Cutting Edge: Priming of NK Cells by IL-181
Julie Chaix,* Marlowe S. Tessmer,†Kasper Hoebe,‡Nicolas Fuse ´ri,* Bernhard Ryffel,§
Marc Dalod,* Lena Alexopoulou,* Bruce Beutler,¶Laurent Brossay,†Eric Vivier,2*?
and Thierry Walzer2*
Recent evidence suggests that NK cells require priming
to display full effector activity. In this study, we dem-
onstrate that IL-18 contributed to this phenomenon.
IL-18 signaling-deficient NK cells were found to be
unable to secrete IFN-? in response to ex vivo stim-
ulation with IL-12. This was not due to a costimula-
tory role of IL-18, because blocking IL-18 signaling
during the ex vivo stimulation with IL-12 did not al-
ter IFN-? production by wild-type NK cells. Rather,
we demonstrate that IL-18 primes NK cells in vivo to
produce IFN-? upon subsequent stimulation with IL-
12. Importantly, IL-12-induced IFN-? transcription
by NK cells was comparable in IL-18 signaling-defi-
cient and -sufficient NK cells. This suggests that
priming by IL-18 leads to an improved translation of
IFN-? mRNA. These results reveal a novel type of
cooperation between IL-12 and IL-18 that requires
the sequential action of these cytokines. The Journal
of Immunology, 2008, 181: 1627–1631.
and the secretion of IFN-? (1, 2). Recent evidence has shown
that, similar to other lymphocytes, NK cells need to be primed
encounter with activated dendritic cells (DC)3in lymphoid or-
Accordingly, NK cells isolated from IL-15-deficient mice dis-
study, we aimed at identifying other molecules involved in NK
atural killer cells are lymphocytes of the innate im-
mune system that play a role in the protection against
pathogens and tumors by means of cell cytotoxicity
cell priming using a candidate gene approach. We focused our
attention on MyD88, a crucial integrator of innate immune re-
of the IL-1 receptor family.
Materials and Methods
Inbred mice were purchased from Charles River. All mice were on a C57BL/6
background. Polyinosinic:polycytidylic acid (poly(I:C); Invivogen) was in-
NK cell culture
NK cells were enriched by negative selection as described (6). They were cul-
NKp46; clone 4E5, anti-Ly49D) and anti-CD107a/GolgiStop (BD Bio-
sciences). IL-2 (3,000 U/ml; Peprotech), IL-12 (20 ng/ml), IL-18 (5 ng/ml;
R&D Systems), anti-IL-18 (20 ?g/ml; MBL International), and anti-IL-18R
(LAK) generation, splenocytes were cultured in medium with recombinant hu-
Systems). In some experiments, NK cells were cultured for 4 h with IL-18 (5
ng/ml), washed three times, and then stimulated as described above.
RNA was extracted with the RNeasy micro kit (Qiagen). Reverse transcriptase
(Invitrogen) was used to generate cDNA. PCR was conducted with a SYBR
Green-based kit (Qiagen) using the following primers: mouse Hprt 5?, GCC
CCAAAATGGTTAAGGTT; mouse Hprt 3?, TTGCGCTCATCTTAGG
CTTT, mouse IFN-? 5?, GAACTGGCAAAAGGATGGTGA; and mouse
IFN-? 3?, TGTGGGTTGTTGACCTCAAAC.
software (Cytel Studio). Two-sided p values are shown as follows: ?, p ? 0.05;
??, p ? 0.001; ???, p ? 0.0001.
*Centre d’Immunologie de Marseille-Luminy, Universite ´ de la Me ´diterrane ´e, Institut Na-
tional de la Sante ´ et de la Recherche Me ´dicale Unite ´, Centre National de la Recherche
Scientifique Unite ´ Mixte de Recherche 6102, Case 906, Campus de Luminy, Marseille,
France;†Department of Molecular Microbiology and Immunology, Division of Biology
and Medicine, Brown University, Providence, RI 02912;‡Cincinnati Children’s Hospital
Research Foundation, University of Cincinnati College of Medicine, Division of Molec-
la Recherche Scientifique Unite ´ Mixte de Recherche 6218, Orle ´ans, France;¶Department
of Immunology, Scripps Research Institute, La Jolla, CA 92037; and?Ho ˆpital de la Con-
ception, Assistance Publique, Ho ˆpitaux de Marseille, Marseille, France
Received for publication February 25, 2008. Accepted for publication June 4, 2008.
