures required prolonged exposure of
the CD4+ T cells to CD8+ DC
in the presence of cognate Ag, as no apoptosis was observed at 24
h. This suggests that temporally distinct events contribute to
activation of the apoptotic response in T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC could be regarded as a multilineage system of leukocytes with
variable function rather than as a homogenous cell type with
predetermined functional properties (35, 36). Because the
DC system as a whole can present Ags in an immunogenic or tolerogenic
fashion, it is possible that the outcome of an immune response
initiated by DC depends on a combination of several factors, including
Ag presentation by a specific type of DC, the nature of the
DC-activating signal, and the stage of DC maturation (37).
IL-6 could be acting at several levels to regulate DC maturation
(38) and function (39). Although
CD8+ DC function has been associated with the
development of Th1 responses to foreign Ags (40, 41), the
protection against potential autoreactivity within inflammatory
contexts dominated by IFN- is also believed to involve specific
actions of the cytokine on DC (14, 42), which might be
able to discriminate self from nonself (8).
While studying the induction of immunity vs tolerance to tumor and/or
self peptides, we have previously found that the outcome of DC
presentation of P815AB (a tumor/self peptide) and NRP-A7 (a peptide
mimotope for autoimmune diabetes) depends on a balance between the
respective immunogenic and tolerogenic properties of
CD8- (myeloid) and
CD8+ (lymphoid) DC (7, 8). IL-12
(6, 7) and IFN- (8) have functionally
opposing effects on peptide presentation by CD8-
DC, and IFN--activated CD8+ DC mediate
tolerogenic activity that can be prevented by CD40 activation on these
cells (10). The effect of CD40 activation is accompanied
by an impaired capacity of the CD8+ DC to
initiate T cell apoptosis in vitro, presumably through modulation of
tryptophan degradation affecting lymphocyte function (10, 43). We demonstrate here that IL-6 has profound effects on the
presentation of tumor or self peptides by CD8-
DC via modulation of the regulatory activity of
CD8+ DC. In addition, we provide evidence that
autocrine IL-6 is involved in the regulatory effect of CD40 activation
in CD8+ DC. Blockade of the tolerogenic activity
of CD8+ DC is observed both under basal
conditions and when the activity of the latter cells is potentiated by
IFN- treatment.
Nonameric P815AB is a synthetic peptide that is related to a major
tumor rejection Ag encoded by the P1A gene in murine
mastocytoma cells (44). Although an efficient target for
rejection responses in immunized mice, this Ag is not sufficiently
immunogenic per se, and induction of protective immunity by
P815AB-pulsed DC requires the use of rIL-12 (3). Effective
priming to P815AB and the detection of class I-restricted responses in
vivo require the presence of an intact CD4+ T
cell compartment in the host (3). Of interest, adoptive
transfer of class II-restricted CD4+ T cell
clones with specificity for P815AB also results in the induction of
protective antitumor immunity (45). Spontaneous autoimmune
diabetes in NOD mice is the result of a CD4+ and
CD8+ T cell-dependent autoimmune process directed
against the pancreatic -cells. NRP-A7 represents a peptide ligand
with strong agonistic activity for CD8+ T cells
in autoimmune diabetes and also induces the deletion of specific
CD8+ T cells under selected conditions of
immunization (4, 5). In both model systems with synthetic
peptides, we found that the pattern of immune response induced by
transfer of peptide-pulsed DC was strongly influenced by IL-6 acting on
CD8+ DC. These findings may be relevant to an
improved understanding of the possible role of IL-6 in avoiding
tolerance induction in cancer immunotherapy, particularly with regard
to the idea that Ag-specific T cell tolerance is known to limit the
efficacy of therapeutic cancer vaccines. Given the exceptional capacity
of DC to induce immunity in vivo, recent reports of the first
successful clinical trials based on vaccination of tumor patients with
autologous blood DC pulsed in vitro with tumor Ags come as no surprise.
However, to maximize antitumor immunity and avoid tolerance induction,
a number of technical questions still need to be addressed, including
the frequency and route of administration, the subset and number of DC
to be used, and the concentration and duration of cytokine treatment.
