Functional Plasticity of Dendritic Cell Subsets
as Mediated by CD40 Versus B7 Activation
This information is current as
of June 15, 2017.
Ursula Grohmann, Roberta Bianchi, Ciriana Orabona,
Francesca Fallarino, Carmine Vacca, Alessandra Micheletti,
Maria C. Fioretti and Paolo Puccetti
J Immunol 2003; 171:2581-2587; ;
doi: 10.4049/jimmunol.171.5.2581
http://www.jimmunol.org/content/171/5/2581
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References
The Journal of Immunology
Functional Plasticity of Dendritic Cell Subsets as Mediated by
CD40 Versus B7 Activation1
Ursula Grohmann, Roberta Bianchi, Ciriana Orabona, Francesca Fallarino, Carmine Vacca,
Alessandra Micheletti, Maria C. Fioretti, and Paolo Puccetti2
D
endritic cells (DCs)3 are potent APCs that possess the
ability to stimulate naive T cells. They exist as distinct
subsets that differ in their lineage affiliation, surface
molecule expression, and biological function. These factors
seem to determine the T cell-polarizing signals and type of T
cell response, namely Th1, Th2, or T regulatory, induced by
DCs (1– 4). Over the last several years, the model of DCs as
natural adjuvants that promote the immune response to foreign
Ags has been modified after the realization that the APCs that
are involved in immunity must also be involved in tolerance to
self Ags. It has become increasingly clear that DCs play an
important role in both central and peripheral tolerance (3, 4).
Two general mechanisms have been proposed by which DCs
might maintain peripheral tolerance. The first is that a subtype
of specialized regulatory DCs is involved (5, 6). The second is
that all DCs have a capacity for initiating tolerance or immunity, the distinction depending on the maturation and/or activation state of the DCs (7–10).
Much evidence suggests now that the capacity of DCs to
orchestrate the immune response is not, in large part, an
intrinsic quality of the cell, but, rather, it is the result of
environmental stimulation. Among the factors that contribute to
environmental conditioning of DCs are cytokine milieu (11),
ligation of pattern recognition receptors for microbial products
(12, 13), dose of Ag (14), and state of maturation (4). An
additional level of DC conditioning may be represented by the
expression of specific ligands by T cells, which signal the DC
through cell-to-cell contact and engagement of surface receptors (15). All these environmental stimuli, either singly or in
combination, may alter the presentation pattern of a DC in the
steady state and after maturation. For example, splenic mature
CD8⫺ DCs mediate host priming to the tumor/self peptide
P815AB (11). In contrast, not only do CD8⫹ DCs inhibit the
induction of immunity by the former cells, but they also initiate
a P815AB-specific tolerant state that may have the characteristics of either anergy or deletional tolerance depending on the
activation state of the CD8⫹ DCs (16, 17). However, these cells
show no inhibitory or tolerogenic activity after CD40 ligation
(17) or exposure to IL-6 (18) or IL-23 (19).
Regulatory T cells are known to express surface CTLA-4, which
mediates suppressive effects via a combination of inhibitory T cell
signaling and blockade of the CD28/B7 costimulatory pathway
(20, 21). We have recently shown that CTLA-4 may function as a
ligand for B7 receptor molecules expressed by DCs, resulting in
tolerogenic effects that are mediated by the induction of tryptophan
catabolism (22). Thus, CD40 ligand and CTLA-4, the expression
of which is reciprocally regulated in T cells (23), might both alter
the presentation programs of DC subsets. In this study we provide
evidence for a complete functional plasticity of tolerogenic and
immunogenic DC subsets, as mediated by the opposing effects of
CD40 and B7 engagement on their surface.
Materials and Methods
Department of Experimental Medicine, University of Perugia, Perugia, Italy
Received for publication April 11, 2003. Accepted for publication July 8, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the Juvenile Diabetes Research Foundation
International (to U.G.) and the Italian Association for Cancer Research (to P.P.).
