994 C. Demangel et al. Eur. J. Immunol. 2002.

32: 994–1002

Autocrine IL-10 impairs dendritic cell (DC)-derived

immune responses to mycobacterial infection by
suppressing DC trafficking to draining lymph nodes
and local IL-12 production
Caroline Demangel1,2, Patrick Bertolino1 and Warwick J. Britton1,3
1
Centenary Institute of Cancer Medicine and Cell Biology, Newtown, Australia
2
Laboratoire d’Ingenierie des Anticorps, Institut Pasteur, Paris, France
3
Department of Medicine, University of Sydney, Sydney, Australia

The production of IL-12 by dendritic cells (DC) early in an immune response is considered
critical for the polarization of CD4+ T lymphocyte response towards a Th1 pattern, a key pro-
cess in the clearance of intracellular pathogens. Infection of bone marrow-derived DC with
Mycobacterium bovis Bacillus Calmette Guérin (BCG) induced a concurrent and dose-
dependent release of IL-10 and IL-12. Here we examined whether the production of IL-10 by
DC affected their IL-12 response to mycobacterial infection and the generation of protective
immune responses in vivo. Compared to wild-type (WT) DC, DC deficient for IL-10 synthesis
(IL-10–/–) showed increased IL-12 production in response to BCG infection and CD40 stimuli
in vitro. Moreover, when transferred into mice, infected IL-10–/– DC were more efficient than
WT DC at inducing IFN- + production to mycobacterial antigens in the draining lymph nodes
(DLN). This effect was associated with increased trafficking of IL-10–/– DC to the DLN and
enhanced IL-12 production by DC within the DLN. These data show that autocrine IL-10
exerts a dual inhibitory effect on the induction of primary immune responses by DC: first, by
down-regulating the migration of infected DC to the DLN and second, by modulating the
IL-12 production by DC in the DLN. Received 5/11/01
Revised 20/12/01
Accepted 21/1/02
Key words: Dendritic cell / Mycobacteria / Cell trafficking / IL-12 / IL-10

1 Introduction IL-12, a pro-inflammatory cytokine governing the devel-

The orientation of the immune response towards Th1 or
Th2 phenotype determines the outcome of parasitic
infections caused by many intracellular pathogens,
including Chlamydia, Mycobacteria and Leishmania. In
these infections, the early priming of antigen-specific
CD4+ T cells producing IFN- + is crucial for the develop-
ment of protective immunity [1–3]. Conversely, redirec-
tion of the immune response towards a Th2 type, as
characterized by CD4+ T cells producing IL-4 and IL-10,
results in increased susceptibility to infection and slower
bacterial clearance in the host [4–6]. The cytokine profile
generated during the early phase of an infection, by
polarizing cellular responses into Th1 or Th2 type, there-
fore has a key impact on the resolution of infectious dis-
eases.
[I 22596]
Abbreviations: CMFDA: 5-Chloromethylfluorescein di-
acetate DC: Dendritic cell DLN: Draining lymph nodes IL-
10–/–: IL-10-deficient MOI: Multiplicity of infection TLR:
Toll-like receptor WT: Wild-type
opment of IFN- + -producing T cells [7], is of particular
importance in this process. The presence of IL-12 during
early infection is associated with expansion of Th1-like T
cells, and as a consequence with resistance to diseases
caused by bacteria, intracellular parasites, fungi, and
viruses [7]. In contrast, IL-10 acts as an immunosuppres-
sive cytokine, exemplified by stronger Th1-like T cell
responses and increased resistance to intracellular path-
ogens in IL-10-deficient (IL-10–/–) animals [5, 8, 9]. Con-
sistently in humans, IL-10 levels correlate with the pro-
gression of leishmaniasis, malaria, leprosy, tuberculosis
and Mycobacterium avium infections [10]. From these
observations, it appears that IL-12 and IL-10 are major
regulators of the development of Th1 cells during the
immune response to infection, which play antagonist
roles. Recently, suppression of the endogenous produc-
tion of IL-10 by APC was found to be a potent means for
boosting Th1 immunity against Chlamydia infection [11].
