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Department of Experimental Medicine, University of Perugia, Perugia, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-) and lymphoid (CD8+) DC. We have
previously shown that CD8- DC can be primed by IL-12 to
overcome inhibition by the CD8+ subset and initiate
immunogenic presentation in vivo when the two types of peptide-pulsed
DC are cotransferred into recipient hosts. IFN-
enhances the
inhibitory activity of CD8+ DC on Ag presentation by the
other subset, blocking the ability of IL-12-treated CD8-
DC to overcome suppression. We report here that CD40 ligation on
lymphoid DC ablated their inhibitory function on Ag presentation as
well as IFN- potentiation of the effect. CD40 modulation of IFN-
action on lymphoid DC involved a reduction in IFN-R expression and
tryptophan-degrading ability. This effect was accompanied in vitro by
an impaired capacity of the CD40-modulated and IFN--treated DC to
initiate T cell apoptosis. In vivo, not only did CD40 triggering on
lymphoid DC abrogate their tolerogenic activity, but it also induced
the potential for immunogenic presentation of P815AB. Importantly, a
pattern similar to P815AB as well as CD40 modulation of lymphoid DC
function were observed on testing reactivity to NRP, a synthetic
peptide mimotope recognized by diabetogenic CD8+ T cells in
nonobese diabetic mice. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and DEC-205 and is considered to
be of lymphoid origin (1, 2). Lymphoid DC are thought to
be important in deletional tolerance, as they appear to be resident,
sedentary cells present in secondary lymphoid organs (3, 4). These cells have the ability to phagocytose other cells,
including myeloid DC, and cross-present Ag derived from phagocytosis,
inducing deletional tolerance (5).
CD8+ DC also produce high amounts of IL-12 when
pulsed with Ag in vitro. When injected into recipient hosts, they
initiate T cell priming, resulting in a Th1-type response
(6). These two apparently conflicting properties of
lymphoid DC, namely their ability to mediate tolerogenic effects and
their Th1-promoting activity, could be reconciled by the idea that the
induction of deletional tolerance is accompanied by very early
production of Th1 (IL-2, IL-3, and IFN-), but not Th2 (IL-4 or
IL-10), cytokines (7), which is consistent with the high
level of IL-12 production by stimulated lymphoid DC (6, 8). Interesting in this regard is the finding that lymphoid DC
may themselves produce significant amounts of IFN- in response to
IL-12 (9).
Using an in vivo model of tumor/self peptide (P815AB) presentation for
induction of class I-restricted skin test reactivity (10, 11), we have previously shown that CD8+ DC
negatively regulate the induction of T cell reactivity by
peptide-loaded myeloid DC. By virtue of their respective actions on
CD8- (12, 13) and
CD8+ (14) DC, IL-12 and IFN- have
functionally opposing effects on peptide presentation by the
CD8- DC subset, and IFN--activated lymphoid
DC mediate tolerogenic effects that prevail over the immunoadjuvant
activity of IL-12 on myeloid DC. Interestingly, the negative regulatory
effect triggered by IFN- in lymphoid DC appears to be restricted to
P815AB, as it is not observed with evolutionarily distant Ags and
involves interference with tryptophan metabolism in vivo. We have
suggested that 1) DC might discriminate self from nonself in an
inflammatory context dominated by IFN- and that 2) IFN- acting on
lymphoid DC might contribute to the maintenance of T cell tolerance to
self Ags via tryptophan degradation affecting T cell responses
(14).
As we expand on this hypothesis, we demonstrate here that triggering of
CD40 on CD8+ DC will abolish their susceptibility
to the tolerogenic activity of IFN- in the induction of T cell
reactivity to P815AB. The effect involves down-regulation of IFN-R
expression and leads to reduced ability of CD8+
DC to metabolize tryptophan and mediate T cell apoptosis in vitro. In
addition, CD40 activation on CD8+ DC makes these
cells capable of immunogenic presentation of P815AB. Finally, a pattern
similar to that of P815AB and a similar effect of CD40 ligation are
observed with a synthetic peptide that acts as a mimotope for
autoimmune diabetes in mice. Collectively, these findings may help to
explain recent evidence in different experimental systems that
CD40-CD40 ligand interactions are crucial in determining whether naive
peripheral T cells are primed or tolerized upon DC presentation of
tumor/self peptides (15).