On the other hand, our findings with the NRP-A7 peptide may help to
explain how DC can cause autoimmune disease and the possible role of
IL-6 in this process.
Cytokines are known to regulate the progression and maintenance of
autoimmunity. Although the roles of most cytokines are generally
controversial (14), the role of IL-6, on the one hand, and
that of TNF-/IFN-, on the other, appear to be more clearly
defined. For example, experiments using knockout mice have demonstrated
an absolute requirement for IL-6 in several models of autoimmunity
(15, 16, 17, 18, 19, 20, 21). The ability of IL-6 to down-modulate the
function of tolerogenic CD8+ DC could provide a
model for explaining at least a portion of the disease-promoting
effects of IL-6 in autoimmunity. In contrast, in systems of
experimental autoimmune encephalomyelitis (23, 24) and
diabetes (25), IFN- exerts a protective role. Although
IFN- may have an essential function in stimulating APC to produce
nitric oxide (an inducer of T cell apoptosis) (46), we
have suggested that IFN--dependent production of IDO by DC may also
result in apoptosis of T lymphocytes (10). We demonstrate
here that IL-6 blocks the stimulatory activity of IFN- on IDO
production by CD8+ DC, an effect that can at
least in part be explained by down-modulation of IFN-R expression.
Interestingly, we have previously observed that CD40 activation also
results in reduced IFN-R expression by CD8+ DC
(10), thus supporting the hypothesis that autocrine IL-6
mediates most of the effects of CD40 activation in
CD8+ DC.
It has been shown that human monocyte-derived macrophages suppress T
cell proliferation in vitro via IFN--mediated induction of IDO
(47). Significant IDO production by human DC has also been
shown to occur (43). Because cells synthesizing IDO
modulate T cell proliferation by reducing the tryptophan concentration
in local tissue microenvironments, tryptophan catabolism may represent
a general mechanism in T cell suppression (48). In our
experimental model with P815AB-specific CD4+ T
cells, we have previously found that the blockade of IDO activity by a
competitive inhibitor would negate the induction of apoptosis in vitro
by IFN--treated DC (8). This suggested that IDO
induction is a major mechanism by which IFN- acts on DC to mediate
apoptosis of T cells and supported the idea that IDO-dependent
modulation of T cell function may involve selective cell death in
addition to inhibition of proliferation. In the present experiments we
found that IL-6 would ablate most of the proapoptotic effect induced by
IFN-, either externally added or putatively produced by the Th1
cells. Although under the adopted experimental conditions (i.e., 18-h
incubation) IL-6 was unable to completely block apoptosis, our current
finding that IL-6 and IFN- exert opposing effects on IDO induction
and T cell apoptosis may be relevant to a better understanding of the
immunological mechanisms governed by IDO-dependent tryptophan
catabolism. On the other hand, the failure of 1-MT to completely negate
the induction of apoptosis may indicate that additional mechanisms,
such as production of NO, may contribute to the proapoptotic effect of
IFN- in our model system. Finally, we are currently evaluating any
possible direct effect of IL-6 on the regulation of IDO expression.
The term lymphoid DC was introduced to describe a mouse DC subset in
the thymus that develops from a population of thymic lymphoid
progenitor cells, and CD8 was considered to be a characteristic
marker for lymphoid DC in mice (49, 50).
CD8+ DC are also found in peripheral lymphoid
organs, and the two types of CD8+ DC are thought
to play important roles in the establishment and maintenance of central
and peripheral tolerance, respectively (11, 51). It is
possible that the murine lymphoid CD8+ DC lineage
corresponds in humans to the progeny of DC precursors with a
characteristic surface phenotype and a plasmacytoid appearance
(52). It has been shown that human DC expressing
significant IDO activity can mediate inhibition of T cell proliferation
(43). Our present and previous observations
(10) provide the first experimental evidence for the
involvement of tryptophan degradation in T cell apoptosis and
regulation of anergy of mature T lymphocytes by
CD8+ DC in the mouse. This may add to our
understanding of the complex role of DC in the control of immunity and
may provide novel mechanistic insights into how DC tolerize T cells to
self Ags and minimize autoimmune reactions (11, 53, 54).