Moreover, when dendritic cells (DC) were engineered to
secrete IL-12, they had an increased capacity to gener-
ate specific IFN- + -producing T cells and to protect mice
against leishmanial challenge [12, 13]. This suggests that

0014-2980/02/0404-994$17.50 + .50/0 © WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2002

Eur. J. Immunol. 2002. 32: 994–1002
IL-12 and IL-10 may exert their regulatory role primarily
at the level of the APC.
DC are major APC in the initiation and the maintenance
of anti-microbial immunity [14]. Numerous in vitro data
have shown that IL-12 is produced by DC following stim-
ulation with microbial products or T cell signals such as
the engagement of surface CD40 [14, 15]. Immunization
with antigen-loaded DC induced the development of
protective Th1-like T cell responses in murine models of
leishmaniasis and tuberculosis [13, 16], suggesting that
interaction of DC with incoming pathogens drives them
to release IL-12 in vivo. This view is supported by the
finding that IL-12 production by DC was detected in
mice early after injection of microbial antigens [17, 18].
Several lines of evidence suggest that DC maturation
and function may be altered by exposure to IL-10 [10].
Addition of IL-10 to DC suppresses DC differentiation
from monocytes [19], and blocks DC maturation in
response to LPS stimulation [20]. Treatment of DC with
exogenous IL-10 down-regulates their expression of
mRNA for IL-12, and impairs their capacity to induce
IFN- + -producing cells to a protein antigen in vivo [21].
Recently, antibody neutralization of DC-derived IL-10
was shown to enhance LPS- and CD40-mediated matu-
ration of DC in vitro [22]. However, whether this mecha-
nism of regulation is relevant in situations were DC are
infected with live organisms, and impairs the develop-
ment of protective immunity remained to be determined.
In the present study, we show that endogenous IL-10
suppresses the induction of antimycobacterial cellular
responses by DC in vivo, and explore the mechanisms
by which IL-10 exerts its regulatory role on DC.
2 Results
2.1 BCG infection induces the production of both
IL-12 and IL-10 by DC
Bacterial stimuli induce DC maturation and up-regulate
their mRNA expression for a number of cytokines,
including IL-12 and IL-10 [14]. To investigate the inter-
play between these two cytokines on DC function follow-
ing mycobacterial infection, bone marrow-derived DC
were incubated with BCG and the release of IL-12 and
IL-10 cytokines measured in culture supernatants.
Fig. 1A shows that secretion of both IL-12 and IL-10 was
induced in BCG-infected DC, as compared to uninfected
DC. The production was augmented in a similar fashion
for IL-12 and IL-10 when the multiplicity of infection
(MOI) was increased from 1 to 10 bacilli per DC. Flow
cytometric analysis revealed that uninfected and BCG-
infected DC equally express the receptor for IL-10 (IL-
10R) (Fig. 1B), demonstrating that IL-10R expression is
Autocrine IL-10 suppresses DC-derived immune responses 995
Fig. 1. IL-12 and IL-10 are co-induced in BCG-infected DC.
(A) IL-12 and IL-10 production by DC following infection with
live BCG is shown as a function of the MOI. Data are mean
and SD of ELISA triplicate measurements and are represen-
tative of two independent experiments. (B) The surface
expression of IL-10R was measured for uninfected DC (DC),
and DC infected with BCG at a MOI of 10 (DC + BCG). FACS
profiles are representative of two independent experiments
and include DC staining with an isotype control Ab (ctrl).
not affected upon infection and that IL-10 can signal in
DC, regardless of their infection status.
2.2 Autocrine IL-10 regulates the IL-12 response
of DC to BCG infection and to CD40
stimulation
To test whether IL-12 production by DC was affected by
endogenous IL-10, DC generated from wild-type (WT) or
IL-10–/– animals were compared for their ability to secrete
IL-12 following BCG infection. As with WT DC, the pro-
duction of IL-12 by IL-10–/– DC increased with increasing
MOI with BCG (Fig. 2A). However, IL-10–/– DC released
significantly higher levels of IL-12 than WT DC. More-
over, when IL-10 signaling was blocked in WT DC by an
antibody to IL-10R, increased IL-12 production was
observed (Fig. 2B).