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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
(12), hamster anti-murine CD40 (HM40-3) mAb used in
combination with goat anti-hamster IgG (16), and
biotinylated rat IgG to murine IFN-R -chain (14)
were previously described. The enzyme inhibitor
1-methyl-D,L-tryptophan (1-MT) was purchased
from Aldrich (Milan, Italy). IL-12 was 98.8% pure, as assessed by
SDS-PAGE, and endotoxin contamination was <0.9 EU/mg in a
Limulus amebocyte assay. The specific activity of the
purified rIL-12 preparation, measured as the ability to stimulate
proliferation in human PHA-activated blasts, was 3.1 x
106 U/mg. Endotoxin was removed from all
solutions containing rIL-12 or anti-CD40 Abs with Detoxi-gel
(Pierce, Rockford, IL), resulting in endotoxin contamination below the
detection limit (0.05 EU/ml) of the assay (Coatest Endotoxin;
Chromogenix, Molndal, Sweden) (16).
Peptides
P815AB (amino acid sequence LPYLGWLVF) and NRP (KYNKANWFL)
were synthesized on solid phase using F-moc for transient
NH2-terminal protection, purified by means of
reverse phase HPLC, and characterized by amino acid analysis. NRP is a
synthetic peptide mimotope recognized in the context of
H-2Kd class I molecules by a prevailing,
pancreatic 
-cell-specific T lymphocyte population in nonobese
diabetic (NOD) mice (17, 18).
DC preparation and treatments and immunization
DC were prepared and fractionated according to CD8 expression
using a positive selection column and CD8 microbeads (Miltenyi
Biotec, Bergish Gladbach, Germany) as previously described
(12, 13, 14). Briefly, DC were obtained from
collagenase-treated spleens (collagenase type IV; Sigma, St. Louis,
MO). The recovered cells were routinely >96%
CD11c+ and appeared to consist of 90–95%
CD8- and 5–10% CD8+
cells. After cell fractionation, the recovered
CD8- cells typically contained <0.8%
contaminating CD8+ DC, whereas the
CD8+ fraction was made up of 
80%
CD8+ DC. In all CD40 stimulations
(16), 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 use of the second cross-linking Ab, as the latter
appears to be necessary for effective DC activation (16).
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. In the experiment shown in Fig. 6
, CD8+ DC (3 x 105)
were instead injected alone, whereas an equal number of unfractionated
DC was included in the experiment in Fig. 7.
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A skin test assay was used for measuring class I-restricted delayed-type hypersensitivity responses to P815AB as previously described (10, 11, 12, 13, 14). 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 (10, 11, 12, 13, 14). 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.
RT-PCR and kynurenine assay
RNA preparation and PCR were previously described in detail
(12, 13). The sequences of 5' sense and 3' antisense
primers of IFN-R -chain were as follows: 5'-GGT TCC TGG ACT GAT
TCC TGC ACC; and 3'-AGT TCT TCC TGT TCT GCT GCT TCG G.
The kynurenine assay was essentially performed as previously described
(19). Briefly, unfractionated DC,
CD8- DC, or CD8+ DC,
either untreated or treated with anti-CD40 mAb and/or IFN-, were
washed and resuspended in HBSS containing 100 µM tryptophan (Sigm).
Cells were incubated for an additional 4 h, followed by harvest of
supernatant and quantitation of kynurenine by HPLC. Indoleamine
2,3-dioxygenase (IDO) activity was expressed as the micromolar
concentration of kynurenine in the sample.