2
Address correspondence and reprint requests to Dr. Paolo Puccetti, Section of Pharmacology, Department of Experimental Medicine, University of Perugia, 06126 Perugia, Italy. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; IDO, indoleamine 2,3-dioxygenase; 1-MT, 1-methyl-D,L-tryptophan; NOD, nonobese diabetic.
Copyright © 2003 by The American Association of Immunologists, Inc.
Mice and reagents
Female DBA/2J (H-2d) mice were obtained from Charles River Breeding
Laboratories (Calco, Milan, Italy). The source and characteristics of the
hamster anti-murine CD40 (HM40-3) mAb used in combination with goat
anti-hamster IgG were previously described (17, 18, 24). Neutralizing, affinity-purified sheep anti-mouse IL-12 p70 polyclonal Ab was provided by
Genetics Institute (Cambridge, MA). Rat mAb 6B4 (anti-mouse IL-6) and
15A7 (anti-mouse IL-6R) were previously described (18). CTLA-4-Ig was
a fusion protein generated from the extracellular domain of murine
CTLA-4 and the Fc portion of a murine IgG3, with native IgG3 representing the control treatment (22). The enzyme inhibitor 1-methyl-D,L-tryptophan (1-MT) was purchased from Sigma-Aldrich (Milan, Italy). The
P815AB (amino acid sequence LPYLGWLVF) and NRP-A7 (KYNKA
0022-1767/03/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
Murine dendritic cells (DCs) can present Ag in an immunogenic or tolerogenic fashion, the distinction depending on either the
occurrence of specialized DC subsets or the maturation or activation state of the DC. Although DC subsets may be programmed
to direct either tolerance or immunity, it is not known whether appropriate environmental stimulation can result in complete
flexibility of a basic program. Using splenic CD8ⴚ and CD8ⴙ DCs that mediate the respective immunogenic and tolerogenic
presentation of self peptides, we show that both the in vivo and in vitro activities of either subset can be altered by ligation of
specific surface receptors. Otherwise immunogenic CD8ⴚ DCs become tolerogenic upon B7 ligation by soluble CTLA-4, a maneuver that initiates immunosuppressive tryptophan catabolism. In contrast, CD40 ligation on tolerogenic CD8ⴙ DCs makes these
cells capable of immunogenic presentation. Thus, environmental conditioning by T cell ligands may alter the default function of
DC subsets to meet the needs of flexibility and redundancy. The Journal of Immunology, 2003, 171: 2581–2587.
2582
FLEXIBILITY OF MOUSE DC SUBSETS IN DIRECTING TOLERANCE OR IMMUNITY
NAFL) peptides were synthesized and purified as previously described (17,
18). All in vivo studies were performed in compliance with National and
Perugia University animal care and use committee guidelines.
DC preparations and treatments and immunization
Skin test assay
A skin test assay was used for measuring class I-restricted, delayed-type
hypersensitivity responses to synthetic peptides as previously described
(17, 18). 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. The data reported
are representative of at least three independent experiments.
Kynurenine assay
Indoleamine 2,3-dioxygenase (IDO) functional activity was measured in
vitro in terms of the ability of DCs to metabolize tryptophan to kynurenine,
whose concentrations were measured by HPLC as previously described
(22).
CTLA-4 ligation of B7 molecules imparts suppressive properties
to CD8⫺ DCs that are dependent on tryptophan catabolism
We have recently proposed a novel model of tolerance induction
by CTLA-4, that occurs through B7-dependent signaling in DCs.