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.
1TheE.V.laboratoryissupportedbyEuropeanUnion(? ?Allostem? ?),LigueNationale
contre le Cancer (“Equipe labelise ´e”), Agence Nationale de la Recherche, INSERM,
CNRS, Ministe `re de l’Enseignement Supe ´rieur et de la Recherche, and Institut Universi-
taire de France. This work was also supported by National Institutes of Health Grant
2Address correspondence and reprint requests to Dr. Eric Vivier and Dr. Thierry Walzer,
Centre d’Immunologie de Marseille-Luminy, Universite ´ de la Me ´diterrane ´e, Institut Na-
tional de la Sante ´ et de la Recherche Me ´dicale Unite ´, Centre National de la Recherche
Scientifique Unite ´ Mixte de Recherche 6102, Case 906, Campus de Luminy, Marseille
13288, France. E-mail addresses: firstname.lastname@example.org and email@example.com
3Abbreviations used in this paper: DC, dendritic cell; IRAK4, IL-1R-associated kinase 4;
KO, knockout; LAK, lymphokine-activated killer; poly(I:C), polyinosinic:polycytidylic
acid; TIR, TLR/IL-1R.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
Results and Discussion
MyD88 is required for IFN-? production by NK cells in response to
We first compared the function of NK cells isolated from un-
challenged wild-type and MyD88 KO mice. For this purpose,
stimulated for 4 h with YAC-1 target cells or cytokines or with
mAb specific to the activating NK cell receptors Ly49D and
NKp46. IFN-? secretion and cytotoxic granule release (which
is proportional to surface CD107a exposure; Ref. 7) were mea-
sured. MyD88 KO NK cells produced four times less IFN-?
than wild-type NK cells in response to stimulation with IL-12
or IL-2 plus IL-12 (Fig. 1, A–C). This was true over a wide
range of IL-12 concentrations (data not shown). Similar results
were also obtained using ELISA and for longer periods of stim-
ulation (Fig. 1D). By contrast, both cell types produced similar
levels of IFN-? in response to all other stimuli tested, i.e., IL-2,
B and data not shown). Wild-type and MyD88 KO NK cells
were equally potent in their degranulation response to YAC1
cells or to Ly49D or NKp46 cross-linking (Fig. 1, B and C).
be normal in MyD88 KO mice (data not shown). Unexpect-
edly, MyD88 KO NK cells produced similar levels of IFN-?
cell IFN-? production induced by IL-12 at the posttranscrip-
We then tested whether MyD88 functioned in a way intrin-
sic to NK cells. We reconstituted irradiated recipient mice with
a 1:1 mixture of wild-type CD45.1?and MyD88-deficient
CD45.1?bone marrow cells. The NK cell response was as-
sessed 8 wk after transplantation. MyD88 KO NK cells dis-
played reduced IFN-? production in response to IL-12 or IL-2
NK cells (Fig. 2A). Their response was otherwise similar to that
of wild-type NK cells (Fig. 2A). These results indicate that
MyD88 has a cell autonomous function in IL-12-induced pro-
duction of IFN-? by NK cells. Next, we analyzed the response
of NK cells in other mutants of the IL-1/TLR/MyD88 path-
way. MyD88 associates with receptors of the TLR and IL-1R
families through a homotypic interaction of their respective
TLR/IL-1R (TIR) domains (8). The Pococurante point muta-
tion in the mouse MyD88 TIR domain abrogates this interac-
IL-1R-associated kinase 4 (IRAK4) (10). We found that both
Pococurante and IRAK4 KO mice phenocopied MyD88 KO
the NK cell response to IL-12 requires the association of
MyD88 with a TIR domain-containing receptor signaling
through the canonical MyD88/IRAK4 signaling pathway.
