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TLR-Dependent Activation Stimuli
Associated with Th1 Responses Confer NK
Cell Stimulatory Capacity to Mouse Dendritic
Cells
Ivan Zanoni, Maria Foti, Paola Ricciardi-Castagnoli and
Francesca Granucci
J Immunol 2005; 175:286-292; ;
doi: 10.4049/jimmunol.175.1.286
http://www.jimmunol.org/content/175/1/286
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References
The Journal of Immunology
TLR-Dependent Activation Stimuli Associated with Th1
Responses Confer NK Cell Stimulatory Capacity to Mouse
Dendritic Cells1
Ivan Zanoni, Maria Foti, Paola Ricciardi-Castagnoli, and Francesca Granucci2
I
nnate immunity is the most ancient line of defense against
microbial infection. It is present and conserved in all animals
throughout evolution (1). Among the cells that are involved
in innate responses, dendritic cells (DCs)3 are able to perceive the
environment and to alert the innate immune system to the presence
of invading microorganisms. During the very early phases after
microbial recognition, DCs acquire the ability to regulate the functions of NK cells (2–7) that potently contribute to infection
eradication by activating a strong inflammatory response and by
exerting the cytotoxic function (8). In the mouse system, only early-stimulated, and not resting, immature myeloid DCs can elicit
inflammatory cytokine production, such as IFN-␥, from NK (7, 9)
cells, whereas immature human myeloid DCs that can prime NK
cells though activated DCs are much more efficient (4). Many different stimuli can induce myeloid DCs activation in both mice and
humans. These stimuli can be generically divided into stimuli that
induce full DC maturation and stimuli that induce a semimature
DC state. Full maturation stimuli are microbial stimuli and inflammatory products, such as PGs (10). Among the microbial stimuli,
some bind TLRs, and others act in a TLR-independent manner.
Ten TLRs have been identified to date, and they recognize constitutive and conserved microbial structures that are absent in host
mammalian cells called microbial-associated molecular patterns
(11). In particular, TLR1, TLR2, and TLR6 interact with peptidoglycan, zymosan, and other microbial products (12, 13). TLR3
binds dsRNA (14), TLR4 binds LPS (15), TLR5 binds flagellin
(16), TLR7 and TLR8 bind imidazoquinolines and ssRNA (17–
19), and TLR9 binds unmethylated CpG DNA (20). Microbial
products that activate DCs in a TLR-independent manner are represented by toxins, such as pertussis toxin (PT) and cholera toxin
(CT) (21, 22). The semimaturation stimuli described to date are
two cytokines, TNF-␣ and IL-4 (9, 23, 24). DCs exposed to TNF-␣
do not become fully mature DCs (23), but have the capacity to
induce the differentiation of regulatory T cells (24), whereas IL4-stimulated DCs, although not able to prime naive T cells, are
able to trigger NK cell functions (9).
Focusing on NK cells, two pathways for DC-mediated NK cell
activation have been described in the mouse. One is dependent on
IL-4, and the other is dependent on microbial stimuli (7, 9). Therefore, an appropriate cytokine milieu containing IL-4 renders DCs
competent for NK cell activation independently of the presence of
microbial stimuli; alternatively, after microbial encounter (Gramnegative bacteria have been tested to date), DCs become able to
efficiently activate NK cells. In this case, one of the mediators
produced by bacterially activated mouse DCs and required to elicit
IFN-␥ from NK cells is IL-2 (7).
In the present work we have investigated the nature of the stimuli that make DCs capable of activating NK cells. In particular, we
have studied whether TLR-dependent and -independent microbial
stimuli and other full maturation stimuli are equally efficient in
rendering DCs able to elicit IFN-␥ production from NK cells and
to recruit NK cells at the draining lymph node.
Materials and Methods
Mice and reagents
Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milan,
Italy
Received for publication December 29, 2004. Accepted for publication April
19, 2005.
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 fellowships and grants from the Italian Ministry of
Education and Research (FIRB and COFIN projects) and the Italian Association
Against Cancer.
2
Address correspondence and reprint requests to Dr. Francesca Granucci, Department
of Biotechnology and Bioscience, University of Milano-Bicocca, Piazza della Scienza
2, Milan, Italy. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; BCG, bacillus CalmetteGuérin; CT, cholera toxin; MOI, multiplicity of infection; PT, pertussis toxin.