Signals from T cells, such as the engagement of surface
CD40 on DC, potentiate the ability of DC to release IL-
12. When DC were infected with BCG at a MOI of 10 and
stimulated with graded amounts of agonistic anti-CD40
antibody [15], a dose-dependent increase in IL-12 pro-
duction was observed (Fig. 3A). IL-12 production was
further augmented in IL-10–/– DC, with CD40 stimulation
of IL-10–/– DC leading to tenfold higher concentrations of
IL-12 as compared to WT DC. Staining for intracyto-
plasmic IL-12 showed that the percentage of IL-12 pro-
ducing cells was higher in IL-10–/– than in WT DC, and
that CD40 stimulation gives rise to DC with higher IL-12-
producing capacity (Fig. 3B and data not shown).
996 C. Demangel et al.
Fig. 2. Endogenous IL-10 regulates the IL-12 response of
DC to BCG infection. (A) IL-12 production by DC derived
from WT or IL-10–/– mice, is shown at various MOI with live
BCG. (B) IL-12 production by DC infected with BCG at an
MOI of 10 in the presence of grading amounts of a neutraliz-
ing anti-IL-10R antibody ( § IL-10R Ab), or an isotype control
(ctrl Ab), are compared. Data are mean and standard error
(SE) of ELISA triplicate measurements on culture superna-
tants, and are representative of two independent experi-
ments. Differences between groups were analyzed using
ANOVA (* p X 0.01).
2.3 IL-10 inhibits the capacity of BCG-infected
DC to generate IFN- q -producing T cells
in vivo
If endogenous IL-10 inhibits the IL-12 response of DC to
microbial stimuli and to CD40 stimulation, IL-10–/– DC
should be more effective than WT DC at priming IFN- + -
secreting T cells against mycobacterial antigens in vivo.
WT and IL-10–/– DC were infected with BCG in vitro and
compared for their ability to activate naive T cells follow-
ing transfer in mice. Seven days following subcutaneous
(s.c.) immunization, cells from pooled draining lymph
Fig. 3. Endogenous IL-10 regulates the IL-12 response of DC to CD40 stimulation. (A) IL-12 production by BCG-infected DC
derived from WT or IL-10–/– mice, in the presence of agonistic anti-CD40 ( § CD40 Ab) or irrelevant (ctrl Ab) antibody, is shown.
Data are mean and SE of triplicate measurements, and are representative of two independent experiments. (B) The percentage
of DC producing IL-12 in response to BCG infection in the presence ( § CD40 Ab), or absence (ctrl Ab), of agonistic anti-CD40
antibody is shown.
Eur. J. Immunol. 2002. 32: 994–1002
nodes (DLN, i.e. inguinal lymph nodes) were restimulated
in vitro with PPD. DLN cells prepared from mice immu-
nized with IL-10–/– DC proliferated to a greater extent to
mycobacterial antigens (Fig. 4A), and generated higher
numbers of IFN- + -producing cells (Fig. 4B), than those
from WT DC injected mice.
2.4 Autocrine IL-10 does not modify the
maturation of DC in vitro
To examine whether this effect of IL-10 on T cell immune
response was due to differences in maturation of IL-10–/–
and WT DC, we investigated the expression of MHC
class II and CD86 costimulatory molecules on DC at the
time of inoculation. As previously reported [23–25], the
expression of both molecules was significantly aug-
mented in WT DC following infection with BCG (Fig. 5).
However, IL-10–/– and WT DC showed equivalent levels
of expression of CD86 and MHC class II molecules,
regardless of the infection with BCG (Fig. 5). Moreover,
no differences in the proportion of apoptotic cells, as
measured by staining with Annexin V, were observed
between BCG-infected IL-10–/– and WT DC (data not
shown), demonstrating that endogenous IL-10 did not
alter DC maturation and viability in this model.