Induction and assay of apoptosis
DC (2.5 x 105) treated overnight
with anti-CD40 mAb 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
(20), in the presence of 5 µM P815AB peptide. At the end
of the coculture, cells were 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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and CD8+ DC induce T lymphocyte tolerance
Our previous studies have shown that IL-12 in vitro confers
priming ability on splenic DC pulsed with an otherwise tolerogenic
tumor/self peptide (P815AB) (10, 12, 13). In fact, the
adjuvant activity of IL-12 on CD8- DC
counteracts a tolerogenic effect of CD8+ DC that
can be detected even when these cells represent as little as 3% of the
DC population (11). In contrast, IFN- acts on
CD8+ DC to abolish the adjuvant activity of IL-12
on myeloid DC (14). Using IL-12-activated
CD8- DC in a model system for assessment of T
cell tolerance (11), we investigated whether IFN-
acting on lymphoid DC would initiate long-lasting effects on host
responsiveness to P815AB. Peptide-loaded CD8-
DC, either alone or in combination with 3% CD8+
DC, were injected into recipient mice to be assayed for skin test
reactivity to the eliciting peptide. Each DC fraction was used either
as such or after treatment with IL-12 (for CD8-
DC) or IFN- (for CD8+ DC; Fig. 1A). As expected, IFN-
completely abolished the adjuvant activity of IL-12 on myeloid DC when
the animals were assayed for skin test reactivity to P815AB at 15 days
after cell transfer. To study the impact of a previous exposure to
IFN--treated CD8+ DC, groups of mice were
further immunized on day 15 with IL-12-activated and peptide-pulsed
CD8- DC. The animals were then assayed for skin
test reactivity to P815AB after 2 wk. Fig. 1B shows that
exposure of mice to IFN--treated CD8+ DC
resulted in a tolerant state that could not be reversed by the use of
IL-12-activated CD8- DC. As unresponsiveness
persisted when the second cell transfer was delayed up to 40, 60, or 90
days after the tolerogenic priming (data not shown), these findings
suggested the occurrence of deletional tolerance initiated by the
action of IFN- on lymphoid DC. This condition appeared to be
different from the anergic state induced by host transfer with
unfractionated DC pulsed with P815AB, as the latter represents a
reversible phenomenon that is no longer observable at 40–60 days after
tolerogenic priming (11).
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on CD8+ DC
In several experimental models it has been shown that the
induction of tolerance vs immunity is determined by resting vs
activated APC (15), which can be effectively modulated via
CD40-CD40 ligand interactions (21). Therefore, we became
interested in ascertaining whether CD40 ligation on
CD8+ DC might affect their negative regulatory
function in the priming to P815AB as well as their susceptibility to
the effect of IFN-. 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. Groups of
CD8+ DC were exposed to anti-CD40 mAb plus
goat anti-hamster Ab as described in Materials and
Methods before IFN- treatment (200 U/ml for 6 h). Fig. 2 shows that CD40 activation on
CD8+ DC blocked their inhibitory function on
peptide presentation by CD8- DC. In addition,
this maneuver reversed the effect of IFN- on
CD8+ DC. These data demonstrated that CD40
ligation on CD8+ DC could prevent T cell anergy
induced by a tolerogenic peptide alone or by a combination of the
peptide with IFN-.
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R expression by anti-CD40 mAb
Final maturation of DC is known to be associated with impaired
responsiveness to IFN- and to be correlated with reduced expression
of IFN-R (22). In addition, CD40 activation is one of
the critical signals that allow the full maturation of DC (21, 23). Therefore, we examined any possible regulation of IFN-R
expression by CD40 activation in lymphoid vs myeloid DC. We first
assayed the qualitative expression of mRNA specific for the -chain
of murine IFN-R. Using RT-PCR, we obtained evidence that CD40
ligation on CD8+ DC completely ablated the
expression of CD119-specific messages (Fig. 3). We next examined the surface
expression of the IFN-R -chain by flow cytometry using
biotinylated rat IgG to murine CD119 (14). The results
showed that triggering of CD40 on lymphoid DC resulted in a marked
decrease (at 24 h) or even disappearance (at 48 h) of the
-chain of the IFN-R. This effect was in contrast with that of
CD40 ligation on CD8- DC, where no significant
changes in CD119 expression were observed at 48 h (Fig. 3).
Although down-regulation of IFN-R -chain expression may be a
major mechanism by which CD40 ligation blocks the effect of IFN- on
CD8+ DC, these data did not exclude the
possibility of a differential effect of CD40 ligation on the survival
of lymphoid vs myeloid DC.
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There is now enough evidence to support a protective role of
IFN- in experimental models of T cell-mediated autoimmunity. In many
of these models, IFN- may have an essential role in stimulating APC
to produce NO (24, 25, 26). NO is an inducer of apoptosis in a
variety of cell types, including T cell clones and activated
CD4+ T cells (27). However, it is
also evident from these models that induction of NO does not account
for all the negative regulatory effect of IFN- (27).