Copyright © 2005 by The American Association of Immunologists, Inc.
C57BL/6 and BALB/c mice were purchased from Harlan Italy. All animals
were housed under pathogen-free conditions. For FACS analysis and cell
purifications, mAbs were purchased from BD Pharmingen. The IFN-␥ and
IL-2 Duo Elisa kit (R&D Systems) were used to measure IFN-␥ and IL-2
in in vitro assays. Quantikine immunoassays (R&D Systems) for CXCL9
and CXCL10 were used to quantify chemokine production by DCs. The
stimuli used were Escherichia coli LPS (Sigma-Aldrich; 10 ␮g/ml), CpG
oligonucleotides (Primm; 1 ␮M), Pam3Cys (EMC Microcollections; 10
␮g/ml), endotoxin free (endotoxin concentration in the stock solution,
⬍0.0007 ng/ml according to the Limulus test) CT (List Biological Laboratory; 1 ␮g/ml), PGE2 (Sigma-Aldrich; 1 ␮M), bacillus Calmette-Guérin
(BCG; Sigma-Aldrich; multiplicity of infection (MOI), 10), Leishmania
mexicana promasitigote (Sigma-Aldrich; MOI, 5), and PT (Sigma-Aldrich;
1 ␮g/ml). Before use, PT was purified on endotoxin removal columns
(Detoxi-Gel; Pierce).
0022-1767/05/$02.00
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Dendritic cells (DCs) have an important role in the activation of NK cells that exert direct antitumor and antimicrobial effects and
can influence the development of adaptive T cell responses. DCs acquire NK cell stimulatory capacity after exposure to various
stimuli. In this study we investigated the nature of the stimuli that confer to DCs the NK cell-activating capacity. After exposure
of DCs to TLR-dependent and -independent microbial stimuli and to nonmicrobial stimuli, we evaluated the ability of activated
DCs to elicit IFN-␥ production from NK cells in vitro and to promote NK cell activation in vivo. We show in this study that only
TLR-dependent microbial stimuli typically associated with Th1 responses confer to DCs the ability to activate NK cells, whereas
stimuli associated with Th2 responses do not have this property. The Journal of Immunology, 2005, 175: 286 –292.
The Journal of Immunology
The studies were reviewed and approved by an appropriate institutional
review committee.
BMDC preparation
Bone marrow cells from C57BL/6 or BALB/c mice were cultured in
IMDM (Euroclone) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 50 ␮M 2-ME (all from Sigma-Aldrich),
10% heat-inactivated FBS (IMDM complete medium), and 10% supernatant
of GM-CSF-transduced B16 tumor cells (25). Fresh medium was added every
3 days. After 7–10 days of culture, cells were analyzed for CD11c expression
and were used in assays when ⬎90% were CD11c positive.
NK cell purification
NK cells were positively selected from splenocytes of BALB/c and
C57BL/6 mice. Cells (108) were stained with biotinylated anti-pan-NK
cell (DX5) Ab (20 ␮g/ml), washed, and incubated with streptavidin
MicroBeads (Miltenyi Biotec). Cells were then positively selected with
Mini-MACS separation columns (Miltenyi Biotec), according to the manufacturer’s recommendations. NK cells were used when ⬎96% were DX5
positive.
Splenic DC purification
DC-NK cell cocultures
DCs were resuspended in complete IMDM without antibiotics, plated in
48-well plates (1.25 ⫻ 105 cells/well), and treated with various stimuli. In
the experiments performed with microorganisms, DCs were treated with
BCG or L. mexicana, respectively, at MOI of 10 and of 5 for 1.5 h, washed
twice with PBS, and supplemented with complete IMDM containing 10%
GM-CSF supernatant, gentamicin (50 ␮g/ml; Sigma-Aldrich), and tetracyclin (30 ␮g/ml; Sigma-Aldrich). In some cases, activated DCs were cultured with rIL-2 (3 ng/ml), ascite-purified anti-IL-2 (S4B6 clone), or rat
IgG2a isotype control mAbs (5 ␮g/ml). After 0.5 h, NK cells (2.5 ⫻ 105
cells/well) were added directly to the culture; 18 h later, clarified supernatants were tested for IFN-␥ production. For the intracellular staining, cells
were incubated with brefeldin A (10 ␮g/ml; Sigma-Aldrich) for 4 h after 4 h
of coculture. Cells were fixed with 2% paraformaldehyde, permeabilized with
PBS containing 5% FBS and 0.5% saponin, and stained with anti-IFN-␥, antiCD11c and anti-DX5 (BALB/c), or anti-NK1.1 (C57BL/6) mAbs.