2.5 IL-10–/– and WT DC differ by their ability to
reach draining lymph nodes following
transfer
We thus compared WT and IL-10–/– DC for their migratory
capacities following transfer in vivo. Following infection
with BCG and prior to s.c. injection, DC were stain-
ed with the fluorescent cell tracker 5-chloromethyl-
Eur. J. Immunol. 2002. 32: 994–1002
Fig. 4. IL-10 inhibits the capacity of BCG-infected DC to
generate IFN- + -producing T cells in vivo. Mice immunized by
BCG-infected DC derived from WT or IL-10–/– mice were
compared for proliferation of inguinal lymph node cells (A),
and frequency of IFN- + producing cells in the lymph nodes
(B), following restimulation with PPD. Mean ± SE for tripli-
cate cultures are shown. Differences between groups were
analyzed using ANOVA (* p X 0.01).
fluorescein diacetate (CMFDA) [26]. Twenty-four hours
later, DLN (inguinal lymph nodes) cell suspensions were
prepared and analyzed by flow cytometry. A CMFDA+
cell subset was detected in the DLN of both mice immu-
nized with WT and IL-10–/– DC (Fig. 6A and B). As
expected, this CMFDA+ cell subset had the CD11c+ CD8–
phenotype of bone marrow-derived DC (data not
shown). However, the frequency (Fig. 6C) and the total
number (Fig. 6D) of CMFDA+ cells in the DLN were signif-
icantly higher in mice injected with IL-10–/– DC than in
Fig. 5. Autocrine IL-10 does not alter DC maturation. Cell
surface expression of MHC class II and CD86 molecules is
shown for uninfected or BCG-infected WT and IL-10–/– DC at
day 5 of culture. Analysis was performed 24 h after infection
with BCG at an MOI of 10. Dashed lines correspond to stain-
ing in the absence of primary specific antibodies (ctrl). FACS
profiles are representative of two independent experiments.
Autocrine IL-10 suppresses DC-derived immune responses 997
Fig. 6. Autocrine IL-10 suppresses the migration of DC in
vivo. Detection of a subset of CMFDA+ CD11c+ cells in the
DLN 24 h after s.c. injection: A representative dot plot is
shown for groups of three mice injected with BCG-infected
WT DC (A) or IL-10–/– DC (B). (C) Percentage of CMFDA+ cells
in total DLN cells, and (D) the total number of CMFDA+ cells
in the DLN 24 h after inoculation of BCG-infected WT or IL-
10–/– DC. Similar findings were obtained in separate experi-
ments. Data are mean and SE measured on groups of three
animals, and differences between groups were analyzed
using ANOVA (* p X 0.05, ** p X 0.01).
those immunized with WT DC. This suggested that IL-10
release by BCG-infected DC limits their ability to traffic to
the DLN.
2.6 Transfer of IL-10–/– DC augments the
production of IL-12 by DC in the DLN
To investigate whether IL-10–/– DC have an enhanced
ability to deliver IL-12 at the site of immune induction,
mice were s.c. immunized with CMFDA-labeled WT or
IL-10–/– DC. Control animals injected with PBS or BCG
were included. After 24 and 40 h, DLN cell suspensions
were prepared and stained for intracytoplasmic IL-12.
Fig. 7 shows that the total number of CD11c+ IL-12+ cells
was significantly higher in the group injected with IL-10–/–
DC than in the other groups 24 h and 40 h post transfer,
with significant differences in the frequencies of these
cells 40 h post transfer.