Using an experimental system of allogeneic islet transplant tolerance, we have shown that B7 engagement by CTLA-4-Ig conditions the DC to produce IFN-␥. The cytokine acts in an autocrine
or paracrine manner to promote induction of the enzyme IDO,
which initiates tolerogenic mechanisms dependent on tryptophan
catabolism (22, 28). We therefore assayed CTLA-4-Ig for possible
effects on the presentation of P815AB by CD8⫺ DCs. Fig. 2A
shows that exposure of these cells to CTLA-4-Ig before peptide
loading and transfer into recipient hosts abolished the induction of
skin test reactivity. The effect was associated with high level production of IFN-␥ in culture supernatants (i.e., ⬎500 pg/ml at 24 h)
in the absence of detectable IL-12 production, which is consistent
with previous data of CTLA-4-Ig treatment of unfractionated DCs
(22). Also, the effect was due to active suppression involving IDO,
Th clones and in vitro assays
The P815AB-specific Th1 cell clone F76 and the NRP-A7-specific Th1 cell
clone FF3 were derived by limiting dilution of cultured lines generated
from the popliteal lymph nodes of DBA/2 mice immunized with P815ABor NRP-A7-pulsed DCs, respectively, as described previously (23) and
were maintained by weekly restimulation of 1 ⫻ 105 cells with 5 ␮M
peptide and 6 ⫻ 106 irradiated spleen cells in complete medium containing
40 U/ml human rIL-2. Proliferation assays were performed in triplicate in
flat-bottom, 96-well microtiter plates in a total volume of 200 ␮l. Cultures
containing T cell clones (5 ⫻ 105 cells/well), purified DCs (104 cells/well),
and 5 ␮M P815AB or NRP-A7 peptide were incubated for 48 h at 37°C.
The proliferation of T cells was determined as previously described (23).
For cytokine determinations, cultures were established using 5 ⫻ 104 T
cells and 5 ⫻ 103 DCs in a 200-␮l volume in the presence of 5 ␮M
P815AB or NRP-A7 peptide, and supernatants were harvested at 24 h for
evaluation of IL-2 contents (23). IL-2 titers (mean ⫾ SD of replicate samples) were expressed as units per milliliter, calculated by reference to standard curves.
Results
CD40 activation and cytokines either enforce or suppress the
presentation programs of DC subsets
The spleens of DBA/2 mice contain a minority fraction (⬃10%) of
mature CD8⫹ DCs that mediate the tolerogenic presentation of the
synthetic tumor/self nonapeptide P815AB, such that peptideloaded CD8⫹ DCs initiate durable Ag-specific anergy upon trans-
FIGURE 1. CD40 activation affects both the priming ability of CD8⫺
DCs and the negative regulatory function of CD8⫹ DCs in the induction of
immunity to P815AB. DCs fractionated according to CD8 expression and
pulsed with P815AB in vitro were transferred into recipient mice to be
assayed for skin test reactivity to the eliciting peptide. The DC fractions
were used either as such (CD8⫺, CD8⫹) or after treatment with anti-CD40
mAb for receptor cross-linking (CD8⫹/CD40:CL, CD8⫺/CD40:CL). Different combinations of DC fractions were injected as indicated. CD40
cross-linking occurred in the presence or the absence of cytokine-neutralizing Abs (indicated). The skin test assay was performed at 2 wk. ⴱ, p ⬍
0.001, experimental vs control footpads.
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DCs were prepared and fractionated according to CD11c/CD8␣ expression
using positive selection columns in combination with CD11c and CD8␣
MicroBeads (Miltenyi Biotec, Bergish Gladbach, Germany) and in the
presence of EDTA to disrupt DC-T cell complexes, as described previously
(18). The recovered cells were ⬎98% CD11c⫹, ⬎99% MHC I-A⫹, ⬎98%
B7-2⫹, ⬍0.1% CD3⫹, ⬍0.5% B220⫹, and appeared to consist of 90 –95%
CD8⫺ and 5–10% CD8⫹ cells. After cell fractionation, the recovered
CD8⫺ cells were ⬃45% CD4⫹ and typically contained ⬍0.5% contaminating CD8⫹ DCs, whereas the CD8⫹ fraction was made up of ⬎95%
CD8⫹ DCs. In all CD40 stimulations (17, 24), DCs were incubated on ice
for 10 min in PBS plus 10% mouse serum, for 20 min with hamster antimouse CD40 mAb (5 ␮g/ml) and then overnight at 37°C with goat antihamster Ab (5 ␮g/ml) in Iscove’s medium plus 10% FCS. To check for
nonspecific effects of anti-CD40 ligation, appropriate controls included incubation of DCs in the presence of the second Ab alone, which appeared
to be devoid of any functional effect. For cytokine neutralization, DCs were
subjected to CD40 activation in vitro in the presence of 6B4 and 15A7
mAbs (for IL-6 neutralization; each at 10 ␮g/ml) or anti-mouse IL-12p70
polyclonal Ab (10 ␮g/ml) as previously described (18, 24). CD8⫺ DCs
were exposed to 40 ␮g/ml CTLA-4-Ig or IgG3 for 24 h at 37°C in the
presence or the absence of 2 ␮M 1-MT. For immunization, cells were
washed between and after incubations before peptide loading (5 ␮M, 2 h
at 37°C), irradiation, and i.v. injection into recipient hosts. CD8⫺ (3 ⫻ 105)
or, where indicated, CD40-modulated CD8⫹ DCs were injected either
alone or in combination with 5% CD8⫹ (or CTLA-4-Ig-treated
CD8⫺) DCs.
