The Natural Cytotoxicity Receptor NKp46 Is Dispensable for
IL-22-Mediated Innate Intestinal Immune Defense against
Naoko Satoh-Takayama,*†Laure Dumoutier,‡Sarah Lesjean-Pottier,*†Vera S. G. Ribeiro,*†
Ofer Mandelboim,§Jean-Christophe Renauld,‡Christian A. J. Vosshenrich,*†
and James P. Di Santo2*†
Natural cytotoxicity receptors (including NKp30, NKp44, and NKp46 in humans and NKp46 in mice) are type I transmembrane
proteins that signal NK cell activation via ITAM-containing adapter proteins in response to stress- and pathogen-induced ligands.
Although murine NKp46 expression (encoded by Ncr1) was thought to be predominantly restricted to NK cells, the identification
of distinct intestinal NKp46?cell subsets that express the transcription factor Rorc and produce IL-22 suggests a broader function
for NKp46 that could involve intestinal homeostasis and immune defense. Using mice carrying a GFP-modified Ncr1 allele, we
found normal numbers of gut CD3?GFP?cells with a similar cell surface phenotype and subset distribution in the absence of
Ncr1. Splenic and intestinal CD3?NKp46?cell subsets showed distinct patterns of cytokine secretion (IFN-?, IL-22) following
activation via NK1.1, NKp46, IL-12 plus IL-18, or IL-23. However, IL-22 production was sharply restricted to intestinal
CD3?GFP?cells with the CD127?NK1.1?phenotype and could be induced in an Ncr1-independent fashion. Because NKp46
ligands can trigger immune activation in the context of infectious pathogens, we assessed the response of wild-type and Ncr-1-
deficient Rag2?/?mice to the enteric pathogen Citrobacter rodentium. No differences in the survival or clinical score were observed
in C. rodentium-infected Rag2?/?mice lacking Ncr1, indicating that NKp46 plays a redundant role in the differentiation of
intestinal IL-22?cells that mediate innate defense against this pathogen. Our results provide further evidence for functional
heterogeneity in intestinal NKp46?cells that contrast with splenic NK cells. The Journal of Immunology, 2009, 183: 6579–6587.
Among the different activating receptors on NK cells, a family of
natural cytotoxicity receptors (NCRs)3has been identified that
play an important role in MHC class I/HLA-independent cytolysis
of target cells (reviewed in Ref. 2). Three NCRs (NKp30, NKp44,
and NKp46) are present in the human genome, while only NKp46
(encoded at the Ncr1 locus) is present in mice. NKp46 demon-
strates predominant NK cell-specific expression in several species
(3, 4), although a small TCR-?? cell population expressing NKp46
has been described in adult mice (5) and in IL-15- expanded hu-
man T cells (6). NKp46 lacks intrinsic signaling motifs and asso-
ciate with ITAM-bearing adaptor molecules (including CD3? and
iverse NK cell effector functions are tightly controlled
via a balance of signals delivered through a diverse set
of activating and inhibitory cell surface receptors (1).
Fc?RI?) to couple ligand binding to the intracellular signaling ma-
chinery (3, 7). Cross-linking NKp46 on mature human or mouse
NK cells with plate-bound Abs elicits cytokine release and en-
hances degranulation of cytotoxic effector molecules (4, 8). Sim-
ilar results have been obtained using anti-NKp30 and anti-NKp44
Abs, suggesting that engagement of NCRs by their respective li-
gands on target cells can modulate NK cell activity (9, 10). Ac-
cordingly, blockade of NCRs, alone or in combination reduces NK
cell cytotoxicity in vitro against susceptible targets (reviewed in
Several NCR ligands have been described, including influenza
virus hemagglutinin (11) and Sendai virus hemagglutinin/neur-
aminidase (12), that bind NKp44 on human NK cells and NKp46
in both mice and humans. More recently, NCR ligands were found
to be expressed by murine lymphoma and myeloma cell lines (13)
and in human primary nevi and melanomas (14, 15). Moreover, a
NKp44-Fc fusion protein binds to the surface of mycobacterium
(Mycobacterium bovis, Mycobacterium tuberculosis)-infected
cells and anti-NKp46 antisera partially inhibit lysis of M. tuber-
culosis-infected monocytes by NK cells, suggesting the existence
of NCR ligands on bacteria or bacterially infected cells (16). Al-
though the nature of certain NCR ligands remains obscure, studies
using NKp46-deficient mice demonstrated increased susceptibility
to lethal influenza infection (17) and aberrant tumor immunosur-
veillance (13), suggesting an important role for triggering NKp46
during innate immune defense.