3 Discussion
Bacteria and their products are potent inducers of DC
activation, promoting profound changes in DC pheno-
type and function [14]. Stimulation of DC with microbial
998 C. Demangel et al.
Fig. 7. DC-derived IL-10 down-regulates the production of
IL-12 by DC in the lymph nodes. The percentage (A) and
total number (B) of IL-12-producing DC in the DLN are
shown 24 h (white) and 40 h (black) after inoculation of
BCG-infected WT and IL-10–/– DC. The IL-12-producing
subset of DC in the DLN was analyzed by gating on CD11c+
cells positive for IL-12, as determined by intracytoplasmic
staining. This cell subset was consistently negative for
CMFDA staining, indicating that the IL-12-producing DC
were of recipient origin (data not shown). Similar findings
were obtained in separate experiments. Data are mean and
SE measured on groups of three animals, and differences
between groups were analyzed using ANOVA (* p X 0.05).
products triggers the up-regulation of a number of mole-
cules specialized in the activation of T cells, including
MHC class I and II, CD1, B7-1, B7-2 and CD40. Acti-
vated DC acquire migration capacity, leave the periph-
eral organs and reach secondary lymphoid tissues. The
production of cytokines is also up-regulated in microbial-
stimulated DC, with release of a range of pro-
inflammatory cytokines such as IL-6, IL-1, TNF- § and IL-
12. The fact that IL-10 is often co-induced with IL-12 in
DC following bacterial stimulation led to the hypothesis
that this cytokine might serve as a counter signal for
blocking exaggerated T cell responses induced by IL-12
[25]. This view is supported by data showing that exoge-
nously added IL-10 inhibits LPS- and CD40-dependent
IL-12 production by DC and impairs their ability to
induce Th1 cells in vivo [20, 21], whereas addition of anti-
IL-10 neutralizing antibody or soluble IL-10R to DC cul-
tures results in opposite effects [22, 27]. Our findings that
IL-10–/– DC have an increased ability to produce IL-12 in
response to mycobacterial and CD40 stimuli, and induce
stronger specific Th1 responses in vivo, further supports
the suppressive role of autocrine IL-10 on the generation
of IL-12-mediated immune responses by DC.
The bacterial molecules and signaling pathways leading
to IL-12 production by DC are incompletely understood;
however, there is evidence that IL-12 is induced by bac-
terial lipoproteins and LPS in human monocytes,
monocyte-derived macrophages and DC in a Toll-like
receptor (TLR)-dependent manner [28–30]. Signaling
Eur. J. Immunol. 2002. 32: 994–1002
through TLR leads to the induction of NF- ‹ B-controlled
genes, including pro-inflammatory cytokines and costi-
mulatory molecules [31]. In contrast, IL-10 cellular pro-
duction is regulated by alteration of RNA degradation
[10], suggesting that IL-10 and IL-12 responses to micro-
bial stimulation are modulated by distinct molecular
mechanisms in DC. Accordingly, activation of TLR2 on
human monocyte-derived DC by exposure to LPS or a
single M. tuberculosis lipoprotein induced IL-12, but not
IL-10 [28]. The interactions between human DC and
intact pathogens are likely to be more complex, with a
variety of bacterial products interacting with specific
DC receptors and contributing to the global cytokine
response of DC. For example, M. tuberculosis can signal
via both human TLR2 and TLR4, leading DC to produce
both IL-10 and IL-12, at low but reproducible levels [23].
Interestingly, although they are closely related mycobac-
teria species, BCG is far more effective at stimulating IL-
12 production by DC than M. avium and M. tuberculosis
(C. Demangel, unpublished). Similarly, different forms of
the same fungus Candida albicans stimulate DC for dis-
criminative production of IL-12 or IL-4 [32], while LPS
extracted from different types of bacteria induce differ-
ential IL-12 production by splenic DC [33]. This suggests
that the innate recognition of bacteria by DC does not
result in a standard profile of cytokine response, but in a
distinctive ‘cytokine pattern’ for each parasite. For vac-
cine development, the measurement of IL-10 and IL-12
production by DC in response to given pathogens or dif-
ferent candidate vaccines may help to predict which are
more likely to elicit Th1-orientated responses in vivo.