fer into recipient hosts (25, 26). The addition of as few as 3–5%
CD8⫹ DC inhibits the induction of immunity to P815AB by purified CD8⫺ DCs in the same model system in vivo, when Agspecific skin test reactivity is measured 2 wk after cell transfer. A
series of cytokines, including IL-12 (11), IFN-␥ (16), IL-6 (18),
and IL-23 (19), either reinforce or ablate the activities of the two
subsets. CD40 activation on CD8⫹ DCs abolishes their tolerogenic
potential, and the same maneuver enables CD8⫺ DCs to overcome
inhibition by unconditioned cells of the other subset (17). In line
with previous data (24, 27), Fig. 1 shows that both effects are
triggered by cytokines acting in an autocrine fashion, with IL-6
mediating the effect of CD40 activation on CD8⫹ DCs, and IL-12
mediating the corresponding effect on CD8⫺ DCs. These data are
consistent with the inflammatory, Th1-promoting, or adjuvant
properties of CD40 activation and the associated cytokine response. However, they do not clarify to which extent a default
program can be varied besides being blocked or implemented in its
expression. This particularly applies to the possible acquisition of
tolerogenic properties by CD8⫺ DCs.
The Journal of Immunology
2583
CD8⫺ DCs or a mixture of unconditioned CD8⫺ DCs plus 5%
CTLA-4-Ig-treated cells, as described for the experiment in Fig.
2A. On day 15 animals received a second cell transfer using peptide-pulsed, unconditioned CD8⫺ DCs. Mice were finally assayed
for skin test reactivity to P815AB after an additional 2 wk. Fig. 3
shows that exposure of mice to CTLA-4-Ig-treated CD8⫺ DCs
resulted in a tolerant state that could not be reversed by the use of
unconditioned CD8⫺ DCs. As unresponsiveness persisted when
the second cell transfer was delayed up to 90 days after the tolerogenic priming (data not shown), these findings suggested the occurrence of deletional tolerance initiated by the action of CTLA4-Ig on CD8⫺ DCs. This condition appeared to be similar to the
state of prolonged unresponsiveness (i.e., deletional tolerance) induced by host transfer with P815AB-pulsed DCs pre-exposed to
IFN-␥ (16, 17). Thus, not only will CTLA-4-Ig-treated CD8⫺ DCs
inhibit priming by unconditioned cells of the same subset, but they
also initiate a state of durable Ag-specific unresponsiveness.
FIGURE 2. B7 activation confers suppressive properties on CD8⫺ DCs
through mechanisms associated with tryptophan catabolism. A, DCs fractionated according to CD8 expression and pulsed with P815AB were transferred into recipient mice to be assayed for skin test reactivity. The CD8⫺
DC fraction was used as such or after treatment with CTLA-4-Ig or control
IgG3. Experimental groups included the use of CD8⫺ DCs treated with 2
␮M 1-MT during exposure to CTLA-4-Ig. An additional group consisted
of the combination of unconditioned CD8⫺ DCs and 5% CD8⫺ DCs
treated with CTLA-4-Ig. ⴱ, p ⬍ 0.001, experimental vs control footpads. B,
The functional activity of IDO produced by CD8⫺ DCs in response to
CTLA-4-Ig treatment was measured in terms of tryptophan degradation to
kynurenine, the levels of which were measured by HPLC. Results are the
mean ⫾ SD of triplicate samples.