In conclusion, the data reported here reinforce our previous findings
that mature, immunologically competent DC may either immunize or
tolerize T cells to tumor or self Ags depending on a combination of
several factors, including Ag presentation by specific types of DC, the
nature of the DC-activating signal, and the balance of cytokine signals
(7, 8, 9, 10). In addition, our current data prospect a critical
role for IL-6 in mediating the effect of CD40 activation on
CD8+ DC, in regulating the susceptibility of
these cells to the effects of IFN-, and, indirectly, in regulating
the tryptophan catabolism pathway that affects T lymphocyte function.
These properties of IL-6 may all influence the ability of DC to
activate or tolerize autoreactive T cells, prime regulatory T cell
subsets, or shift a Th1/Th2 balance, thus confirming the critical role
that IL-6 plays in immune reactions to self Ags.
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Acknowledgments |
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2 Address correspondence and reprint requests to Prof. Paolo Puccetti, Department of Experimental Medicine, Pharmacology Section, University of Perugia, Via del Giochetto, I-06122 Perugia, Italy. E-mail address: plopcc{at}tin.it
3 Abbreviations used in this paper: DC, dendritic cells; IDO, indoleamine 2,3-dioxygenase; 1-MT, 1-methyl-DL-tryptophan; NOD, nonobese diabetic; PI, propidium iodide.
Received for publication February 2, 2001. Accepted for publication May 7, 2001.
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B and primes DC for IL-12 production. Immunity 9:315.[Medline]
- dendritic cells to enhance presentation of a tumor peptide in vivo. J. Immunol. 163:3100. inhibits presentation of a tumor/self peptide by CD8- dendritic cells via potentiation of the CD8+ subset. J. Immunol. 165:1357. gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE). J. Immunol. 156:5.[Abstract]
confers resistance to experimental allergic encephalomyelitis. Eur. J. Immunol. 26:1641.[Medline]
(IFN-) down-modulates autoimmune diabetes in non-obese diabetic (NOD) mice. Clin. Exp. Immunol. 117:38.[Medline]
, IL-12, and IFN-, and induction of IL-10. J. Immunol. 164:4197.-positive dendritic cells in vivo. Blood 96:1865.-positive dendritic cells from a common myeloid progenitor. Science 290:2152.+ and CD8- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J. Exp. Med. 189:587. eliminates responding CD4 T cells during mycobacterial infection by inducing apoptosis of activated CD4 T cells. J. Exp. Med. 192:117.This article has been cited by other articles:
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  F. Qian, J. Villella, P. K. Wallace, P. Mhawech-Fauceglia, J. D. Tario Jr., C. Andrews, J. Matsuzaki, D. Valmori, M. Ayyoub, P. J. Frederick, et al. Efficacy of Levo-1-Methyl Tryptophan and Dextro-1-Methyl Tryptophan in Reversing Indoleamine-2,3-Dioxygenase-Mediated Arrest of T-Cell Proliferation in Human Epithelial Ovarian Cancer Cancer Res., July 1, 2009; 69(13): 5498 - 5504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Curti, S. Trabanelli, V. Salvestrini, M. Baccarani, and R. M. Lemoli The role of indoleamine 2,3-dioxygenase in the induction of immune tolerance: focus on hematology Blood, March 12, 2009; 113(11): 2394 - 2401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. B. Ganesh, D. M. Cheatem, J. R. Sheng, C. Vasu, and B. S. Prabhakar GM-CSF-induced CD11c+CD8a--dendritic cells facilitate Foxp3+ and IL-10+ regulatory T cell expansion resulting in suppression of autoimmune thyroiditis Int. Immunol., March 1, 2009; 21(3): 269 - 282. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-I. Jeong, S. W. Kim, I. D. Jung, J. S. Lee, J. H. Chang, C.-M. Lee, S. H. Chun, M.-S. Yoon, G. T. Kim, S. W. Ryu, et al. Curcumin Suppresses the Induction of Indoleamine 2,3-Dioxygenase by Blocking the Janus-activated Kinase-Protein Kinase C{delta}-STAT1 Signaling Pathway in Interferon-{gamma}-stimulated Murine Dendritic Cells J. Biol. Chem., February 6, 2009; 284(6): 3700 - 3708. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