Constitutive expression of IL-10R has been observed on
murine bone marrow-derived DC (Fig. 1B), lymph node
DC of both CD8+ and CD8– subsets (data not shown),
and on human PBMC-derived DC [22], suggesting that
IL-10 and IL-10R constitute a general modulatory loop
for the regulation of DC function. Interestingly, in vitro
stimulation of DC with agonistic anti-CD40 antibody sig-
nificantly up-regulated the expression of IL-10R (data not
shown). This modulation of IL-10R surface expression
was observed regardless of infection with BCG, and sug-
gests that IL-10 might play a regulatory effect more
selectively on CD40-activated DC. This may represent a
mechanism by which DC having migrated to the second-
ary lymphoid organs and contacted T cells can regulate
their own production of IL-12. We are currently examin-
ing if the up-regulation of IL-10R is a relevant mechanism
in vivo for the control of IL-12 synthesis by DC in the
lymph nodes.
In addition to its effect on IL-12 synthesis by DC, IL-10
has been described to inhibit the maturation of blood-
derived and dermal DC, as measured by the expression
of costimulatory molecules and their capacity to pro-
Eur. J. Immunol. 2002. 32: 994–1002
mote alloantigen-specific T cell responses [10]. However,
the expression of CD86 and MHC class II molecules was
comparable in IL-10–/– DC and WT DC (Fig. 5), and was
not modified in splenic DC by treatment with IL-10 [21],
suggesting that the effect of IL-10 on DC maturation
might vary with the type of DC. Moreover, WT and IL-
10–/– DC did not differ in the percentage of apoptotic or
necrotic cells. Therefore, the differences in the immune
responses driven by IL-10–/– and WT DC following trans-
fer in mice were essentially due to differential production
of IL-12 and migratory capacities in this model.
Tissue damage, presence of stressed or necrotic cells
and microbial products are danger signals triggering the
release of locally important concentrations of inflamma-
tory mediators of DC migration [34, 35]. The capacity of
IL-10–/– or WT DC to migrate to the DLN was examined
24 h after inoculation. Previous studies have shown that
the number of splenic DC trafficking to the DLN after s.c.
injection reached a maximum within 24 h [26, 36].
Accordingly, migrating DC could be detected in DLN fol-
lowing loading of splenic [37] or bone marrow-derived
[38] DC in the trachea within 24 h and were no longer
detectable 48 h later, supporting the rapid migration of
DC. However, the proportion of DC migrating to the DLN
always constituted a small percentage of the injected
pool (0.1–1.0%). This proportion of migratory DC is con-
sistent with other studies [26, 36], and questions the fate
of the remaining cells. It is likely that some of the injected
DC die at the site of inoculation, and released antigens
are recaptured and processed by resident DC. Interest-
ingly, T cell priming in the DLN occurs with viable but not
with killed antigen-pulsed DC following transfer [39].
Therefore, despite the fact that the proportion of traffick-
ing cells is relatively small, the direct migration of
antigen-bearing DC appear to be essential for the initia-
tion of specific T cell responses. If so, then the manipula-
tion of DC to increase their migration properties before
transfer may be a way to improve the efficacy of DC-
based therapies.
The trafficking of DC from inflammatory sites to lymphoid
tissues has been associated with a chemokine receptor
switch on DC surface, with down-regulation of inflamma-
tory receptors and induction of CCR7 [40]. Recently,
it was demonstrated that concomitant exposure of
monocyte-derived DC with LPS and IL-10 blocks this
chemokine receptor switch on DC and impairs their abil-
ity to respond to inflammatory chemokines [41], sug-
gesting that IL-10 can suppress this migration process.
Consistently, FITC-induced epidermal Langerhans cell
migration from skin to DLN was enhanced in IL-10–/–
mice [42]. In the present study, BCG-infected IL-10–/– DC
migrated to the DLN more efficiently than WT DC follow-
ing transfer into mice, suggesting that autocrine IL-10
Autocrine IL-10 suppresses DC-derived immune responses 999
is an important regulator of DC migration in vivo in
response to microbial stimuli.
Enhanced IL-12 production was observed in the DLN of
mice immunized with IL-10–/– DC, as compared to WT
DC. Intriguingly, the CMFDA+ subset of DC in the DLN
was consistently negative for IL-12 production, indicat-
ing that the IL-12-producing cells were of recipient ori-
gin. This raises the question as to how the information
transfer between the different DC populations occurs. It
is possible that although most injected DC do not
migrate, they deliver a cytokine message and/or anti-
genic peptides to surrounding uninfected DC, which
induces these to migrate to secondary lymphoid organs.