We have previously shown that CD40 activation in CD8⫹ DCs
inhibits the tolerogenic potential of these cells (17). This occurs
through induction of autocrine IL-6, which blocks IFN-␥-induced
activation of IDO by down-regulating the expression of IFN-␥
receptors on the cell surface (18). We therefore wanted to investigate whether B7 engagement, which results in the release of
IFN-␥ (22), and CD40 engagement, which ultimately prevents intracellular signaling of the cytokine (17, 18), would exert reciprocal influence on the suppressive capacity of CD8⫹ DCs. Experiments were conducted using a combination of P815AB-pulsed
CD8⫺ and 5% CD8⫹ DCs in the experimental model illustrated
above. The CD8⫹ DCs were either untreated or subjected to B7
and/or CD40 activation. As expected, Fig. 4 shows that CD40
activation blocked the baseline suppressive effect of the CD8⫹
subset. However, the copresence of CTLA-4-Ig during CD40 activation fully restored this activity. Thus, the two maneuvers,
CD40 activation and B7 activation, have opposing effects on
CD8⫹ DC function, and the impact of B7 activation appears to be
as it could be reversed by the addition of the enzyme inhibitor
1-MT during cell exposure to CTLA-4-Ig. The latter treatment and
1-MT also had opposing effects on IDO activity in vitro, as measured by the conversion of tryptophan to kynurenines (Fig. 2B).
Of particular interest, Fig. 2A shows that the addition of 5%
CD8⫺ DCs treated with CTLA-4-Ig to a population of untreated
cells completely blocked the induction of immunity by the latter
cells. Thus, the presence of a minority fraction of cells exposed to
CTLA-4-Ig within a population of otherwise immunogenic CD8⫺
DCs will mimic the suppressive effects of the other subset. In
experiments not reported here we found that the minimal percentage of CD8⫺ DCs treated with CTLA-4-Ig required for effective
suppression was 2–3%.
CTLA-4 ligation of B7 molecules on CD8⫺ DCs induces specific
tolerance
To further explore the effect induced by CD8⫺ DCs treated with
CTLA-4-Ig, we used an experimental design previously adopted to
ascertain the nature of the suppressive properties imparted by
IFN-␥ to CD8⫹ DCs (17). We studied the impact of a previous
exposure to CTLA-4-Ig-treated CD8⫺ DCs on the priming ability
of subsequent, otherwise immunogenic, vaccination to P815AB.
Groups of mice were first injected with either CTLA-4-Ig-treated
FIGURE 3. Peptide-pulsed CD8⫺ DCs treated with CTLA-4-Ig induce
Ag-specific tolerance. DCs fractionated according to CD8 expression and
pulsed with P815AB were transferred into recipient mice. The CD8⫺ DC
fraction was used as such or after treatment with CTLA-4-Ig, as indicated
in Fig. 2. On day 15 after primary cell transfer, mice were treated with an
otherwise effective priming consisting of P815AB-pulsed CD8⫺ DCs.
Mice were assayed for skin test reactivity after an additional 2 wk. Controls
included the use of mice injected on day 15 with CD8⫺ DCs pulsed with
the antigenically unrelated P91A peptide (26) as a second cell transfer, and
then assayed after 2 wk for skin test reactivity to the same peptide. ⴱ, p ⬍
0.001, experimental vs control footpads.