IL-18 signaling is required for optimal IL-12-induced IFN-?
production by NK cells
We sought to determine which MyD88-coupled receptor(s)
were involved in the NK cell response to IL-12. NK cells iso-
lated from TLR1, TLR2, TLR3, TLR4, TLR 5, TLR7, or
TLR9 knockout mice responded normally to stimulation with
also responded normally to stimulation with IL-12 (data not
sponse to IL-12. NK cells from wild-type (WT) and MyD88 KO mice were
stimulated under the indicated conditions. A–C, The expression of NK1.1,
CD3, CD107a, and IFN-? were measured. A, Representative FACS anal-
ysis of NK1.1 and IFN-? expression by gated CD3?cells. Cytokine stim-
ulation did not induce CD107a surface exposure (data not shown). B, Rep-
resentative FACS analysis of IFN-? expression and CD107a surface
exposure by gated NK1.1?CD3?cells. C, Mean production of IFN-? (left)
and CD107a surface exposure (right) by gated MyD88 NK cells, expressed
as the percentage of the wild-type value in each condition. Data are mean ?
SD of n ? 8 mice. D, IFN-? level was measured by ELISA in the culture
supernatant over time. Data are mean? SD for n ? 4 MyD88 KO mice and
n ? 3 WT mice. E, Detection of IFN-? transcripts as assessed by quanti-
tative RT-PCR upon 4 h of stimulation. The relative quantity of IFN-?
transcripts was determined by normalization to Hprt1 mRNA. Data are
mean ? SD of n ? 3.
MyD88 is required for IFN-? production by NK cells in re-
1628CUTTING EDGE: PRIMING OF NATURAL KILLER CELLS BY IL-18
shown). By contrast and similarly to MyD88 KO NK cells, IL-
18R KO NK cells responded poorly to IL-12 and IL-2 plus
IL-12 but normally to Ly49D and NKp46 cross-linking (Fig.
ex vivo IFN-? production by NK cells in response to IL-12. In
support of this conclusion, NK cells isolated from IL-18 KO
IL-12 and IL-18 are well-known to synergize in inducing
IFN-? production by NK cells when coadministered in vitro
culture of splenocytes with exogenous IL-12, endogenous pro-
duction of IL-18 can occur and subsequently costimulate IL-
12-induced IFN-? production (14). This phenomenon could
potentially explain why, in our experiments, NK cells deficient
for the IL-18 pathway were impaired for IFN-? production in
response to stimulation by exogenous IL-12. However, the ad-
dition of blocking mAbs to IL-18 and IL-18R or NF-?B inhib-
itors did not block the production of IFN-? by wild-type NK
cells induced by IL-12 or IL-2 plus IL-12 stimulation (Fig. 4
and data not shown). As a control, these reagents efficiently
blocked the production of IFN-? by wild-type NK cells in-
IL-12 during a 4-h in vitro assay but it is mandatory in vivo to
prime NK cells to respond to a subsequent stimulation with
In vitro or in vivo NK cell preactivation restores IL-12-induced IFN-?
production by MyD88 KO NK cells
observed in IL-18 signaling-deficient mice could be overcome
by prior in vivo or in vitro NK cell activation. We first cultured
WT and IL-18 KO NK cells with IL-18 before the stimulation
KO NK cells produced IFN-? as efficiently as WT NK cells in
response to both stimuli (Fig. 5A). Second, MyD88 KO and
WT mice were injected with poly(I:C) to activate NK cells in
vivo. IFN-? production by in vivo activated MyD88 KO NK
cells in response to IL-12 or IL-2/IL-12 was lower than that of
WT NK cells (Fig. 5B), but the difference was less than that
between resting WT and MyD88 KO NK cells (Fig. 1A).
Third, NK cells from MyD88 KO and WT mice were cultured
for 6 days in vitro with IL-2 to generate LAK. The response of
to IL-12. NK cells from mixed bone marrow chimera mice (wild-type CD45.1
with MyD88 KO or Pococurante or IRAK4 KO) were stimulated as indicated.