IL-2 production by activated DCs
IL-2 was measured by ELISA in the supernatants of 18-h activated DCs.
For the intracellular staining, DCs were incubated with brefeldin A (10
␮g/ml; Sigma-Aldrich) for 4 h after 2 h of activation. Cells were fixed with
2% paraformaldehyde, permeabilized with PBS containing 5% FBS and
0.5% saponin, and stained with anti-IL-2 and anti-CD11c mAbs.
In vivo NK cell recruitment and activation
Immature DCs and DCs (106) exposed for 2 h in vitro to various activation
stimuli were injected s.c. at the tail base. The number of NK cells in the
draining inguinal lymph nodes was enumerated 24 h later by FACS analysis. For intracellular staining, single cell suspensions of draining lymph
nodes were prepared and incubated with brefeldin A (10 ␮g/ml; SigmaAldrich), ionomycin (100 ng/ml; Sigma-Aldrich), and PMA (50 ng/ml;
Sigma-Aldrich) for 4 h. Cells were fixed with 2% paraformaldehyde, permeabilized with PBS containing 5% FBS and 0.5% saponin, and stained
with FITC-labeled anti-IFN-␥ mAb. In BALB/c mice, NK cells were identified by staining with DX5 and anti-asialo-GM1 Abs; in C57BL/6 mice,
NK cells were identified as NK1.1⫹CD3⫺ cells.
Results
Only DCs activated with TLR-dependent microbial stimuli are
able to induce NK cells to produce IFN-␥
The process of DC maturation depends on the nature of the stimulus. Indeed, DCs show diverse responses to different stimuli and
this might influence the outcome of the immune reaction. To investigate the stimuli that could render DCs capable of activating
NK cells, BALB/c and C57BL/6 bone marrow-derived DCs activated with diverse full-maturation stimuli were cocultured with
syngeneic NK cells. The ability of activated DCs to prime NK cells
was verified by testing the capacity of NK cells to produce IFN-␥.
Coculture supernatants were collected 18 h later, and IFN-␥ production by NK cells was measured. In this experiment we used full
maturation, TLR-dependent and -independent microbial stimuli
and full-maturation, nonmicrobial stimuli. The TLR-dependent
microbial stimuli used were: LPS (TLR4), CpG (TLR9), Pam3Cys
(TLR2) (26), BCG, and L. mexicana promastigote (27, 28). The
TLR-independent microbial stimuli were CT and PT. The nonmicrobial inflammatory stimulus we used was PGE2.
As shown in Fig. 1, only DCs activated by TLR-dependent microbial stimuli were able to induce IFN-␥ production by NK cells,
whereas DCs exposed to TLR-independent microbial stimuli or to
inflammatory nonmicrobial stimuli could not elicit IFN-␥ production from NK cells. The only exception was Pam3Cys, which conferred to DCs the ability to activate NK cells only in the context of
FIGURE 1. IFN-␥ production by NK cells cocultured with DCs stimulated with various activation stimuli. Immature DCs or DCs activated with the
indicated stimuli were cultured together with syngeneic NK cells for 18 h. Levels of IFN-␥ in the supernatants were then quantified by ELISA. Left panel,
DCs and NK cells on the BALB/c background; right panel, DCs and NK cells on the C57BL/6 background. DC⫹NK, DCs activated with the indicated
stimuli cocultured with NK cells; DC, DCs stimulated with the indicated stimuli without NK cells; NK, NK cells stimulated with the indicated stimuli
without DCs; iDC, immature DCs; iDC⫹NK, immature DCs cocultured with NK cells. The experiment was repeated three times with similar results. Insets
represent the intracellular staining performed in the mixed DC-NK populations after 4 h of coculture. DCs were activated with the indicated stimuli.