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CD40 engagement on CD8⫹ DCs blocks suppressive activity,
and the effect is countered by concurrent activation of B7
2584
FLEXIBILITY OF MOUSE DC SUBSETS IN DIRECTING TOLERANCE OR IMMUNITY
FIGURE 4. CD40 engagement on CD8⫹ DCs blocks their suppressive activity in the priming to P815AB and NRP-A7, which is abolished by B7
activation. DCs fractionated according to CD8 expression were pulsed with a peptide and transferred into recipient mice to be assayed for reactivity to the
eliciting peptide. The CD8⫹ fraction was used as such or after treatment with the CD40 cross-linker or a combination of CD40 cross-linker and CTLA-4-Ig.
Exposure of CD8⫹ DCs to the CD40 cross-linker and CTLA-4-Ig was also performed in the presence of 2 ␮M 1-MT. After pulsing with P815AB or
NRP-A7, the different fractions were injected in different combinations. ⴱ, p ⬍ 0.001, experimental vs control footpads.
CD40 engagement on CD8⫹ DCs and B7 engagement on CD8⫺
DCs alter the immune function of these cells in vitro
The skin test response to P815AB that is triggered by transfer of
peptide-pulsed DCs is a class I-restricted response that requires
class II-restricted CD4⫹ T cells for afferent induction in vivo (25,
26). Using P815AB-specific CD4⫹ T cell clones, we have recently
shown that CD8⫺ and CD8⫹ DCs manifest differential ability to
sustain Th1 cell proliferation and cytokine production in vitro (23).
We therefore extended the exam of DC conditioning to this model
of secondary response in vitro. We measured the proliferation and
IL-2 production of a Th1 clone cultured with either type of DC
subset in the presence of P815AB. Fig. 5 shows that the poor
response sustained by unconditioned CD8⫹ DCs was converted
into a strong response by CD40 activation in the latter cells. In
contrast, activation of B7 by CTLA-4-Ig in CD8⫺ DCs dramati-
cally reduced their ability to stimulate Th1 cell proliferation and
IL-2 production. A similar pattern of baseline reactivity and the
induction of similar changes by CD40 or B7 activation were observed on assaying the proliferative response and IL-2 production
of a Th1 clone specific for NRP-A7 (Fig. 5). Thus, CD40 and B7
activation will produce changes in DC subsets that may alter the
immune function of these cells in a primary as well as in a secondary response.
CD40 engagement on CD8⫹ DCs makes these cells
immunogenic, which is countered by CD8⫺ DCs treated with
CTLA-4-Ig
Complete flexibility of DC programs would require that each subset be able to substitute for the other upon appropriate conditioning
in vitro. We therefore investigated the combined effects of CD40activated CD8⫹ DCs and B7-activated CD8⫺ DCs on the induction of skin test reactivity to P815AB. Fig. 6 shows that CD40activated CD8⫹ DCs would present P815AB in an immunogenic
fashion when transferred into recipient hosts in sufficient amounts
(e.g., with an inoculum size similar to that of untreated CD8⫺ DCs
in the induction of immunity, i.e., of at least 105 cells). However,
the addition of 5% CD8⫺ DCs treated with CTLA-4-Ig completely
blocked the induction of reactivity by CD40-activated CD8⫹ DCs.
Similar to the results in Fig. 3, the coinjection of CD40-activated
CD8⫹ DCs and B7-activated CD8⫺ DCs resulted in a state of
specific unresponsiveness that could not be reversed by the injection of unconditioned CD8⫺ DCs or CD40-activated CD8⫹ DCs
for at least 90 days after the first cell transfer (data not shown).
This demonstrates that environmental stimulation can condition
each subset to mimic the default function of the other subset.