Recently, several groups described NKp46 expression on a
novel innate cell subset in the murine intestine (18–20). Using
mice carrying a GFP reporter inserted into the Ncr1 locus, we were
able to identify CD3?GFP?cells from the intestinal lamina pro-
pria and intraepithelial compartments that differentially expressed
*Cytokines and Lymphoid Development Unit, Institut Pasteur, Paris, France;
†INSERM Unite ´ 668, Paris, France;‡Ludwig Institute for Cancer Research, Experi-
mental Medicine Unit, Universite Catholique de Louvain, Brussels, Belgium; and
§The Lautenberg Center for General and Tumor Immunology, Hebrew University-
Hadassah Medical School, Jerusalem, Israel
Received for publication June 19, 2009. Accepted for publication September 5, 2009.
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.
1This work was supported by grants to J.P.D. from the Institut Pasteur, INSERM, and
as an “Equipe Labelise ´” by the Ligue Nationale Contre le Cancer. N.S.-T. was the
recipient of fellowship from The Uehara Memorial Foundation, Japan.
2Address correspondence and reprint requests to Dr. James P. Di Santo, Cytokines
and Lymphoid Development Unit, Institut Pasteur, Paris, France, F-75724. E-mail
3Abbreviations used in this paper: NCR, natural cytotoxicity receptor; LTi, lymphoid
tissue inducer; LPL, lamina propria lymphocyte; WT, wild type.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
CD127 and NK1.1 (18). All intestinal NKp46?subsets developed
independently of the thymus and were found in recombinase-ac-
tivating gene 2 (Rag2)-deficient mice. A minor subset of gut
(NK1.1?CD127?) with expression of typical NK cell markers (in-
cluding NKG2D, DX5, CD122, CD11b, and Ly49 family mem-
bers). Surprisingly,a predominant
(CD127?NK1.1?) failed to express the above-cited NK markers
and, furthermore, lacked typical effector functions (perforin,
IFN-?) associated with mature NK cells (18).
Analyses of the gene expression profiles revealed the expression
of the transcription factor Rorc in subsets of lamina propria
CD3?NKp46?cells (18–20, 31). Rorc has been previously shown
to be essential for thymocyte survival, development of lymphoid
tissue inducer (LTi) cells and for the differentiation of polarized T
cells that express IL-17 family cytokines (21). Surprisingly, intes-
tinal NKp46?cells failed to express constitutive or inducible IL-
17A, although gut CD3?NKp46?cells expressing CD127 har-
bored IL-22 transcripts. Moreover, the homeostasis of these
NKp46?IL-22?cells required the presence of intestinal microflora
(18, 19) and an intact Rorc gene (18–20). These results identify
novel NKp46?cell subsets in the gut that appear hard-wired for
rapid IL-22 production in response to microbial signatures.
IL-22 expression by gut CD3?NKp46?cells appears to play a
role in immune defense against intestinal pathogens. Initial im-
mune control of the Gram-negative bacteria Citrobacter rodentium
infection is T and B cell independent and critically depends on
IL-22 (22). IL-22 is proposed to “cross-talk” with intestinal epi-
thelial cells and promote innate defense through the elaboration of
antimicrobial peptides (22, 23). Although earlier studies suggested
that innate IL-22 production under these conditions was derived
from CD11c?dendritic cells (22), we showed that mice lacking
intestinal CD3?NKp46?cells were unable to produce IL-22 and
rapidly succumbed to C. rodentium (18). These studies suggest
that IL-22-producing NKp46?cells play an important role in in-
testinal homeostasis and in the early innate response that provides
protection against pathogens.
Cytokine production by NK cells is controlled by cell surface
ligands that trigger NK cell receptors as well as by soluble factors
elaborated in the local microenvironment (24). Little is known
about the mechanisms that control IL-22 production from
CD3?NKp46?cells in the gut. Signaling through NKp46 could
impact on the development, differentiation, anatomical localiza-
tion, homeostasis, and/or activation of intestinal IL-22?cells. In
this study, using Ncr1GFP/?reporter mice, we examine the devel-
opment and differentiation of intestinal IL-22?cell subsets in the
absence of Ncr1 and assess the capacity of Ncr1-deficient Rag2?/?
mice to resist early infection by C. rodentium.
Materials and Methods
C57BL/6 mice were purchased from Janvier. Ncr1GFP/?mice on the
C57BL/6 background (17) were bred to homozygosity to create Ncr1-de-
ficient mice (Ncr1GFP/GFP) and with Rag2-deficient mice on the C57BL/6
background to generate Rag2?/?Ncr1GFP/?and Rag2?/?Ncr1GFP/GFP
mice. Rag2?/?Il2rb?/?and Rag2?/?Il2rg?/?mice on the C57BL/6 back-
ground have been previously described (18). Il22?/?mice on the C57BL/6
background have been previously reported (25). All mice were housed
under specific pathogen-free conditions at the Laboratory Animal Facilities
of the Institut Pasteur. Mice were used for experiments at 6–12 wk of age,
and experiments were conducted following the guidelines provided by the
Animal Care and Use Committee of the Institut Pasteur and in accordance
with French law.