Cocultured cells were triple-stained with anti-IFN-␥, anti-CD11c, and anti-DX5 (for BALB/c cells) or anti-NK1.1 (for C57BL/6 cells) mAbs and analyzed
by FACS. NT, nonactivated DCs cocultured with NK cells.
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DCs were obtained from BALB/c and C57BL/6 mice by negatively selecting CD19⫹, CD3⫹, panV␤⫹, F4/80⫹, DX5⫹, and GR1⫹ cells using the
MicroBeads (Miltenyi Biotec) system. The resulting population was ⬎80%
CD11c⫹ and did not show any T or B cell contamination (data not shown).
287
288
DC-MEDIATED NK CELL ACTIVATION
BALB/c, but not C57BL/6, cells. To verify that IFN-␥ was actually
produced by NK cells, the presence of IFN-␥-positive NK cells
was confirmed by intracellular staining after 4 h of coculture (Fig.
1, insets). We detected no IFN-␥ production by CD11c⫹ cells (Fig.
1, insets).
IL-2 derived from DCs activated with TLR-dependent microbial
stimuli is required to elicit IFN-␥ production from NK cells
NK cell activation in vivo
It has been recently shown that during an inflammatory process,
NK cells can be recruited at the lymph nodes, where they play a
role in T cell priming. This process is regulated by IL-4 and LPSstimulated DCs that, when injected s.c., migrate to the draining
lymph nodes (9, 30) and recruit and activate NK cells (9, 31). NK
cell activation in the lymph nodes can be tested by measuring the
up-regulation of CD69 (9). We thus considered this phenomenon
as a parameter to investigate in vivo the type of stimuli that could
confer to DCs NK cell stimulatory capacity and involve them in
the immune response. To perform this experiment, DCs activated
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We have previously shown that, in the case of E. coli infections,
IL-2 produced early by bacterially activated mouse DCs is required for the activation of NK cell-mediated immunity in vitro
and in vivo (7). Thus, we tested whether the stimuli used in the
previous experiment could induce DCs to produce IL-2. The presence of IL-2 in the supernatants of activated DCs was measured
24 h after the stimulus encounter. In agreement with our previous
observation (7, 29), we found that only DCs activated with TLRdependent microbial stimuli were able to produce IL-2, whereas
DCs activated with all other stimuli did not demonstrate this property (Fig. 2A). To verify that IL-2 was actually produced by DCs
and not by bone marrow-contaminating cells, the presence of IL2-positive, CD11c-positive cells was confirmed by intracellular
staining 2 h after activation (Fig. 2A, lower panels).
The stimuli that could not induce IL-2 production by DCs could,
however, induce activation, as shown by the up-regulation of activation markers (Fig. 2B). The results were similar for BALB/c
and CB57BL/6 DCs as well as for DCs after incubation with NK
cells (data not shown).
To test whether DC-derived IL-2 was required to elicit IFN-␥
from NK cells, the effects of IL-2 secreted by activated DCs were
blocked in DC-NK cell cocultures using a blocking anti-IL-2 Ab.
As shown in Fig. 3A, blocking IL-2 completely inhibited the capacity of DCs to activate NK cells. Conversely, the addition of
exogenous rIL-2 to the DC-NK cell cocultures in which DCs were
activated with molecules unable to stimulate IL-2 production was
not sufficient to induce NK cell activation. The only exception was
Pam3Cys in the C57BL/6 background, suggesting that the inability
of C57BL/6 DCs activated with Pam3Cys to elicit IFN-␥ production from NK cells was due to their inability to produce IL-2 in this
context. Together, these data suggest that IL-2 is not the only factor required for IFN-␥ production by NK cells in DC-NK cell
interactions.
The requirement of DC-derived IL-2 for NK cell activation was
confirmed in freshly purified splenic CD11c⫹ DCs. In this experiment CD11c⫹ cells were obtained from spleens by negative selection, activated with TLR-dependent microbial stimuli, and
cocultured with syngeneic NK cells for 18 h in the presence or the
absence of IL-2-blocking Ab. As shown in Fig. 3B, IFN-␥ was
produced by NK cells in an IL-2-dependent manner. The lower
efficiency of NK cell activation observed under these conditions
could be due to the fact that most of the ex vivo splenic DCs were
already in a mature state and did not respond to additional stimulation (data not shown).