IFN--dependent production of IDO by macrophages is known to result
in inhibition of T cell proliferation through tryptophan degradation
(28). Recent evidence indicates that IDO production by
human DC may suppress T cell proliferation (19). We have
already shown that the effect triggered by IFN- in
CD8+ DC involves interference with tryptophan
metabolism in vivo upon transfer of P815AB-pulsed DC (14).
Therefore, we sought to determine whether CD40 ligation on
CD8+ DC might interfere with IDO activity as
induced by IFN- treatment. The functional activity of IDO produced
by activated CD8+ DC was measured in terms of the
ability to metabolize tryptophan to kynurenine. Although not entirely
quantitative, this assay provides a reliable means of measuring
functional IDO activity, reflecting a multifactored combination of IDO
expression, tryptophan transport into the cells, and intracellular
conditions that post-translationally affect enzyme activity
(28). CD8+ DC were treated overnight
with IFN- or were exposed sequentially to anti-CD40 mAb and
IFN-. Cells were then washed and resuspended in HBSS containing 100
µM tryptophan and incubated at 37°C for 4 h. Supernatants were
harvested, and kynurenine was measured by HPLC. Fig. 4 shows that virtually no kynurenine was
detected in supernatants of DC unexposed to IFN-. However, DC
activation with the cytokine alone resulted in high levels of
tryptophan breakdown product. This effect of IFN- was completely
blocked by pre-exposure of CD8+ DC to
anti-CD40 mAb. These data demonstrated that at least one functional
property of IFN-, namely, the induction of IDO activity, is
abolished by CD40 activation that results in down-modulation of
IFN-R expression.
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We next examined whether expression of IDO might contribute to the
negative regulatory and tolerogenic properties of IFN- via induction
of T cell apoptosis in our model system with P815AB. We used a
P815AB-specific CD4+ T cell clone
(20) for measurement of apoptosis upon coculture of the
latter cells with unfractionated DC exposed to IFN-, either alone or
in combination with anti-CD40 mAb. Fig. 5 shows that the addition of
IFN--treated DC to clone F76 cells cultured in the presence of
P815AB caused a significant proportion (40%) of these cells to
undergo apoptosis. The effect was inhibited either by pre-exposure of
the DC to anti-CD40 mAb or by the addition of the competitive
inhibitor of IDO, 1-MT, to the coculture of DC and
CD4+ T cells. Fig. 5 also shows that no effect
was induced by IFN- when the purified CD8-
fraction was used in the place of unfractionated DC. These data suggest
that IFN- may act through expression of IDO in
CD8+ DC to regulate T cell apoptosis and could
explain our previous observation that inhibition of IDO will abrogate
the negative regulatory role of CD8+ DC in vivo
(14).
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We next examined whether triggering of CD40 on lymphoid DC might
not only abolish their tolerogenic activity but also render the cells
capable of effective presentation of P815AB in the absence of the
CD8- component. Recipient mice were transferred
with 3 x 105 CD8- or
CD8+ DC; the latter cells were used either as
such or after treatment with anti-CD40 mAb. Mice were assayed for
skin test reactivity to P815AB at 2 wk after DC transfer. Fig. 6
shows that in contrast to untreated
cells, the CD40-modulated lymphoid DC were fully capable of presenting
P815AB in an immunogenic fashion. Although the number of CD40-modulated
CD8+ DC injected per mouse was considerably
higher than that occurring in a DBA/2 spleen, these data indicated that
CD40 activation might induce the potential for immunogenic presentation
of P815AB in lymphoid DC. Furthermore, these data tend to rule out the
possibility that selective death of CD8+ DC is a
major mechanism through which CD40 ligation exerts its effect on
presentation of P815AB in vivo and breakdown of tryptophan into
kynurenine in vitro.
Inhibitory activity of CD8+ DC and opposing effect of CD40 ligation on presentation of a peptide mimotope for autoimmune diabetes
To investigate whether the distinct patterns of activity observed
with different DC subsets and the effect of CD40 ligation could be
observed with self peptides other than P815AB, we used a peptide
mimotope recognized by diabetogenic CD8+ T cells
in NOD mice. NRP is a synthetic nonapeptide that elicits the
proliferation, cytokine secretion, differentiation, and cytotoxicity of
a diabetogenic H-2Kd-restricted
CD8+ T cell specificity that uses a TCR
rearrangement frequently expressed by CD8+ T
cells propagated from the earliest insulitic lesions of NOD mice
(17, 18). In an experimental model analogous to that of
P815AB, NRP-pulsed DC were transferred into recipient hosts either as
such or after cell fractionation and treatment with anti-CD40 mAb.