Cell isolation and flow cytometric analysis
Total splenocytes were prepared by gentle dissociation using mesh filters fol-
lowed by erythrocyte lysis using NH4Cl. Intestinal Peyer’s patches were re-
moved and intestinal epithelial cells eliminated by incubation in 0.5 mM
EDTA. Subsequently, lamina propria lymphocytes (LPL) were isolated using
0.5 mg/ml collagenase (Sigma-Aldrich) and were further purified using
discontinuous Percoll gradients (40 and 75%). To analyze cell pheno-
type, FITC- or PE-conjugated NKp46 mAb (29A1.4; eBioscience), anti-
CD3 (eBioscience), anti-NK1.1 (BD Pharmingen), anti-CD127 (BD
Pharmingen), anti-CD94 (Serotec), anti-2B4 (BD Pharmingen), anti-
Ly49A/D (BD Pharmingen), anti-CD11b (eBioscience), anti-CD122
(eBioscience), and anti-NKG2D (BD Pharmingen) were used as previously
described (18). Purified polyclonal goat Abs against mouse NKp46 were
available from R&D Systems. All washings and reagent dilutions were
made with PBS containing 2% FCS. A Canto II flow cytometer interfaced
to the FACSDiva software (BD Biosciences) was used to acquire data that
was analyzed using FlowJo software Tree Star).
In vitro cytokine stimulation and intracellular staining
Cross-linking Abs (NK1.1 clone PK135, BD PharMingen; 2B4, BD Pharm-
ingen; NKp46 clone 29A1.4, a gift from E. Vivier, CIML, Marseille, France)
was used to coat 96-well tissue culture plates for 4 h at 37°C. After three
washes with PBS, total splenocyte or LPL preparations diluted in RPMI 1640
(BD Pharmingen). In some cases, cells were left unstimulated or stimulated
with cytokines (IL-12 at 5 ng/ml plus IL-18 at 100 ng/ml; IL-23 at 40 ng/ml)
for 6 h. After surface staining and incubation with LIVE/DEAD Fixable re-
agent (Invitrogen), cells were fixed in 4% paraformaldehyde and processed for
intracellular cytokine detection. Cells were stained with PE-conjugated anti-
IFN-? (BD Pharmingen) and Alexa Fluor 647- labeled anti-IL-22 mAb
(MH22B2.2; Ref. 26) in permeabilization buffer (0.1% saponin in HBSS) be-
fore extensive washing in PBS and analysis. For ROR?t detection, an anti-
mouse/human ROR?t-PE (clone AFKJS-9; eBioscience) was used with fixa-
tion and permeabilization (eBioscience).
Immunohistology was performed as previously described (18, 27). Tissues
(lymph node and small intestine) were fixed in 4% paraformaldehyde over-
night at 4°C, equilibrated with 30% sucrose in PBS, before extensive di-
alysis against PBS. Rabbit anti-GFP mAb (Invitrogen) was used for GFP
detection followed by an anti-rabbit Alexa Fluor 488 mAb (Invitrogen).
Sections were stained with PE-conjugated anti-B220 mAb (BD Pharmin-
gen). To detect CD3 expression, hamster anti-CD3 mAb (BD Pharmingen)
and anti-hamster Alexa Fluor 647 mAb were used. Control staining with
fluorochrome-conjugated anti-rabbit or anti-hamster secondary Abs alone
gave only nonspecific background staining (data not shown).
C. rodentium infection
Groups of five Ncr1GFP/GFPand Ncr1GFP/?mice (male, 8 wk of age) were
gavaged with 2 ? 109CFU C. rodentium (CDC1843-73; ATCC 51116).
Mice were weighed and checked for diarrhea/bloody stool on a daily basis.
Survival was monitored for the first 40 days after infection.
The statistical significance of differences between groups was determined
by the unpaired Student t test. Values of p ? 0.05 were considered
Normal development, distribution, and homeostasis of intestinal
CD3?GFP?cell subsets in homozygous Ncr1GFP/GFPreporter
mice lacking NKp46 expression
Mice bearing a targeted insertion of the GFP reporter into the Ncr1
locus show specific GFP expression in CD3?NK cells (NK1.1?,
DX5?) in the bone marrow, lymph nodes, spleen, and peripheral
blood (17). To demonstrate that GFP faithfully recapitulates
NKp46 expression, we further assessed cell surface NKp46 protein
expression in CD3?GFP?cells from Ncr1GFP/?and Ncr1GFP/GFP
mice. The anti-NKp46 mAb (clone 29A1.4; eBioscience) uni-
formly stained splenic CD3?GFP?cells from Ncr1GFP/?mice but
not from Ncr1-deficient Ncr1GFP/GFPmice (Fig. 1A). In contrast,
purified polyclonal goat Abs raised against mouse NKp46
6580INNATE INTESTINAL IL-22 PRODUCTION IS Ncr1 INDEPENDENT
(BAB225 and FAB225P; R&D Systems) showed poor specificity,
with unexpected staining on splenic and intestinal GFP?cells
from both Ncr1GFP/?and Ncr1GFP/GFPmice and no selective
staining of GFP?cells in Ncr1GFP/?mice compared with Ncr1-
deficient Ncr1GFP/GFPmice (supplemental Fig. 14and data not
shown). NK cell markers, including NK1.1, DX5, NKG2D,
CD122, and CD244 (2B4), were normally expressed on splenic
CD3?GFP?cells in the absence of Ncr1 (Fig. 1A and data not
shown). Similar resultswere
CD3?GFP?cells isolated from the small intestinal lamina propria
and intraepithelial compartments (Fig. 1A, supplemental Fig. 2,
and data not shown).
