Unraveling the Heterogeneity and Specialization of ILCs.

In this issue of Immunity, Zeis et al. report the generation of a single-cell atlas of lung innate lymphoid cells (ILCs). Their findings provide insight into the how ILCs are locally maintained, revealing in situ differentiation and diversification as mechanisms of ILC renewal and function.

Bacteria-Induced Group 2 Innate Lymphoid Cells in the Stomach Provide Immune Protection through Induction of IgA.

The intestinal microbiota shapes and directs immune development locally and systemically, but little is known about whether commensal microbes in the stomach can impact their immunological microenvironment. Here, we report that group 2 innate lymphoid cells (ILC2s) were the predominant ILC subset in the stomach and show that their homeostasis and effector functions were regulated by local commensal communities. Microbes elicited interleukin-7 (IL-7) and IL-33 production in the stomach, which in turn triggered the propagation and activation of ILC2. Stomach ILC2s were also rapidly induced following infection with Helicobacter pylori. ILC2-derived IL-5 resulted in the production of IgA, which coated stomach bacteria in both specific pathogen-free (SPF) and H. pylori-infected mice. Our study thus identifies ILC2-dependent IgA response that is regulated by the commensal microbiota, which is implicated in stomach protection by eliminating IgA-coated bacteria including pathogenic H. pylori.

RORγt+ innate lymphoid cells regulate intestinal homeostasis by integrating negative signals from the symbiotic microbiota.

Lymphoid cells that express the nuclear hormone receptor RORγt are involved in containment of the large intestinal microbiota and defense against pathogens through the production of interleukin 17 (IL-17) and IL-22. They include adaptive IL-17-producing helper T cells (T(H)17 cells), as well as innate lymphoid cells (ILCs) such as lymphoid tissue-inducer (LTi) cells and IL-22-producing NKp46+ cells. Here we show that in contrast to T(H)17 cells, both types of RORγt+ ILCs constitutively produced most of the intestinal IL-22 and that the symbiotic microbiota repressed this function through epithelial expression of IL-25. This function was greater in the absence of adaptive immunity and was fully restored and required after epithelial damage, which demonstrates a central role for RORγt+ ILCs in intestinal homeostasis. Our data identify a finely tuned equilibrium among intestinal symbionts, adaptive immunity and RORγt+ ILCs.

The chemokine receptor CXCR6 controls the functional topography of interleukin-22 producing intestinal innate lymphoid cells.

Interleukin-22 (IL-22) plays a critical role in mucosal defense, although the molecular mechanisms that ensure IL-22 tissue distribution remain poorly understood. We show that the CXCL16-CXCR6 chemokine-chemokine receptor axis regulated group 3 innate lymphoid cell (ILC3) diversity and function. CXCL16 was constitutively expressed by CX3CR1(+) intestinal dendritic cells (DCs) and coexpressed with IL-23 after Citrobacter rodentium infection. Intestinal ILC3s expressed CXCR6 and its ablation generated a selective loss of the NKp46(+) ILC3 subset, a depletion of intestinal IL-22, and the inability to control C. rodentium infection. CD4(+) ILC3s were unaffected by CXCR6 deficiency and remained clustered within lymphoid follicles. In contrast, the lamina propria of Cxcr6(-/-) mice was devoid of ILC3s. The loss of ILC3-dependent IL-22 epithelial stimulation reduced antimicrobial peptide expression that explained the sensitivity of Cxcr6(-/-) mice to C. rodentium. Our results delineate a critical CXCL16-CXCR6 crosstalk that coordinates the intestinal topography of IL-22 secretion required for mucosal defense.

Human gain-of-function STAT1 mutation disturbs IL-17 immunity in mice.

Gain-of-function (GOF) mutations in the gene for signal transducer and activator of transcription 1 (STAT1) account for approximately one-half of patients with chronic mucocutaneous candidiasis (CMC) disease. Patients with GOF-STAT1 mutations display a broad variety of infectious and autoimmune manifestations in addition to CMC, and those with severe infections and/or autoimmunity have a poor prognosis. The establishment of safe and effective treatments based on a precise understanding of the molecular mechanisms of this disorder is required to improve patient care. To tackle this problem, we introduced the human R274Q GOF mutation into mice [GOF-Stat1 knock-in (GOF-Stat1R274Q)]. To investigate the immune responses, we focused on the small intestine (SI), which contains abundant Th17 cells. Stat1R274Q/R274Q mice showed excess phosphorylation of STAT1 in CD4+ T cells upon IFN-γ stimulation, consistent with the human phenotype in patients with the R274Q mutation. We identified two subpopulations of CD4+ T cells, those with 'normal' or 'high' level of basal STAT1 protein in Stat1R274Q/R274Q mice. Upon IFN-γ stimulation, the 'normal' level CD4+ T cells were more efficiently phosphorylated than those from WT mice, whereas the 'high' level CD4+ T cells were not, suggesting that the level of STAT1 protein does not directly correlate with the level of pSTAT1 in the SI. Inoculation of Stat1R274Q/R274Q mice with Candida albicans elicited decreased IL-17-producing CD4+RORγt+ cells. Stat1R274Q/R274Q mice also excreted larger amounts of C. albicans DNA in their feces than control mice. Under these conditions, there was up-regulation of T-bet in CD4+ T cells. GOF-Stat1R274Q mice thus should be a valuable model for functional analysis of this disorder.