After 2 wk the animals were assayed for skin test reactivity (Fig. 7). Analogous to the pattern of P815AB,
unfractionated DC pulsed with NRP failed to induce a delayed-type
hypersensitivity response. In contrast, a strong response was induced
by the CD8- cell fraction alone, an effect that
was negated by the addition of 3% CD8+ DC.
Remarkably, treatment of the CD8+ fraction with
anti-CD40 mAb restored the ability of CD8-
DC to present NRP in an immunogenic fashion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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may limit the T cell response to a tumor/self peptide by
inducing IDO expression in lymphoid DC (14). Furthermore,
we demonstrate that CD40 activation in these cells 1) ablates their
basal inhibitory activity on peptide presentation by
CD8- DC, 2) down-regulates IFN-R expression
and makes lymphoid DC unresponsive to IFN-, and 3) induces the
potential for immunogenic presentation of P815AB in this otherwise
tolerogenic DC subset.
Lymphoid DC are considered to be of primary importance in deletional
tolerance (2, 3, 4). These cells have the ability to
phagocytose other cells and may be able to cross-present Ag obtained
from phagocytosis, inducing deletional tolerance (5, 29, 30). The hypothesis that CD8- DC are
stimulatory whereas CD8+ DC may restrict the
immune response and play a role in tolerance induction is further
supported by the observation that DC within the T cell areas (the
majority of which expresses CD8) present high levels of self
peptides (31). However, two recent studies have revealed
that both CD8- and CD8+ DC
can sensitize naive T cells and direct the development of distinct Th
subpopulations (32), thus calling attention to a possible
major role of DC in regulating Th1 vs Th2 development. In a study by
Maldonado-López et al. (6), splenic
CD8+ DC transferred intrafootpad were found to
prime an immune response to keyhole limpet hemocyanin that was
dominated by Th1 cytokines. In contrast, administration of
CD8- DC induced a Th2 response. In another study
using DC subsets pulsed with an OVA peptide (33),
CD8+ DC were found to induce high concentrations
of the Th1 cytokines IFN- and IL-2, but little or no Th2 cytokines.
In contrast, myeloid DC induced large amounts of the Th2 cytokines IL-4
and IL-10, in addition to IFN- and IL-2. The reason for the
different regulations of IFN- and IL-12 in these studies is not
clear, but could be related to differences in the purification
procedures, the maturation state of the transferred DC, the form of Ag,
and/or the frequency of Ag-specific T lymphocytes in the recipients
(32).
The apparent contradiction between the tolerogenic properties of
lymphoid DC and their ability to produce large amounts of IL-12
(6, 8) could be explained in several ways. In
autoimmunity, it has been proposed that proinflammatory cytokines may
be required at an early time to induce self-reactive responses by
priming inflammatory Th1 responses; however, the late expression of the
same cytokines could drive the terminal differentiation and death of T
cells, including those engaged in autoreactive responses
(34). Smith et al. (7) recently reported that
tolerance induction may indeed require an early production of Th1
cytokines, accounting for the ability of lymphoid DC to produce IL-12.
DC themselves may produce significant amounts of IFN- in response to
IL-12, with the cytokine being released mainly by
CD8+ DC (9). It has been recently
shown that paralysis of IL-12 production by CD8+
DC may prevent infection-induced immunopathology in response to
microbial products (35). In our model system with a
tumor/self peptide, we have proposed the occurrence of a negative
feedback loop in which IFN- down-regulates the presentation of self
Ags via a direct action on lymphoid DC (14). The
hypothesis of such a loop is not in conflict with the widely accepted
idea that IFN- activates different types of myeloid APC to produce
IL-12, according to a positive feedback system that is probably
required for optimal production of IL-12 in the early response to
foreign entities, such as conserved molecules on bacteria and other
evolutionarily distant organisms (36).