We found that the transcription factor Rorc (encoding ROR?t) was
expressed in the CD127?NK1.1?subset of intestinal lamina propria
CD3?GFP?cells in a Ncr1-independent manner (Fig. 1B). Immuno-
histological analysis confirmed a normal distribution of GFP?cells in
4The online version of this article contains supplemental material.
expression by splenic and small intestinal (SI) CD3?GFP?cells in Ncr1GFP/?and Ncr1GFP/GFPmice. NKp46 vs 2B4 expression on gated CD3?GFP?cells
from spleen or small LPL in Ncr1GFP/?(shaded), Ncr1GFP/GFPmice (black line), and isotype control (dashed line) are shown. Representative results in one
of three independent experiments are shown. B, ROR?t expression by CD127?NK1.1?subset of CD3?GFP?cells in Ncr1GFP/?and Ncr1GFP/GFPmice.
Staining with isotype control Ab (green) vs ROR?t-specific Ab in Ncr1GFP/?(blue) and Ncr1GFP/GFPmice (red) are shown. Representative results in one
of two independent experiments are shown. C, Immunohistological staining of CD3?GFP?cells in the lymph nodes (LN) and small intestine from
Ncr1GFP/?and Ncr1GFP/GFPmice. Sections were stained with 4?,6-diamidino-2-phenylindole (DAPI; blue), anti-GFP (green), anti-B220 (red), and anti-
CD3 (white). Original magnification, ?20. PP, Peyer’s patch. Representative results in one of two independent experiments are shown.
Normal development and distribution of CD3?GFP?cells in Ncr1GFP/?(WT) and Ncr1GFP/GFP(knockout) mice. A, Comparison of NKp46
6581 The Journal of Immunology
the secondary lymphoid organs and small intestine in Ncr1GFP/?and
Ncr1GFP/GFPmice (Fig. 1C). In the lymph node, Ncr1-expressing
(GFP?) cells were found in the paracortex, the perifollicular regions,
and the medulla, whereas GFP?cells were predominantly identified
in the lamina propria in the small intestine with scattered cells in the
epithelium (Fig. 1C). Overall, these results suggest that NKp46 ex-
pression is not required for normal development or anatomical distri-
bution of ROR?t?intestinal Ncr1-expressing cells.
Although CD3?NKp46?cells from the bone marrow and
spleen appear as relatively homogeneous populations of NK1.1?
NK cells, previous studies have identified substantial diversity in
intestinal CD3?NKp46?cells (17). Using CD127 and NK1.1, we
previously characterized three subsets of gut CD3?NKp46?cells
that showed distinct cell surface phenotypes and functional at-
tributes (18). To assess a role for the NKp46 receptor in the de-
velopment of these diverse gut NKp46?cell subsets, we compared
CD127 vs NK1.1 expression on splenic and intestinal CD3?GFP?
cells from Ncr1GFP/?and Ncr1GFP/GFPmice. No statistically sig-
nificant differences were detected in the frequencies and absolute
numbers of splenic and small intestinal CD3?GFP?cell subsets in
the absence of NKp46 (Fig. 2 and Table I). These results suggest
that NKp46 expression is not essential for the development and
homeostasis of diverse NKp46?cell subsets in mice.
Cytokine production from diverse splenic and intestinal
CD3?GFP?cell subsets in Ncr1GFP/?and Ncr1GFP/GFPmice
following triggering via activating receptors or cytokines
Previous studies had demonstrated that the triggering of cell sur-
face NK1.1 or NKp46 could elicit cytokine production (IFN-?)
from splenic NK cells (8, 28). Although plate-bound anti-NK1.1 or
NKp46 Abs could stimulate IFN-? production from splenic
NK1.1?cells, cross-linking NK1.1, 2B4, or NKp46 on intestinal
CD3?GFP?cells did not generate appreciable IFN-? production
(Fig. 3 and supplemental Fig. 3), even though a subset of gut
NKp46?cells expressed IFN-? transcripts as previously demon-
strated (18) using “Yeti” reporter mice (29). Intestinal CD3?GFP?
cells were not blocked in their capacity to produce IFN-?, as stim-
ulation of intestinal LPL with a combination of IL-12 plus IL-18
clearly induced IFN-? secretion (Fig. 3).