Discussion
There are several aspects of the immunobiology of murine DC
subsets that are incompletely understood. Examples are the ontogenetic and functional relationships between CD8⫹ and CD8⫺
DCs, a portion of which in the spleen expresses the CD4 marker
(3). Although CD8 cannot be used as a lymphoid DC marker for
peripheral DCs, the evidence for a CD8⫹ lymphoid DC lineage
within the thymus is substantial, and CD8 is a valuable marker for
the separation of two functionally distinct DC subsets (2). CD8⫹
DCs are thought to be important in deletional tolerance, as they
appear to be resident, sedentary cells present in secondary lymphoid organs. These cells have the ability to phagocytose other
cells, including CD8⫺ DCs, and cross-present Ags derived from
phagocytosis, a process that has been referred to as cross-tolerance
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dominant when CD8⫹ DCs are coexposed to the cross-linkers in
vitro. This could be due to a greater latency in the induction of
downstream effects by CD40 activation. In experiments not reported here, we found that sequential, rather than concurrent, exposure to the CD40 cross-linker and CTLA-4-Ig would result in no
suppression by the CD8⫹ subset. Of interest, Fig. 4 also shows that
when CD8⫹ DCs were coexposed to the CD40 cross-linker and
CTLA-4-Ig in the presence of 1-MT, the suppressive activity of
these cells was again lost. Therefore, once IDO activity is blocked,
the combined effects of CD40 and B7 activation do not result in an
effective inhibitory action by the CD8⫹ DC subset.
To investigate whether the distinct patterns of activity observed with DC subsets and the effect of CD40 or B7 ligation
could be observed with self peptides other than P815AB, we
used a peptide mimotope recognized by diabetogenic CD8⫹ T
cells in nonobese diabetic (NOD) mice (17, 18). NRP-A7 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. In an
experimental model analogous to that of P815AB, CD8⫺ DCs
pulsed with NRP-A7 were transferred into recipient hosts either
alone or in combination with CD8⫹ DCs. The latter cells were
used either as such or after B7 and/or CD40 activation (Fig. 4).
Under these conditions, CD8⫹ and CD8⫺ DCs showed patterns
of reactivity similar to those observed with P815AB. Changes
identical with those seen with P815AB were induced by the
activation of B7 and/or CD40 molecules on CD8⫹ DCs.
The Journal of Immunology
2585
FIGURE 5. Presentation of peptide Ag by CD8⫺ and CD8⫹ DCs to the
Th1 clones F76 (for P815AB) and FF3 (for NRP-A7). The proliferative
response of T cells exposed to peptide-loaded DCs was measured in terms
of radiolabel uptake at 48 h. Counts are expressed as the mean counts per
minute of replicate samples ⫾ SE. IL-2 production was measured in parallel culture supernatants harvested at 24 h. CD8⫹ and CD8⫺ DC fractions
were used either as such or after CD40 activation (CD8⫹/CD40:CL) or B7
activation (CD8⫺/CTLA-4-Ig).
(10, 29). The same DC subset, however, has been previously implicated in cross-priming (30), which appears to require CD40 expression (31, 32). These data indicate that CD8⫹ DCs play a critical role in both tolerance and immunity to cell-associated Ags.
Although this may provide a potential mechanism by which CTL
can be immunized to viral Ags while maintaining tolerance to self
(29), how a single DC subset can switch between these two modes
of Ag presentation is unclear.
Using self peptides that express class I- and class II-restricted
epitopes, we show that appropriate stimuli can alter the default
function of DC subsets, resulting in immunity or tolerance according to the type of prevailing environmental conditioning. Not only
does CD40 activation enhance priming by CD8⫺ DCs and ablate
suppression by the other subset, as previously reported (17, 18),
but it also makes CD8⫹ DCs capable of immunogenic presentation
of self peptides. In contrast, CTLA-4-Ig engagement of B7 confers
suppressive properties on CD8⫺ DCs, mimicking the qualitative
and quantitative expression of the inhibitory activity of unconditioned CD8⫹ DCs. A significant portion of these regulatory effects
is probably mediated by the release of specific cytokines, most
notably IL-12 and IFN-␥ from CD8⫺ DCs (in response to CD40
and B7 activation, respectively) and IL-6 from CD8⫹ DCs (in
response to CD40 ligation). The suppressive activity induced by
CTLA-4-Ig in CD8⫺ DCs is dependent upon effective tryptophan
catabolism, thus suggesting that the occurrence of IDO-mediated
regulatory effects involving the DC is the principal mediator of
tolerance (4, 7), as previously described (22).