A. J. Muller, M. D. Sharma, P. R. Chandler, J. B. DuHadaway, M. E. Everhart, B. A. Johnson III, D. J. Kahler, J. Pihkala, A. P. Soler, D. H. Munn, et al. Chronic inflammation that facilitates tumor progression creates local immune suppression by inducing indoleamine 2,3 dioxygenase PNAS, November 4, 2008; 105(44): 17073 - 17078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. L. Belladonna, C. Volpi, R. Bianchi, C. Vacca, C. Orabona, M. T. Pallotta, L. Boon, S. Gizzi, M. C. Fioretti, U. Grohmann, et al. Cutting Edge: Autocrine TGF-{beta} Sustains Default Tolerogenesis by IDO-Competent Dendritic Cells J. Immunol., October 15, 2008; 181(8): 5194 - 5198. [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]
|
|
|
|
|
|
L. Romani, T. Zelante, A. De Luca, F. Fallarino, and P. Puccetti IL-17 and Therapeutic Kynurenines in Pathogenic Inflammation to Fungi J. Immunol., April 15, 2008; 180(8): 5157 - 5162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Giroux, E. Yurchenko, J. St.-Pierre, C. A. Piccirillo, and C. Perreault T Regulatory Cells Control Numbers of NK Cells and CD8{alpha}+ Immature Dendritic Cells in the Lymph Node Paracortex J. Immunol., October 1, 2007; 179(7): 4492 - 4502. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M Blois, U. Kammerer, C. A. Soto, M. C Tometten, V. Shaikly, G. Barrientos, R. Jurd, D. Rukavina, A. W Thomson, B. F Klapp, et al. Dendritic Cells: Key to Fetal Tolerance? Biol Reprod, October 1, 2007; 77(4): 590 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Feunou, S. Vanwetswinkel, F. Gaudray, M. Goldman, P. Matthys, and M. Y. Braun Foxp3+CD25+ T Regulatory Cells Stimulate IFN-{gamma}-Independent CD152-Mediated Activation of Tryptophan Catabolism That Provides Dendritic Cells with Immune Regulatory Activity in Mice Unresponsive to Staphylococcal Enterotoxin B J. Immunol., July 15, 2007; 179(2): 910 - 917. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Fallas, W. Yi, N. A. Draghi, H. M. O'Rourke, and L. K. Denzin Expression Patterns of H2-O in Mouse B Cells and Dendritic Cells Correlate with Cell Function J. Immunol., February 1, 2007; 178(3): 1488 - 1497. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D.-Y. Hou, A. J. Muller, M. D. Sharma, J. DuHadaway, T. Banerjee, M. Johnson, A. L. Mellor, G. C. Prendergast, and D. H. Munn Inhibition of Indoleamine 2,3-Dioxygenase in Dendritic Cells by Stereoisomers of 1-Methyl-Tryptophan Correlates with Antitumor Responses Cancer Res., January 15, 2007; 67(2): 792 - 801. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Agaugue, L. Perrin-Cocon, F. Coutant, P. Andre, and V. Lotteau 1-Methyl-Tryptophan Can Interfere with TLR Signaling in Dendritic Cells Independently of IDO Activity J. Immunol., August 15, 2006; 177(4): 2061 - 2071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
J. I. Bleier, S. C. Katz, U. I. Chaudhry, V. G. Pillarisetty, T. P. Kingham III, A. B. Shah, J. R. Raab, and R. P. DeMatteo Biliary obstruction selectively expands and activates liver myeloid dendritic cells. J. Immunol., June 15, 2006; 176(12): 7189 - 7195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Orabona, P. Puccetti, C. Vacca, S. Bicciato, A. Luchini, F. Fallarino, R. Bianchi, E. Velardi, K. Perruccio, A. Velardi, et al. Toward the identification of a tolerogenic signature in IDO-competent dendritic cells Blood, April 1, 2006; 107(7): 2846 - 2854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A Wallet, P. Sen, and R. Tisch Immunoregulation of Dendritic Cells Clin. Med. Res., August 1, 2005; 3(3): 166 - 175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Gordon, F. Li, A. Nayyar, J. Xiang, and X. Zhang CD8{alpha}+, but Not CD8{alpha}-, Dendritic Cells