There is now substantial evidence to indicate a protective role for
proinflammatory cytokines in experimental models of T cell-mediated
autoimmunity (34, 37). In many of these models, IFN-
has been shown to act via induction of NO by APC (24, 25, 26),
although NO may not account for all the observed regulatory effects of
IFN- (27). Although IFN--dependent production of IDO
by macrophages is known to result in inhibition of T cell proliferation
through tryptophan degradation (28), recent evidence
suggests that IDO production by human DC may also suppress T cell
proliferation (19). In our experimental model of
P815AB-specific CD4+ cells, we found that the
blockade of IDO activity by a competitive inhibitor would negate the
induction of apoptosis in vitro by IFN--treated DC. This suggests
that IDO induction is a major mechanism by which IFN- acts on DC to
mediate apoptosis of T cells. Our current data may represent the first
experimental evidence for the involvement of tryptophan degradation in
the regulation of T cell apoptosis. A recent study indicates that a
metabolite of the tryptophan/kynurenine pathway, 3-hydroxyanthranilic
acid, can act as an endogenous inducer of apoptosis in
monocyte/macrophage cell lines (38).
Triggering of DC in vivo through CD40 is a powerful activation
stimulus, causing these cells to express the full array of
Ag-presenting/costimulatory molecules (21, 39) and
regulating their migration in vivo (40). Moreover,
injection of CD40-modulated DC restores Ag-specific CTL responses in
CD4+ T cell-depleted mice (41).
These data indicate that the function of CD4+ Th
cells is mediated through CD40-dependent activation of APC. Recent
evidence suggests that the CD40-CD40 ligand pair can act as a switch in
vivo, determining whether naive peripheral CTL are primed or tolerized
(42) and accounting for the ability of CD40 ligation to
convert tumor-specific CD4+ T cell tolerance into
T cell priming (43). In line with these observations, we
demonstrate here that CD40 activation on lymphoid DC overcomes T cell
unresponsiveness to an otherwise tolerogenic tumor/self peptide even
when the potentiating effect of IFN- on lymphoid DC would result in
the induction of peripheral tolerance. Under these conditions,
triggering of CD40 is sufficient to prevent tolerization of T cells as
assessed by CD4+ T cell-dependent induction of
skin test reactivity mediated by CD8+ T cells
(10, 11). These findings may be important for improved
understanding of the requirements for Th cell function in avoiding
tolerance induction, particularly in regard to the idea that
Ag-specific T cell tolerance is known to limit the efficacy of
therapeutic cancer vaccines.
Spontaneous autoimmune diabetes in NOD mice is the result of a
CD4+ and CD8+ T
cell-dependent autoimmune process directed against the pancreatic
-cells. In an attempt to extend our current results with P815AB to
different peptides, we have resorted to a recently described peptide
mimotope recognized by CD8+ T cells from NOD mice
(17, 18). Besides representing a peptide ligand for
CD8+ T cells in autoimmune diabetes, NRP will
induce the deletion of specific CD8+ T cells
under selected conditions of immunization (18). We found
that the pattern of immune response induced by transfer of NRP-pulsed
DC was similar to that of P815AB when different DC subsets were
injected either singly or in combination. Most importantly, CD40
ligation on lymphoid DC negated their ability to modulate the
immunogenic presentation of NRP by myeloid DC.
In conclusion, the data reported here reinforce our previous suggestion
that murine DC are able to discriminate self from nonself in an
inflammatory context dominated by IFN-, calling attention to the
possibility that tryptophan degradation may be one mechanism by which
IFN- acting on CD8+ DC contributes to the
maintenance of peripheral T cell tolerance. As it is known that
CD40/CD40 ligand interactions are crucial in conditioning immunogenic
vs tolerogenic presentation of tumor/self peptides to T cells by DC
(15, 21, 42, 43, 44), the finding that CD40 activation on
CD8+ DC abrogates their tolerogenic potential in
vivo may have important implications for autoimmunity and immunotherapy
of cancer. It is likely that a variety of different effects contributes
to the ability of CD40 activation to prevent IFN--driven tolerance
mediated by lymphoid DC, including down-regulation of IFN-R
expression. However, the finding of antagonistic effects of IFN-
treatment and CD40 activation on lymphoid DC suggests that IFN-
modulation of these cells may be a useful means of limiting autoimmune
disease sustained by activated T cells and further supports the
clinical use of CD40-stimulating agents as components of
anti-cancer vaccines.
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Acknowledgments |
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Footnotes |
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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.
3 Abbreviations used in this paper: DC, dendritic cell(s); IDO, indoleamine 2,3-dioxygenase; 1-MT, 1-methyl-D,L-tryptophan; NOD, nonobese diabetic; PI, propidium iodide.