A, Mean production of IFN-? (left) and CD107a surface exposure (right) by
gated MyD88 KO NK cells, expressed as the percentage of the wild-type value
in each condition. Data are mean ? SD of n ? 8 mice. B, Mean production of
IFN-? (left) and CD107a surface exposure (right) by gated Pococurante or
IRAK4 KO NK cells, expressed as the percentage of the wild-type value in each
condition. Data are mean ? SD of n ? 4 mice.
MyD88 and IRAK4 have an intrinsic role in NK cell response
stimulated as indicated. The expression of NK1.1, CD3, and IFN-? were mea-
sured. Results show the mean production of IFN-? by gated NK cells from
IL-18R KO mice (A) or IL-18 KO mice (B). Results are expressed as the per-
centage of the wild-type value in each condition. Data are mean ? SD of n ?
10 mice for each genotype.
IL-18 signaling is required for IL-12-induced IFN-? produc-
with IL-12. NK cells isolated from wild-type mice were stimulated 4 h in vitro
as indicated in the presence or absence of anti-IL-18 Ab (A) or anti-IL-18R?
mAbs (B). Results show the mean production of IFN-? by gated NK cells, ex-
pressed as the percentage of the control (no antibody) value. Data are mean ?
SD of n ? 7 (A) or n ? 8 (B).
Endogenous IL-18 does not costimulate NK cells stimulated
1629 The Journal of Immunology
MyD88 KO LAK in response to IL-12 or IL-2/IL-12 was com-
parable to the response of WT LAK (Fig. 5C). Thus, the re-
sponsiveness of MyD88 KO NK cells to IL-12 can be restored
by in vitro activation with IL-2 and partially recovered by in
vivo activation with poly(I:C). Altogether, these results show
that the defective production of IFN-? by NK cells in IL-18
signaling-deficient mice is not due to a developmental defi-
ciency but rather to a defect of priming.
Our results reveal a novel type of cooperation between IL-12
and IL-18 that requires a sequential activity of these cytokines.
What is the molecular basis of this phenomenon? IL-12 and
IL-18 have been previously reported to act synergistically to in-
duce IFN-? secretion by T cells and NK cells (12, 13). Synergy
scription factors induced and by the cooperation between these
factors. For instance, IL-12-induced STAT4 up-regulates the
(15). However, we found that the IFN-? gene was transcribed
at a similar level in WT and MyD88 KO NK cells in response
to IL-12 stimulation. Accordingly, we found the following: 1)
IL-12R chains were expressed normally in MyD88 KO NK
cells at both the mRNA and protein levels; and 2) STAT4 was
stimulation with IL-12 (data not shown). These results suggest
an unexpected role of the IL-18 pathway in the posttranscrip-
tional regulation of IFN-? induced by IL-12. Posttranscrip-
tional regulation of IFN-? has been shown to occur at different
stages, from the localization of IFN-? mRNA (16) to the sta-
results do not favor a role for IL-18 in IFN-? mRNA stabiliza-
tion, as the level of IFN-? mRNA is similar in wild-type and
MyD88 KO NK cells stimulated with IL-12. They are, how-
ever, compatible with the two other possibilities. Interestingly,
Hodge et al. found that IL-12 induces IFN-? mRNA accumu-
lation in the nucleus of cells of the NK92 line. They also found
translation of this mRNA (16). Thus, it is possible that the
stimulation of NK cells with IL-12 induces a suboptimal pro-
duction of IFN-? by NK cells, because translation is limiting.
In this model, a prior stimulation by IL-18 could result in a
other NK cell effector functions such as cytotoxicity have been
forin and granzyme B mRNA are translated upon IL-15-medi-
ated priming (20). Thus, the control of mRNA translation
could be central in NK cell priming. Interestingly, although we
degranulate, others previously reported that NK cells isolated
from IL-18 KO mice had defective cytotoxicity against YAC1
cells (21). One possible explanation for this discrepancy could
be that the level of granule proteins are also limiting in IL-18
KO NK cells.