We next assessed whether cytokines or cell surface receptor
cross-linking could elicit IL-22 production from intestinal
CD3?NKp46?cell subsets. We used a recently described anti-
IL-22 Ab (MH22B2.2; Ref. 26) that specifically detects mouse
IL-22 (supplemental Fig. 4). Neither NK1.1 nor NKp46 cross-
linking was capable of inducing IL-22 production from splenic or
intestinal CD3?NKp46?cells (Fig. 3 and supplemental Fig. 3).
Interestingly, IL-12 plus IL-18 stimulation also resulted in IL-22
production from intestinal but not splenic CD3?GFP?cells, while
IL-23 stimulation induced IL-22 from CD3?GFP?cells and only
in the gut (Fig. 3).
Our previous transcriptional analysis suggested that although
IL-22 expression was detectable in the CD127?NK1.1?subset,
?10-fold higher levels of IL-22 transcripts were present in intes-
tinal NKp46?cells that were CD127?but completely lacked
NK1.1 expression (18). Despite the presence of IL-22 transcripts
by CD3?NKp46?that coexpress CD127, constitutive IL-22 pro-
tein expression was not obvious in intestinal CD3?GFP?CD127?
cell subsets in Ncr1GFP/?or Ncr1GFP/GFPmice (Fig. 4).
To define which intestinal CD3?GFP?subsets could produce
IFN-? and/or IL-22 in Ncr1GFP/?and Ncr1GFP/GFPmice, we ana-
lyzed intracellular cytokine staining patterns in intestinal CD3?GFP?
subsets that differentially express CD127 and NK1.1 following cyto-
kine stimulation. IL-23 can induce IL-22 production in Th17-polar-
ized T cells (30). IL-23 also induces IL-22 production from small
intestinal LPL of wild-type (WT) (31) and Rag2-deficient mice (data
not shown), consistent with a non-T cell source in the gut. Following
stimulation with IL-23, we detected IL-22 production in intestinal
CD3?GFP?cells that was negatively correlated with NK1.1 expres-
sion and sharply restricted to the NK1.1?fraction of CD3?GFP?
cells (Fig. 4A). This result contrasts with previous reports indicating
cell subsets in the absence of Ncr1. NK1.1 vs CD127 expression on gated
CD3?GFP?cell subsets from spleen and small LPL of Ncr1GFP/?and
Ncr1GFP/GFPmice. Subset frequencies are indicated. Representative results
in one of six independent experiments are shown.
Normal development of splenic and intestinal CD3?GFP?
Table I. Homeostasis of splenic and intestinal CD3?GFP?cell subsets in Ncr1GFP/?and Ncr1GFP/GFP
1,174,883 ? 616,293
126,645 ? 48,932
78,882 ? 93,371
22,991 ? 7,871
21,832 ? 5,219
1,004,992 ? 120,287
156,277 ? 67,374
93,371 ? 42,210
36,302 ? 23,190
21,875 ? 12,689
aAbsolute numbers of splenic and intestinal CD3?GFP?cells (total and subsets defined by differential expression of CD127
and NK1.1) in Ncr1GFP/?and Ncr1GFP/GFPmice (n ? 6 of each genotype) were assessed. No statistically significant differences
were observed in the absence of Ncr1.
6582 INNATE INTESTINAL IL-22 PRODUCTION IS Ncr1 INDEPENDENT
that IL-22 protein could be detected in intestinal NKp46?cells ex-
pressing intermediate levels of NK1.1, but not in NKp46?NK1.1?
cells following stimulation with phorbol ester and calcium
ionophore (20) or IL-23 (19). IL-23 failed to induce IFN-? from
any gut CD3?GFP?cell subset, and IL-22 production from
intestinal CD3?GFP?cells was independent of NKp46 expres-
sion and equally detected in Ncr1GFP/?and Ncr1GFP/GFPmice
Following IL-12 plus IL-18, a different pattern of cytokine pro-
duction was observed among intestinal CD3?GFP?subsets (Fig. 4).
As expected, NK1.1?cells could produce IFN-? under these condi-
tions (similar to splenic NK1.1?cells), which was Ncr1 independent
(Fig. 4B). Interestingly, the CD127?NK1.1?subset of intestinal
CD3?GFP?cells could produce both IFN-? and IL-22 under these
conditions with cells generally producing only IL-22 or IFN-?, al-
though a small subset of cells clearly produced both cytokines (Fig.
4A). Stimulation with either IL-12 or IL-18 alone was capable of
inducing IL-22 production, while IFN- ? production appeared to re-
obtained in intestinal cells from mice lacking Ncr1 (Fig. 4B). These
results indicate that different cytokine signals can elicit distinct cyto-
kine production patterns from intestinal CD3?GFP?subsets.