FIGURE 2. IL-2 production by DCs stimulated with various activation
stimuli and up-regulation of activation markers. A, DCs were activated
with the indicated stimuli. A, Upper panels, IL-2 levels were quantified by
ELISA in the supernatants of 18-h activated DCs. A, Lower panels, Intracellular staining performed on DCs activated with the indicated stimuli.
Nonactivated (NT) and activated DCs were double stained with anti-IL-2
and anti-CD11c Abs. B, The up-regulation of activation markers at the
surface of DCs stimulated with the indicated activation stimuli was measured by FACS analysis at 24 h. Thin lines indicate the expression levels
of activation markers on immature DCs; thick lines represent the expression levels of activation markers on mature DCs. A representative experiment of three is shown.
with different stimuli were injected s.c., and, 24 h later, the number
of NK cells in the draining lymph nodes was enumerated. We also
evaluated the percentage of activated, CD69⫹, NK cells. As shown
in Fig. 4, A and B, only TLR-dependent stimuli conferred to DCs
NK cell stimulatory capacity in vivo. In agreement with this observation, DCs exposed to these activation stimuli were able to
The Journal of Immunology
289
induce IFN-␥ production by NK cells, as indicated by the presence
of IFN-␥-positive NK cells in the draining lymph nodes (Fig. 4C).
As previously observed, Pam3Cys had an effect only in BALB/c,
not in C57BL/6, mice (Fig. 4).
In contrast, concerning NK cell recruitment/proliferation,
among the TLR-dependent stimuli, Pam3Cys did not show any
effect in BALB/c or C57BL/6 mice, whereas LPS and CpG were
very effective (Fig. 5A). Among the TLR-independent microbial
stimuli, PT, although unable to confer to DCs the ability to
activate NK cells, was efficient in making DCs capable of inducing recruitment/proliferation of NK cells at the draining
lymph nodes (Fig. 5A).
It has been shown that NK cell recruitment at the draining
lymph nodes is partially dependent on CXCR3 (31). Therefore, we
evaluated whether the various activation stimuli could induce different CXCR3 ligand production by mouse DCs. To this purpose,
CXCL9 and CXCL10 were measured in the supernatants of activated DCs 18 h after stimulation. Similar to the previous observation, LPS and CpG very efficiently induced the production of
both chemokines by DCs, whereas Pam3Cys, CT, and PGE2 did
not have any effect (Fig. 5B), suggesting that CXCR3L may be
involved in NK cell recruitment. In contrast, PT, although able to
induce NK cell accumulation, led to only a weak release of
CXCL10 (Fig. 5B), suggesting that in this case, NK cell enrichment at the draining lymph nodes might be due to different
mechanisms.
Discussion
We have shown in this study that TLR-dependent, and not TLRindependent, full-maturation stimuli confer to DCs the ability to
elicit IFN-␥ production by NK cells. Moreover, migrating DCs
activated with specific full maturation stimuli are able to recruit or
induce NK cell proliferation at the draining lymph nodes.
It has been recently observed that activated NK cells can provide in the lymph nodes an early source of IFN-␥ necessary for
subsequent polarization of T cell responses toward Th1 (31). The
activation of the Th1 program depends on the activity of the transcription factor T-bet, which is induced by TCR and IFN-␥R triggering (32). Consistent with this observation, the stimuli that we
found able to confer to DCs NK cell stimulatory capacity were
typically associated in vivo with Th1 responses. In particular, LPS
and CpG were able to induce DC-mediated NK cell activation
(IFN-␥ production) and recruitment in vitro and in vivo, whereas
the Th2 stimulus Pam3Cys, although it could stimulate DCs to
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FIGURE 3. DC-derived IL-2 is a key molecule for DC-mediated NK cell activation. Bone marrow-derived DCs (A) and freshly isolated splenic DCs
(B) activated with the indicated inflammatory stimuli were cultured together with syngeneic NK cells in the presence of anti-IL-2-blocking or isotype control
Abs or in the presence of exogenous rIL-2 (A). IFN-␥ in the supernatant was measured by ELISA after 18 h of coculture. Left panels, DCs and NK cells
on the BALB/c background; right panels, DCs and NK cells on the C57BL/6 background. A, A representative experiment of three is shown. B, The mean ⫾
SD of triplicate wells are shown.