Another open question is the source of IL-18 in vivo. IL-18
has been shown to be produced at the basal level (14) by differ-
ent cell types including macrophages (22) and DC (23). Inter-
actions between macrophages or DC and NK cells have been
to be essential for IL-15-mediated NK cell priming (3). There-
fore, they could also potentially prime NK cells through IL-18
production. This could occur during NK cell development, as
bone marrow NK cells were found to produce IFN-? in re-
sponse to stimulation with IL-12 (24). The absence of one cy-
tokine could be partly compensated by the others during in-
flammation. Indeed, we found that in vitro stimulation with
IL-2 or in vivo stimulation with poly(I:C) restored at least par-
tially the ability of MyD88 KO NK cells to produce IFN-? in
response to stimulation with IL-12. Similarly, IL-18 deficiency
has been shown to be compensated by other pathways in the
production of IFN-? in the liver upon infection with mouse
CMV (25). Redundancy in different cytokine pathways might
thus contribute to the robustness of NK cell responses.
The authors have no financial conflict of interest.
1. Yokoyama, W. M. 2005. Natural killer cell immune responses. Immunol. Res. 32:
2. Vivier, E., E. Tomasello, M. Baratin, T. Walzer, and S. Ugolini. 2008. Functions of
natural killer cells. Nat. Immunol. 9: 503–510.
cells prime natural killer cells by trans-presenting interleukin 15. Immunity 26:
4. Nakazato, K., H. Yamada, T. Yajima, Y. Kagimoto, H. Kuwano, and Y. Yoshikai.
TCR?? intestinal intraepithelial T lymphocytes in IL-15-deficient mice. J. Immunol.
and J. P. Di Santo. 2005. Roles for common cytokine receptor ?-chain-dependent
peripheral NK cells in vivo. J. Immunol. 174: 1213–1221.
6. Walzer, T., L. Chiossone, J. Chaix, A. Calver, C. Carozzo, L. Garrigue-Antar,
Y. Jacques, M. Baratin, E. Tomasello, and E. Vivier. 2007. Natural killer cell traffick-
ing in vivo requires a dedicated sphingosine 1-phosphate receptor. Nat. Immunol. 8:
7. Alter, G., J. M. Malenfant, and M. Altfeld. 2004. CD107a as a functional marker for
the identification of natural killer cell activity. J. Immunol. Methods 294: 15–22.
8. Adachi, O., T. Kawai, K. Takeda, M. Matsumoto, H. Tsutsui, M. Sakagami,
K. Nakanishi, and S. Akira. 1998. Targeted disruption of the MyD88 gene results in
loss of IL-1- and IL- 18-mediated function. Immunity 9: 143–150.
9. Jiang, Z., P. Georgel, C. Li, J. Choe, K. Crozat, S. Rutschmann, X. Du, T. Bigby,
S. Mudd, S. Sovath, et al. 2006. Details of Toll-like receptor:adapter interaction re-
vealed by germ-line mutagenesis. Proc. Natl. Acad. Sci. USA 103: 10961–10966.
IFN-? production by MyD88 KO NK cells. A, NK cells from wild-type (WT)
and IL-18 KO mice were cultured for 4 h in vitro with IL-18, extensively
washed, and then stimulated for 4 h as indicated. The percentage of IFN-??
and MyD88 KO mice were stimulated for 4 h in vitro as indicated. The per-
centage of IFN-??NK cells was measured; n ? 4. C, LAK cells from wild-type
and MyD88 KO NK cells were stimulated for 4 h in vitro as indicated. The
percentage of IFN-??LAK cells was measured; n ? 4.
In vitro or in vivo NK cell preactivation restores IL-12-induced
1630CUTTING EDGE: PRIMING OF NATURAL KILLER CELLS BY IL-18
10. Suzuki, N., N. J. Chen, D. G. Millar, S. Suzuki, T. Horacek, H. Hara, D. Bouchard, Download full-text
K. Nakanishi, J. M. Penninger, P. S. Ohashi, and W. C. Yeh. 2003. IL-1 receptor-
associated kinase 4 is essential for IL-18-mediated NK and Th1 cell responses. J. Im-
munol. 170: 4031–4035.