Although the exact mechanisms by which DC exposure to 1-MT
in vitro can affect their activity in vivo once the cells have been
separated from the enzyme inhibitor are unclear, we have consistently observed prolonged IDO inhibition in this type of experimental setting. This suggests that the inhibition of IDO by 1-MT
is both a time-dependent and slowly reversible phenomenon (17).
One important feature of the biological activity of CD8⫺ DCs in
our model systems with P815AB and NRPA-7 is that the action of
this subset, upon conditioning by CTLA-4-Ig treatment, is not limited to impaired priming by unconditioned CD8⫺ DCs. Similar to
the effect of IFN-␥-treated CD8⫹ DCs (17), host transfer with
CD8⫺ DCs exposed to CTLA-4-Ig, either alone or in combination
with untreated cells, will result in specific unresponsiveness that is
not reversed by a subsequent (i.e., at 90 days), otherwise effective
priming with the peptide. Also, the cotransfer of CD40-activated
CD8⫹ DCs and B7-activated CD8⫺ DCs resulted in a state of
unresponsiveness that could not be overcome by later transfer of
unconditioned CD8⫺ DCs or CD40-activated CD8⫹ DCs. This
indicates that either subset can, under specific conditions, initiate a
state of durable tolerance to the peptide.
Several considerations can be made in this regard. First, analogous to the condition of IFN-␥-treated CD8⫹ DCs (17), the effect
of CD8⫺ DCs treated with CTLA-4-Ig appears to be different from
the anergic state induced by host transfer with unfractionated DCs
pulsed with P815AB, because the latter represents a reversible
phenomenon that is no longer observable 40 – 60 days after tolerogenic priming (26). Second, the recent observation that selected
tryptophan catabolites, namely kynurenine derivatives, are strong
inducers of apoptosis in T cells is compatible with an important
role for DCs as mediators of deletional tolerance occurring by
IDO-dependent effects (28, 33). Finally, and perhaps more importantly in the present context, the demonstration of specific tolerance in mice receiving CD8⫺ DCs exposed to CTLA-4-Ig indicates that B7 activation in these cells is an effective means of
inducing a switch between immunity and tolerance. Although
switching between different types of immunity has been reported
as a result of DC flexibility in directing Th cell development (14),
this effect is unlikely to contribute to the tolerant state observed in
mice receiving CTLA-4-Ig-treated DCs.
Another interesting observation in our current data may be represented by the opposing effects of CD40 activation and B7 activation on CD8⫹ DC function, with the impact of B7 activation
being dominant when CD8⫹ DCs were coexposed to the cross-
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 6. Cross-regulation of DC subsets after conditioning via CD40
or B7 activation. Otherwise suppressive CD8⫹ DCs were assayed for priming ability to P815AB following CD40 activation according to the experimental conditions illustrated in Fig. 1. The priming ability of CD40-activated CD8⫹ DCs (3 ⫻ 105) was also assayed in the presence of a minority
fraction (5%) of CD8⫺ DCs rendered suppressive by treatment with
CTLA-4-Ig, IgG3 representing the control treatment. ⴱ, p ⬍ 0.001, experimental vs control footpads.
2586
FLEXIBILITY OF MOUSE DC SUBSETS IN DIRECTING TOLERANCE OR IMMUNITY
ined in our model, the present data can improve our understanding
of the functional plasticity, cooperation, and cross-regulation of
DC subsets in light of the cross-talk between these cells and T
lymphocytes.
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Ag, the state of maturation of the DC, and environmental signals
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maturation and activation state of the DC and environmental signals. Thus, besides default programming, which is necessary to
face the challenges of their usual setting, each DC subset can acquire disparate abilities in an instructive mode. This might provide
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CTLA-4 are reciprocally regulated in their expression by a Th1
clone (23) raises the issue of the effects of T cells, including regulatory T cells, on unconditioned CD8⫺ and CD8⫹ DCs. Although
such possible bidirectional influences are currently being exam-
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