A role for NK1.1?cells but not the NKp46 receptor in the innate
immune response to C. rodentium infection in Rag2-deficient mice
Although IL-22 production from in vitro-activated NKp46?cells
appeared independent of Ncr1 expression, a more rigorous test of
NKp46?cell function would involve the capacity of these cells to
produce IL-22 in vivo following infection with the Gram-negative
bacterium C. rodentium (18, 31). Consistent with IL-22 production
from innate cells, we found that CD3?NKp46?NK1.1?cells (but
not CD3?cells) were IL-22?during the first week after C. ro-
dentium infection (Fig. 5A and data not shown). To assess a role
for NKp46 in the innate intestinal defense, we generated T and B
cell-deficient mice that were competent or incompetent for NKp46
triggering (Rag2?/?Ncr1GFP/?and Rag2?/?Ncr1GFP/GFPmice)
and orally infected them with C. rodentium. Rag2?/?Il2rg?/?
mice were used as a positive control because these mice lack all
NKp46?cells, produce no IL-22, and succumb rapidly after C.
rodentium infection (18). We found no difference in the capac-
ity of Rag2?/?mice with or without NKp46 to resist early C.
rodentium infection (Fig. 5, B and C). Both Rag2?/?Ncr1GFP/?
and Rag2?/?Ncr1GFP/GFPmice showed similar clinical scores and
maintained body weight up to 40 days after infection, whereas all
infected Rag2?/?Il2rg?/?mice died within 8 days. Thus, innate
immune responses to C. rodentium are intact in the absence of
We previously reported that the early immune response to C. ro-
dentium infection was intact in Rag2?/?Il2rb?/?mice that harbor
normal numbers of CD3?NKp46?cells that express CD127 but are
intact IL-22 production in Rag2?/?Il2rb?/?mice (18) that was likely
produced by the CD127?NK1.1?subset of intestinal CD3?NKp46?
cells as demonstrated above (Fig. 4A). In this study, we extend
these initial observations by analyzing C. rodentium infection
at later time points in Rag2?/?Il2rb?/?mice. Interestingly, we
found that Rag2?/?Il2rb?/?mice (but not Rag2?/?mice) suc-
micelackNK1.1?cells(18)that can produce IFN-? (Fig. 4A), these
results underline the important role for intestinal NK1.1?cells in
the defense against C. rodentium. These results are also in accor-
dance with the previous report from the Colonna laboratory using
NK1.1 depletion in C. rodentium-infected Rag2?/?mice (31).
Nevertheless, the kinetics of susceptibility to C. rodentium in the
production following NK1.1 cross-
linking or cytokine stimulation of
splenic and intestinal CD3?GFP?
cells. A, Splenocytes and small intes-
tine LPL (SI) were stimulated in vitro
with plate-bound NK1.1 Ab, IL-12 ?
IL-18, IL-23 or without stimulation as
control. Intracellular staining of IL-22
vs IFN-? on gated CD3?GFP?cells
is shown. One representative experi-
ment of four is shown. B, The per-
centage of IFN-? (left)- or IL-22
in the spleen (?) vs SI (f) are shown.
Results are means ? SD from four
independent determinations. ? and
???, p ? 0.01 and ??, p ? 0.05, com-
parison of spleen vs SI for NK1.1, IL-
12-IL-18, or IL-23 stimulation.
Activation of cytokine
6583The Journal of Immunology
absence of NK1.1?cells is clearly distinct and delayed (?30 days)
in comparison to infected Rag2?/?Il2rg?/?mice (Fig. 5, B and C)
or to infected Rag2?/?mice when endogenous IL-22 is neutral-
ized (22). Under these conditions, mice rapidly succumb to infec-
tion (?7 days). These results point to distinct roles for
CD127?NK1.1?and NK1.1?subsets of CD3?NKp46?cells in
the innate intestinal defense against C. rodentium.
NCRs comprise a set of transmembrane signaling receptors in
human NK cells (NKp30, NKp44, NKp46) and in mice (NKp46
encoded at the Ncr1 locus) that interact with specific ligands
and modulate NK cell effector functions (reviewed in Ref. 32).
Although NCR expression was initially characterized as NK
specific (4, 7) and later proposed as part of a “universal” def-
inition of NK cells (33), we now appreciate that NKp46 can be
expressed by some T cell subsets (34, 35) and by intestinal cells
that have only a marginal resemblance to classical NK cells
(18–20, 31). As such, the functional heterogeneity within
CD3?NKp46?cells of a given tissue may vary considerably. In
some organs (e.g., spleen), CD3?NKp46?cells are quite ho-
mogeneous and comprise cytolytic, IFN-?-producing NK cells.