11. Tabeta, K., K. Hoebe, E. M. Janssen, X. Du, P. Georgel, K. Crozat, S. Mudd,
N. Mann, S. Sovath, J. Goode, et al. 2006. The Unc93b1 mutation 3d disrupts ex-
ogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9. Nat.
Immunol. 7: 156–164.
12. Robinson, D., K. Shibuya, A. Mui, F. Zonin, E. Murphy, T. Sana, S. B. Hartley,
S. Menon, R. Kastelein, F. Bazan, and A. O’Garra. 1997. IGIF does not drive Th1
development but synergizes with IL-12 for interferon-? production and activates
IRAK and NF?B. Immunity 7: 571–581.
13. Shibuya, K., D. Robinson, F. Zonin, S. B. Hartley, S. E. Macatonia, C. Somoza,
C. A. Hunter, K. M. Murphy, and A. O’Garra. 1998. IL-1? and TNF-? are required
for IL-12-induced development of Th1 cells producing high levels of IFN-? in
BALB/c but not C57BL/6 mice. J. Immunol. 160: 1708–1716.
14. Fantuzzi, G., D. A. Reed, and C. A. Dinarello. 1999. IL-12-induced IFN-? is depen-
dent on caspase-1 processing of the IL-18 precursor. J. Clin. Invest. 104: 761–767.
15. Nakahira, M., H. J. Ahn, W. R. Park, P. Gao, M. Tomura, C. S. Park, T. Hamaoka,
gene expression: IL-12-induced STAT4 contributes to IFN-? promoter activation by
up-regulating the binding activity of IL-18-induced activator protein 1. J. Immunol.
16. Hodge, D. L., A. Martinez, J. G. Julias, L. S. Taylor, and H. A. Young. 2002. Regu-
lation of nuclear ? interferon gene expression by interleukin 12 (IL-12) and IL-2 rep-
resents a novel form of posttranscriptional control. Mol. Cell Biol. 22: 1742–1753.
and molecular mechanisms of IFN-? production induced by IL-2 and IL-12 in a hu-
man NK cell line J. Leukocyte Biol. 58: 225–233.
18. Ben-Asouli, Y., Y. Banai, Y. Pel-Or, A. Shir, and R. Kaempfer. 2002. Human inter-
feron-? mRNA autoregulates its translation through a pseudoknot that activates the
interferon-inducible protein kinase PKR. Cell 108: 221–232.
19. Khabar, K. S., and H. A. Young. 2007. Post-transcriptional control of the interferon
system. Biochimie 89: 761–769.
20. Fehniger, T. A., S. F. Cai, X. Cao, A. J. Bredemeyer, R. M. Presti, A. R. French, and
T. J. Ley. 2007. Acquisition of murine NK cell cytotoxicity requires the translation of
a pre-existing pool of granzyme B and perforin mRNAs. Immunity 26: 798–811.
21. Takeda, K., H. Tsutsui, T. Yoshimoto, O. Adachi, N. Yoshida, T. Kishimoto,
H. Okamura, K. Nakanishi, and S. Akira. 1998. Defective NK cell activity and Th1
response in IL-18-deficient mice. Immunity 8: 383–390.
22. Dinarello, C. A. 1999. Interleukin-18. Methods 19: 121–132.
23. Andrews, D. M., A. A. Scalzo, W. M. Yokoyama, M. J. Smyth, and
M. A. Degli-Esposti. 2003. Functional interactions between dendritic cells and NK
cells during viral infection. Nat. Immunol. 4: 175–181.
24. Kim, S., K. Iizuka, H. S. Kang, A. Dokun, A. R. French, S. Greco, and
W. M. Yokoyama. 2002. In vivo developmental stages in murine natural killer cell
maturation. Nat. Immunol. 3: 523–528.
selective IL-18 requirements for induction of compartmental IFN-? responses during
viral infection. J. Immunol. 165: 4787–4791.
1631 The Journal of Immunology