290
DC-MEDIATED NK CELL ACTIVATION
elicit IFN-␥ production from NK cells in vitro in the BALB/c
background, was not able to promote in vivo DC-mediated NK cell
recruitment/proliferation at the draining lymph nodes. Because under steady state conditions, NK cells are present in very low numbers in mouse lymph nodes (9), their recruitment/proliferation and
activation could be relevant to have enough IFN-␥ for Th1
polarization.
PT has also been associated with Th1 responses observed during
Bordetella pertussis infections (33). In agreement with this, we
found that PT was able to induce DC-mediated NK cell recruitment/proliferation. Nevertheless, it was not able to confer to DCs
NK cell activation capacity in vitro and in vivo. Because we observed that this activity is attributable only to TLR-dependent, fullactivation stimuli, we could hypothesize that in Bordetella infections, Bordetella microbial-associated molecular patterns are
required for NK cell activation, and PT is necessary to optimize
NK cell recruitment. It has been shown that NK cell recruitment at
the draining lymph nodes is partially dependent on CXCR3 (31).
In line with this observation, we have found that DCs activated
with the microbial stimuli (LPS and CpG) able to guarantee NK
cell accumulation produce large amounts of two CXCR3Ls,
CXCL9 and CXCL10. This suggests that the accumulation of NK
cells at the draining lymph nodes might be due to DC-mediated,
CXCR3-dependent NK cell recruitment. Conversely, PT induces
very low CXCL9 and CXCL10 expression; this indicates that in
this case either other chemokines are involved in the NK cell recruitment process, or PT-matured DCs stimulate local NK cell
proliferation.
TLR-dependent, full-activation stimuli can make DCs able to
induce IFN-␥ production by NK cells. This function is dependent
on IL-2 secreted by activated DCs and other mediators produced in
this context. In fact, blocking the effects of IL-2 in DC-NK cocultures in presence of TLR-dependent, full-activation stimuli resulted in a lack of NK cell priming, whereas the addition of exogenous IL-2 in DC-NK cell cocultures when DCs were activated
with TLR-independent, full-activation stimuli was not sufficient to
elicit IFN-␥ secretion by NK cells. We have observed in E. coliactivated DCs (7) that ICOS ligand and CX3CR1 were not additional mediators. In agreement with a previous work, we can also
exclude the effect of costimulatory molecules, such as B7.1, B7.2,
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FIGURE 4. NK cell activation in the lymph nodes. A, CD69 up-regulation at the surface of NK cells in the draining lymph nodes 24 h after s.c. injection
of DCs activated with the stimuli described in the figure. In BALB/mice, lymph node cell suspensions were triple stained with anti-DX5, anti-asialo-GM1,
and anti CD69 Abs; in C57BL/6 mice, activated NK cells were identified as NK1.1⫹CD3⫺CD69⫹. A representative experiment of three is shown. B,
Quantification of the percentage of CD69⫹ NK cells in BALB/c and C57BL/6 lymph nodes. Data represent the mean ⫾ SD from six mice. C, Intracellular
IFN-␥ expression by DX5⫹asialo-GM1⫹ (BALB/c) or NK1.1⫹CD3⫺ (C57BL/6) NK cells recovered from DC-draining lymph nodes. DCs were activated
with the indicated stimuli. NT, nonactivated DCs. The percentages of IFN-␥-producing cells are indicated.
The Journal of Immunology
and CD40, in inducing IFN-␥ production by NK cells (34). These
molecules are, indeed, efficiently up-regulated at the DC surface in
the presence of all full-activation stimuli we used, but TLR-independent stimuli did not confer to DCs the ability to activate NK
cells, even in the presence of exogenous IL-2.