In contrast, in the gut, a subset of CD3?NKp46?cells include
classical NK cells, but the majority are characterized by the
absence of most NK cell markers and effector functions and
instead express the transcription factor Rorc and produce IL-22
(18–20, 31). Given that the homeostasis of this latter population
is dependent on microbial flora (18, 19) and is implicated in
IL-22 production in response to the intestinal pathogen C. ro-
dentium (18, 22, 31), we assessed whether NKp46 expression
was required for the development, differentiation, and function
of intestinal innate lymphocytes that produce IL-22 and protect
against C. rodentium.
By analyzing mice harboring a GFP-modified Rag2?/?allele
(17) in the heterozygous (WT) or homozygous (knockout) state,
we were able to assess whether the absence of surface NKp46
expression influenced the development and function of intesti-
nal CD3?GFP?cells. We found that the subset distribution of
CD3?GFP?cells expressing CD127 and/or NK1.1 as well as
their overall homeostasis (cell numbers and tissue distribution)
and IL-22 production by intestinal
Ncr1GFP/?(A) and Ncr1GFP/GFP(B)
mice. IFN-? vs IL-22 production
from intestinal CD3?GFP?subsets
of Ncr1GFP/?and Ncr1GFP/GFPmice
gated according to CD127 vs NK1.1
expression. Small intestinal LPL were
stimulated with IL-12 ? IL-18 or
IL-23 for 4–6 h or left unstimulated
before processing for intracellular cy-
tokine staining. Representative results
in one of three independent experi-
ments are shown. No significant dif-
ferences in percentages of IL-22- or
IFN-?-producing subsets were ob-
served in the absence of Ncr1 (IL-23-
CD127?NK1.1?cells: 37.3 ? 9.5%
in Ncr1GFP/?mice vs 29 ? 4.6% in
Ncr1GFP/GFPmice; IL-12 ? IL-18-in-
duced IL-22 production
CD127?NK1.1?cells: 26.7 ? 2.9%
in Ncr1GFP/?mice vs 25 ? 6.1% in
Ncr1GFP/GFPmice; and IL-12 ? IL-
18-induced IFN- ? production from
CD127?NK1.1?cells: 50.6 ? 1.2%
in Ncr1GFP/?mice vs 46 ? 6.1% in
Comparison of IFN-?
6584INNATE INTESTINAL IL-22 PRODUCTION IS Ncr1 INDEPENDENT
was normal in the absence of NKp46. Previous studies have
shown that ROR?t is expressed in a subset of intestinal
CD3?NKp46?cells (18–20), and it has been postulated that
elevated ROR?t expression by this subset may indicate that
these cells derive from or may function as LTi-like cells (20,
CD3?NKp46?cells was unaltered in the absence of NKp46 and
moreover, no defects in LTi function were apparent in mice
deficient in Ncr1 (Ncr1GFP/GFPmice showed normal develop-
ment of lymph nodes and Peyer’s patches; data not shown).
Thus, if CD3?NKp46?cells expressing ROR?t have an LTi-
like role, this function appears intact in the absence of Ncr1.
As indicated above, intestinal CD3?NKp46?cells exhibit
substantial phenotypic and functional diversity (18–20). Using
CD127and NK1.1,we have
(CD127?, CD127?) appear related to classical NK cells by
many criteria, including expression of NK markers (NKG2D,
CD122, DX5, Ly49 family members), perforin and IFN-? ef-
fector functions, and IL-15 dependence. In contrast, the
CD127?NK1.1?subset lacks NK markers and is noncytotoxic,
but expresses transcripts for Rorc and IL-22 (18). Using IL-22-
specific Abs, we demonstrated that IL-22 protein is barely de-
tectable in unstimulated gut CD3?NKp46?cells, but is rapidly
produced in activated cells. Thus, IL-22 protein expression may
be controlled posttranscriptionally with rapid translation in
cells containing a preformed pool of IL-22 transcripts in a fash-
ion analogous to IFN-? (37). This regulation would be consis-
tent with the role for IL-22-producing cells during initial innate
phases of the immune response.
The signals that control cytokine production from intestinal
CD3?NKp46?cells are poorly understood and could include
receptors that recognize cell-associated as well as soluble li-
gands. In this report, we assessed the contribution of cell sur-
face receptors (NK1.1, NKp46, 2B4) and soluble cytokines (IL-
12, IL-18, IL-23) to IL-22 and IFN-? production by intestinal
CD3?NKp46?cell subsets. In contrast to splenic NK cells,
intestinal NKp46?cells failed to produce cytokines after
NK1.1, 2B4, or NKp46 cross-linking, but were competent for
cytokine secretion after stimulation with soluble factors. We
found that several intestinal CD3?NKp46?