Alternatively, one can speculate that the few migrating
DC of donor origin are able to activate local DC to
secrete IL-12 when they reach the lymph nodes, result-
ing in a local amplification of the initial response. This
could be mediated by cytokine release or cell/cell con-
tact with resident DC or by recruiting other circulating DC
in the nodes. Taken together, our results support the
emerging concept (Maraskovsky et al., unpublished) that
in physiological situations of infection, only a small sub-
set of the DC population activated by microbial agents in
peripheral tissues migrates effectively to the DLN. In
contrast, the vast majority of infected DC do not leave
the site of infection. Our experiments suggest that this
retention may be IL-10 dependent. Further, IL-10
appears to be an important factor regulating the genera-
tion of the appropriate type of immune responses
against microbial infections. Blocking DC-derived IL-10
appears to be a potent way to stimulate enhanced Th1
immune responses and may contribute to more efficient
anti-microbial vaccine strategies.
4 Materials and methods
4.1 Mice
Six- to eight-week-old C57BL/6 mice were obtained
from the Animal Resources Centre (Perth, Australia). IL-
10–/– mice on a C57BL/6 background [43] were kindly
provided by Dr. Donna Rennick (DNAX Research Insti-
tute, Palo Alto, CA) and kept under specific pathogen-
free conditions at the Centenary Institute animal facility.
4.2 DC cultures
Bone marrow-derived DC were generated by a modifica-
tion of the method previously described by Inaba et al.
[44]. Briefly, murine bone marrow cell suspensions were
incubated with a mixture of M5.114 (anti-MHC class II),
RA3-6B2 (anti-B220), 53-6.7 (anti-CD8), GK1.5 (anti-
1000 C. Demangel et al.
CD4) and RB6-8C5 (anti-Ly-6G) mAb. Cells bound to
antibodies were eliminated using Dynabeads M-450
coated with sheep anti-rat IgG. Remaining cells were
cultured in complete medium (CM) of RPMI 1640 con-
taining 5% FCS, 50 ? M 2-ME, 2 mM glutamine supple-
mented with 2.5 ng/ml recombinant murine granulocyte/
macrophage colony-stimulating factor and 5 ng/ml
recombinant murine IL-4 (both from Peprotech, Rocky
Hill, NJ). To test the effect of CD40 ligation on DC, a rat
anti-CD40 IgG (clone 1C10) [15, 45] or an irrelevant rat
IgG (GL113, ATCC HB11679) was added to the CM. For
BCG infection, day 4 DC cultures were incubated with
live BCG (Tokyo strain, ATCC 35737) at various MOI
(from 1 to 10). Cells were washed after 16 h to eliminate
free mycobacteria, and cultured in fresh medium for an
additional 48 h before harvesting culture supernatants
for cytokine assays. For transfer experiments, DC were
harvested 4 h after infection with BCG at a MOI of 10,
and washed twice in PBS before inoculation.
4.3 DC tracking in vivo
Before inoculation, DC were labeled with fluorescent cell
tracker green CMFDA (Molecular Probes, Eugene, OR)
as previously described [26]. DC were then washed
twice, resuspended in PBS (107 cells/ml), and 200 ? l
(equivalent 2×106 DC) were injected s.c. into the base of
the tail of recipient animals. Mice injected with an equiv-
alent volume of PBS, or PBS containing 106 live BCG,
were used as controls. Twenty-four hours later, cell sus-
pensions were prepared from the DLN (inguinal lymph
nodes) by a modification of the procedure previously
described [46]. Briefly, DLN fragments from three mice
per group were digested for 20 min at room temperature
(RT) with DNase-collagenase, and treated with EDTA to
disrupt T cell–DC complexes. The resulting DLN cell
preparations were then enriched in DC by centrifugation
in a Nycodenz medium (Nycomed, Oslo, Norway) and
elimination of the high-density cells. The remaining frac-
tion was used directly for flow cytometry analysis with
gating on CD11c+ cells.