Tolerize Th2 Responses via Contact-Dependent and -Independent Mechanisms, and Reverse Airway Hyperresponsiveness, Th2, and Eosinophil Responses in a Mouse Model of Asthma J. Immunol., August 1, 2005; 175(3): 1516 - 1522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Orabona, M. L. Belladonna, C. Vacca, R. Bianchi, F. Fallarino, C. Volpi, S. Gizzi, M. C. Fioretti, U. Grohmann, and P. Puccetti Cutting Edge: Silencing Suppressor of Cytokine Signaling 3 Expression in Dendritic Cells Turns CD28-Ig from Immune Adjuvant to Suppressant J. Immunol., June 1, 2005; 174(11): 6582 - 6586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. H. Tan, S. C. Beutelspacher, S.-A. Xue, Y.-H. Wang, P. Mitchell, J. C. McAlister, D. F. P. Larkin, M. O. McClure, H. J. Stauss, M. A. Ritter, et al. Modulation of human dendritic-cell function following transduction with viral vectors: implications for gene therapy Blood, May 15, 2005; 105(10): 3824 - 3832. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Terness, J.-J. Chuang, T. Bauer, L. Jiga, and G. Opelz Regulation of human auto- and alloreactive T cells by indoleamine 2,3-dioxygenase (IDO)-producing dendritic cells: too much ado about IDO? Blood, March 15, 2005; 105(6): 2480 - 2486. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. O'Keeffe, T. C. Brodnicki, B. Fancke, D. Vremec, G. Morahan, E. Maraskovsky, R. Steptoe, L. C. Harrison, and K. Shortman Fms-like tyrosine kinase 3 ligand administration overcomes a genetically determined dendritic cell deficiency in NOD mice and protects against diabetes development Int. Immunol., March 1, 2005; 17(3): 307 - 314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
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|
|
|
|
|
|
D. Winsor-Hines, C. Merrill, M. O'Mahony, P. E. Rao, S. P. Cobbold, H. Waldmann, D. J. Ringler, and P. D. Ponath Induction of Immunological Tolerance/Hyporesponsiveness in Baboons with a Nondepleting CD4 Antibody J. Immunol., October 1, 2004; 173(7): 4715 - 4723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
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|
|
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|
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|
|
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A. L. Mellor and D. H. Munn Tryptophan Catabolism and Regulation of Adaptive Immunity J. Immunol., June 15, 2003; 170(12): 5809 - 5813. [Full Text] [PDF]
|
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|
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|
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|
 D. H. Munn, M. D. Sharma, J. R. Lee, K. G. Jhaver, T. S. Johnson, D. B. Keskin, B. Marshall, P. Chandler, S. J. Antonia, R. Burgess, et al. Potential Regulatory Function of Human Dendritic Cells Expressing Indoleamine 2,3-Dioxygenase Science, September 13, 2002; 297(5588): 1867 - 1870. [Abstract] [Full Text] [PDF]
|
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J. Mann, F. Oakley, P. W. M. Johnson, and D. A. Mann CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells. REGULATION BY TRAF2, AP-1, NF-kappa B, AND CBF1 J. Biol. Chem., May 3, 2002; 277(19): 17125 - 17138. [Abstract] [Full Text] [PDF]
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A. L. Mellor, D. B. Keskin, T. Johnson, P. Chandler, and D. H. Munn Cells Expressing Indoleamine 2,3-Dioxygenase Inhibit T Cell Responses J. Immunol., April 15, 2002; 168(8): 3771 - 3776. [Abstract] [Full Text] [PDF]
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F. Fallarino, C. Vacca, C. Orabona, M. L. Belladonna, R. Bianchi, B. Marshall, D. B. Keskin, A. L. Mellor, M. C. Fioretti, U. Grohmann, et al. Functional expression of indoleamine 2,3-dioxygenase by murine CD8{alpha}+ dendritic cells Int. Immunol., January 1, 2002; 14(1): 65 - 68. [Abstract] [Full Text] [PDF]
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