Received for publication August 2, 2000. Accepted for publication October 10, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is critical to the control of murine autoimmune encephalomyelitis and regulates both in the periphery and in the target tissue: a possible role for nitric oxide. J. Immunol. 163:5278.-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis. J. Exp. Med. 192:123. 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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  H. L. Stevenson, E. C. Crossley, N. Thirumalapura, D. H. Walker, and N. Ismail Regulatory Roles of CD1d-Restricted NKT Cells in the Induction of Toxic Shock-Like Syndrome in an Animal Model of Fatal Ehrlichiosis Infect. Immun., April 1, 2008; 76(4): 1434 - 1444. [Abstract] [Full Text] [PDF]
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 H. R. Turnquist, G. Raimondi, A. F. Zahorchak, R. T. Fischer, Z. Wang, and A. W. Thomson Rapamycin-Conditioned Dendritic Cells Are Poor Stimulators of Allogeneic CD4+ T Cells, but Enrich for Antigen-Specific Foxp3+ T Regulatory Cells and Promote Organ Transplant Tolerance J. Immunol., June 1, 2007; 178(11): 7018 - 7031. [Abstract] [Full Text] [PDF]
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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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 P. J. O'Connell, X. Wang, M. Leon-Ponte, C. Griffiths, S. C. Pingle, and G. P. Ahern A novel form of immune signaling revealed by transmission of the inflammatory mediator serotonin between dendritic cells and T cells Blood, February 1, 2006; 107(3): 1010 - 1017. [Abstract] [Full Text] [PDF]
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 B. Baban, A. M. Hansen, P. R. Chandler, A. Manlapat, A. Bingaman, D. J. Kahler, D. H. Munn, and A. L. Mellor A minor population of splenic dendritic cells expressing CD19 mediates IDO-dependent T cell suppression via type I IFN signaling following B7 ligation Int. Immunol., July 1, 2005; 17(7): 909 - 919. [Abstract] [Full Text] [PDF]
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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]
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M. G. Cusi, M. T. Del Vecchio, C. Terrosi, G. G. Savellini, G. Di Genova, M. La Placa, F. Fallarino, C. Moser, C. Cardone, G. Giorgi, et al. Immune-Reconstituted Influenza Virosome Containing CD40L Gene Enhances the Immunological and Protective Activity of a Carcinoembryonic Antigen Anticancer Vaccine J. Immunol., June 1, 2005; 174(11): 7210 - 7216. [Abstract] [Full Text] [PDF]
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 C. Haase, K. Skak, B. K. Michelsen, and H. Markholst Local Activation of Dendritic Cells Leads to Insulitis and Development of Insulin-Dependent Diabetes in Transgenic Mice Expressing CD154 on the Pancreatic {beta}-Cells Diabetes, October 1, 2004; 53(10): 2588 - 2595. [Abstract] [Full Text] [PDF]
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A. L. Mellor, P. Chandler, B. Baban, A. M. Hansen, B. Marshall, J. Pihkala, H. Waldmann, S. Cobbold, E. Adams, and D. H. Munn Specific subsets of murine dendritic cells acquire potent T cell regulatory functions following CTLA4-mediated induction of indoleamine 2,3 dioxygenase Int. Immunol., October 1, 2004; 16(10): 1391 - 1401. [Abstract] [Full Text] [PDF]
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F. Fallarino, C. Asselin-Paturel, C. Vacca, R. Bianchi, S. Gizzi, M. C. Fioretti, G. Trinchieri, U. Grohmann, and P. Puccetti Murine Plasmacytoid Dendritic Cells Initiate the Immunosuppressive Pathway of Tryptophan Catabolism in Response to CD200 Receptor Engagement J. Immunol., September 15, 2004; 173(6): 3748 - 3754. [Abstract] [Full Text] [PDF]
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 S.-i. Fujii, K. Liu, C. Smith, A. J. Bonito, and R. M. Steinman The Linkage of Innate to Adaptive Immunity via Maturing Dendritic Cells In Vivo Requires CD40 Ligation in Addition to Antigen Presentation and CD80/86 Costimulation J. Exp. Med., June 21, 2004; 199(12): 1607 - 1618. [Abstract] [Full Text] [PDF]
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