(NK1.1?cells but also CD127?NK1.1?cells) can produce
IFN-? following stimulation. Moreover, IL-12 plus IL-18-in-
duced production of IFN-? by the CD127?NK1.1?subset was
accompanied by IL-22 production, with a small population of
IL-22?IFN-??double producers. The explanation for the ex-
istenceof distinct cytokine-producing
CD127?NK1.1?subset is not yet known, but several hypoth-
eses can be forwarded. One possibility is that further heteroge-
neity exists in the CD127?NK1.1?subset with subsets that are
selectively tuned toward either IL-22 or IFN-? production. An
alternative explanation is that cytokine profiles are dynamically
regulated within CD127?NK1.1?cells (e.g., starting out as ma-
jor IL-22 producers that evolve toward IFN-? producers as cells
become chronically stimulated). Collectively, these results sug-
gest that intestinal CD3?NKp46?cells may have the potential
to secrete different cytokine profiles depending on the environ-
mental context. As such, intestinal CD3?NKp46?cell subsets
could have varied roles throughout the course of an evolving
IL-22-expressing CD3?NKp46?cells are present in the small
and large intestine under steady-state conditions (18–20) and
cultured colonic lamina propria cells from C. rodentium-in-
fected mice produce IL-22 that is correlated with the presence
of CD3?NKp46?NK1.1?cells (18, 22). Although other labo-
ratories detected IL-22 protein in a subset of intestinal NK1.1?
cells (19, 20), in our hands, IL-22 production following cyto-
kine stimulation was sharply restricted to the CD127?NK1.1?
subset of CD3-NKp46?cells. IL-22 production by intestinal
NK1.1?cells would be consistent with the normal early innate
immune response (up to 30 days) after C. rodentium infection in
mice that genetically lack NK1.1?cells (18) or are treated with
depleting anti-NK1.1 Abs (31). Nevertheless, NK1.1?cells
clearly play an important role in the ultimate defense against
this pathogen as illustrated by the reduced survival of C. ro-
Rag2?/?mice. Our results are consistent with the previous re-
port showing that anti-NK1.1 treatment reduced overall sur-
vival of C. rodentium-infected WT mice (31). Because the
NK1.1?subset of intestinal CD3?NKp46?cells is a poor IL-22
producer, but can produce IFN-? following stimulation, it re-
mains possible that the major role for intestinal NK1.1?cells in
the course of C. rodentium infection would be at a latter stage,
C. rodentium-infected C57BL/6 mice (day 5 after infection) were analyzed for
spontaneous IL-22 production. Body weight changes (B) and survival (C) of
Rag2?/?Il2rg?/?(diamond), Rag2?/?Il2rb?/?(triangle), Rag2?/?Ncr1GFP/?
(circle), and Rag2?/?Ncr1GFP/GFP(square) mice after C. rodentium infection.
Representative results (means ? SD) from one experiment (n ? 5 mice of
each genotype infected in an experiment). One representative experiment of
two is shown.
Survival of Rag2?/?Ncr1GFP/?and Rag2?/?Ncr1GFP/GFP
6585The Journal of Immunology
complementing the IL-22-mediated restriction of bacterial
spread into the host. Further studies will be required to test this
Lastly, by comparing C. rodentium infection in T and B cell-
deficient mice that were competent or incompetent for NKp46
mice), we could provide clear evidence that NKp46 was not
required for innate immune defense against this pathogen. Sev-
eral explanations may account for this result. First, NKp46 li-
gands may not be induced during the course of C. rodentium
infection. Little is known about the nature of NKp46 ligands
outside of the identified viral hemagglutinins (11). Studies us-
ing NKp46-Fc fusion proteins may help to further investigate
whether C. rodentium (or other bacterial infections) induce
NKp46 ligands. Second, IL-22 production from intestinal
CD3?NKp46?cells may be elicited by triggering of other cell
surface receptors or via soluble factors. Stimulation with IL-12,
IL-18, or IL-23 resulted in IL-22 production in vitro, and IL-12,
IL-18 and IL-23 production by monocytes or dendritic cells has
been documented following triggering of pathogen-associated
molecular pattern receptors during infection (38). The critical
role of IL-12 and/or IL-23 in vivo for immunity to C. rodentium
was reported by MacDonald and colleagues (39), who found
increased mortality and compromised C. rodentium control in
mice lacking the IL-12p40 subunit of IL-12 and IL-23. Release
of IL-12 and/or IL-23 during C. rodentium infection may be
important for IL-22 production from CD3?NKp46?cells re-
quired for immune defense. Finally, while IL-22 production by
CD3?NKp46?cells may still play a critical role during C. ro-
dentium infection, other IL-22-producing cells may also be en-
gaged during this process and compensate in the absence of
NKp46. Further analysis of the critical signals that control
IL-22 production in vivo during steady state and after infections
challenge will help to unravel the biological roles for distinct
CD3?NKp46?cell subsets in intestinal immune protection and
We thank G. Eberl for critical comments and expert immunohistology ad-
vice and M. Be ´rard and C. Bizet (Collection de l’Institut Pasteur) for sup-
plying C. rodentium.
The authors have no financial conflict of interest.
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