4.4 Flow cytometry
Expression of surface markers was examined by pre-
incubating DC with mouse serum and staining with anti-
IL-10R (1B13a, kindly provided by Dr. Kevin Moore,
DNAX Research Institute, Palo Alto, CA) [47, 48], or the
isotype control GL113, anti-MHC class II Ab (M5.114) or
anti CD86 (GL1), plus FITC-conjugated mouse-adsorbed
anti-rat Ig (Caltag). Flow cytometric analysis was then
performed on cells gated for CD11c+ (PE-conjugated
HL3) propidium iodide– (PI) (both from PharMingen, San
Eur. J. Immunol. 2002. 32: 994–1002
Diego, CA). For DC migration assays, CMFDA fluores-
cence was used to distinguish donor-derived DC from
recipient DC. For intracytoplasmic staining, DC puri-
fied from the lymph nodes were fixed with 4% (w/v)
paraformaldehyde 20 min at RT and permeabilized
in 0.1% BSA, 0.5% saponin PBS. Cells were then stain-
ed with PE-conjugated rat anti-mouse IL-12 (C15.6,
PharMingen), and with biotinylated anti-CD11c (HL3,
PharMingen) followed by streptavidin coupled to PE-
Cy5. Analysis was then performed on CD11c+-gated
cells. Pre-incubation of the fixed/permeabilized cells
with unlabeled C15.6 antibody (20 ? g/ml) prior to stain-
ing with PE-C15.6 abolished the detection of intracyto-
plasmic IL-12, confirming the specificity of the staining
(data not shown). Samples were analyzed on a FACScali-
bur (Becton Dickinson, San Jose, CA).
4.5 Cytokine production assays
IL-12 production by DC was measured in culture super-
natants using an ELISA kit for IL-12p70 heterodimer
detection (PharMingen). IL-10 release was measured by
ELISA using the mAb SXC-4 and SXC-1 [49].
4.6 Proliferation assay and ELIspot for cytokine-
producing cells
Seven days post immunization by s.c. route, DLN from
three mice were pooled, and a single-cell suspension
prepared by sieving and re-suspending the cells in cul-
ture medium. For proliferation assay, cell suspensions
were plated in 96-well plates at 5×105 cells per well with
either medium alone, 10 ? g/ml PPD of M. tuberculosis
(Statens Seruminstitut, Copenhagen, Denmark) or 10 ? g/
ml Con A (Sigma, St. Louis, MO). [3H]Thymidine was
added after 48 h of culture and the difference between
PPD and medium-supplemented wells ( ¿ cpm) was mea-
sured 6 h later. ELIspot assays were conducted as previ-
ously described [15].
Acknowledgements: We thank Dr. Kevin Moore (DNAX,
Palo Alto, CA) for giving us anti-IL-10R antibody reagents,
Dr. Donna Rennick (DNAX) for providing us with the IL-10–/–
mice, and Dr. Andrew W. Heath (University of Sheffield Medi-
cal School, Sheffield, GB) for giving us the anti-CD40 anti-
body. We also wish to thank Dr. Adrian L. Smith (Centenary
Institute) for assistance with the staining of DC with CMFDA,
Dr. Jamie A. Triccas (Centenary Institute) and Dr. Andrew G.
Bean (CSIRO, Geelong, Australia) for helpful discussions.
The support of the NSW Health Department through its
research and development infrastructure grants program is
gratefully acknowledged. This study was funded by the
National Health and Medical Research of Australia.
Eur. J. Immunol. 2002. 32: 994–1002 Autocrine IL-10 suppresses DC-derived immune responses
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Correspondence: Warwick J. Britton, Centenary Institute of
Cancer Medicine and Cell Biology, Locked Bag No. 6, New-
town, NSW 2042, Australia
Fax: +61-2-9351-3968
e-mail: wbritton — medicine.usyd.edu.au