Other cytokines, such as IL-12 and IL-15, have been shown to
be involved in NK cell activation (35, 36). In our system we can
exclude a role for IL-15, because IL-15-deficient DCs activated
with LPS and CpG are able to induce efficient IFN-␥ production by
NK cells in an IL-2-dependent manner (data not shown). Moreover, concerning IL-12, mouse and human DCs efficiently secrete
bioactive IL-12 only if exposed to IL-4 (37). As mentioned in the
introduction, two pathways for DC-mediated NK cell activation
have been described: one dependent on IL-4, and the other dependent on microbial stimuli (7, 9). IL-4 is, indeed, a DC semimaturation stimulus, and DCs exposed to IL-4 acquire the ability to
activate NK cells independently from the presence of full-activation microbial stimuli and IL-2, although the presence of microbial
stimuli increases the efficiency of this process (4). In addition, the
exposure of DCs to IL-4 inhibits microbial-induced IL-2 production (38). Thus, it is possible that IL-12 contributes to DC-medi-
ated NK cell activation when IL-4 and microbial stimuli are involved. Moreover, in the experiment mentioned above, showing
the involvement of IL-15, DCs were exposed to IL-4 (36). This
could explain the discrepancy with our results and may suggest
that, as in the case of IL-12, IL-15 is also involved in the IL-4dependent pathway. In this regard, the residual NK cell activation
that we observed with freshly isolated splenic DCs in the presence
of blocking IL-2 Ab (Fig. 3B) could be due to previous exposure
of some of the purified DCs to IL-4.
Consistent with the observation that the TLR-dependent, fullactivation stimuli able to render DCs efficient stimulators of NK
cells are stimuli associated with Th1 responses, L. mexicana-activated DCs could not elicit IFN-␥ production from NK cells. Infections with L. mexicana have been associated with the development of Th2 responses in many mouse strains (39), in contrast to
L. major infections that lead to the development of a protective
Th1 response (40). In the context of L. major infections, the development of a Th1 response could be due to the activation of NK
cells in lymph nodes. In fact, it has been shown that the IFN-␥
required to activate protective CD4⫹ T cells in response to L.
major is produced by NK cells (41).
Besides L. mexicana, the other Th2-associated, TLR-independent, inflammatory stimuli we used, CT and PGE2, could not confer to DCs the ability to activate and recruit NK cells at the draining lymph node.
The activation of NK cells is important during microbial (bacterial) infection not only to influence subsequent T cell responses,
but also to provide a direct early antimicrobial effect exerted by
IFN-␥ that potently activates phagocytes (4, 42). This is consistent
with the activity of the TLR-dependent activation stimuli described in this study.
Direct involvement of NK cells has been also demonstrated in
antitumor responses in different experimental systems (43), and the
role of NK has been unequivocally observed in patients with cancer (44). The ability of TLR-dependent microbial stimuli to render
DCs capable of activating NK cells may explain the efficacy of
bacterially based antitumor therapies. In clinical treatments of
bladder cancer, BCG therapy is considered the most effective immunotherapy to date (45). Moreover, other than its application in
nonspecific immunotherapy for cancer, BCG has also been used as
an immune adjuvant for active specific immunotherapy in tumor
vaccines in melanoma patients with measurable clinical responses
(46, 47). The BCG therapy has often been associated with the
activation of NK cells (45, 48). Because both mouse and human
DCs activated with BCG are very efficient in activating NK cells
(4), the antitumor NK cell responses observed in vivo in BCG
treatments could be DC mediated.
In conclusion, various stimuli with different adjuvant effects
have specific consequences on the activation of DCs and on their
subsequent influence on innate NK responses. Given the direct
involvement of NK cells in antitumor and antimicrobial functions
and their role in determining Th1 immunity, adjuvant choice in
DC-based therapies should be performed taking into account the
effect on NK cells, because this could influence the final outcome
of the clinical response.
Acknowledgments
We thank N. Winter for supplying BCG and T. Aebischer for providing
L. mexicana.
Disclosures
The authors have no financial conflict of interest.
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FIGURE 5. NK cell recruitment/proliferation in the lymph nodes. A,
Absolute numbers of NK cells in the draining lymph nodes 24 h after s.c.
injection of DCs activated with the indicated stimuli. NK cells were identified by double staining with anti-DX5 and anti-asialo-GM1 Abs in
BALB/c mice and with anti-NK1.1 and anti-CD3 Abs in C57BL/6 animals.
A representative experiment of three is shown. NT, untreated mice. B,
CXCL9 and CXCL10 production by DCs. Supernatants of DCs 18 h after
activation with the indicated stimuli were tested by ELISA for the presence
of CXCL9 and CXCL10.
291
292
DC-MEDIATED NK CELL ACTIVATION
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