Regulation of cytokine secretion in human CD127(+) LTi-like innate lymphoid cells by Toll-like receptor 2.

Lymphoid tissue inducer cells are members of an emerging family of innate lymphoid cells (ILC). Although these cells were originally reported to produce cytokines such as interleukin-17 (IL-17) and IL-22, we demonstrate here that human CD127(+)RORC(+) and CD56(+)CD127(+) LTi-like ILC also express IL-2, IL-5, and IL-13 after activation with physiologic stimuli such as common γ-chain cytokines, Toll-like receptor (TLR) 2 ligands, or IL-23. Whereas TLR2 signaling induced IL-5, IL-13, and IL-22 expression in a nuclear factor κB (NF-κB)-dependent manner, IL-23 costimulation induced only IL-22 production. CD127(+) LTi-like ILC displayed clonal heterogeneity for IL-13 and IL-5 production, suggesting in vivo polarization. Finally, we identified a role for autocrine IL-2 signaling in mediating the effects of TLR2 stimulation on CD56(+)CD127(+) and CD127(+) LTi-like ILC. These results indicate that human LTi-like ILC can directly sense bacterial components and unravel a previously unrecognized functional heterogeneity among this important population of innate lymphoid cells.

Phenotypic and Functional Plasticity of Murine Intestinal NKp46+ Group 3 Innate Lymphoid Cells.

Group 3 innate lymphoid cells (ILC3) actively participate in mucosal defense and homeostasis through prompt secretion of IL-17A, IL-22, and IFN-γ. Reports identify two ILC3 lineages: a CCR6(+)T-bet(-) subset that appears early in embryonic development and promotes lymphoid organogenesis and a CCR6(-)T-bet(+) subset that emerges after microbial colonization and harbors NKp46(+) ILC3. We demonstrate that NKp46 expression in the ILC3 subset is highly unstable. Cell fate mapping using Ncr1(CreGFP) × Rosa26(RFP) mice revealed the existence of an intestinal RFP(+) ILC3 subset (Ncr1(FM)) lacking NKp46 expression at the transcript and protein levels. Ncr1(FM) ILC3 produced more IL-22 and were distinguishable from NKp46(+) ILC3 by differential CD117, CD49a, DNAX accessory molecule-1, and, surprisingly, CCR6 expression. Ncr1(FM) ILC3 emerged after birth and persisted in adult mice following broad-spectrum antibiotic treatment. These results identify an unexpected phenotypic instability within NKp46(+) ILC3 that suggests a major role for environmental signals in tuning ILC3 functional plasticity.

Understanding the immune signature fingerprint of peritoneal dialysis-related peritonitis.

Although acute peritonitis is a common and severe complication associated with peritoneal dialysis, the culture-based test used as the diagnostic criterion for this disease is often too slow to allow appropriate point-of-care diagnosis of specific bacterial infection. To address this problem, Zhang et al. report the efficacy of a novel set of immune biomarkers derived from a machine-learning algorithm applied to patient data. This fingerprint could predict major pathogenic causes of peritonitis.

Heterogeneity and diversity of group 3 innate lymphoid cells: new cells on the block.

Innate lymphoid cells (ILCs) are a newly identified subset of innate cells that play fundamentally crucial roles for early immune defense at mucosal and non-mucosal sites. ILCs consist of ILC1s, ILC2s and ILC3s, which each have distinct transcription factors controlling their development and function. Interestingly, each of the ILC subsets represents the innate counterparts of CD4(+) helper T-cell subsets T(h1), T(h2) and T(h17) on the basis of transcriptional regulation. ILC1s that produce IFN-γ or TNF-α, ILC2s that produce T(h2)-type cytokines mainly such as IL-5 or IL-13 and ILC3s have been recently reported and reviewed in terms of IL-22- or IL-17-producing function and cell development. However, in this relatively new field, it remains likely that additional functional and regulatory mechanisms remain to be explored. More recent findings show that ILC3s are regulated by RORγt, which plays an important role for the mucosal barrier and surface protection against pathogenic bacterial infection. ILC3s might cooperate with other cells (e.g. T cells or dendritic cells) directly or indirectly, and subsequently ILC3s have impact on tissues with prompt regulation. Especially, ILC3s in mucosal site are well known to protect the intestinal surface barrier through inducible anti-microbial peptides via IL-22. Here, I will summarize and discuss the roles, function and heterogeneity of ILC3s in mucosal tissues.

Microbial flora drives interleukin 22 production in intestinal NKp46+ cells that provide innate mucosal immune defense.

Natural killer (NK) cells are innate lymphocytes with spontaneous antitumor activity, and they produce interferon-gamma (IFN-gamma) that primes immune responses. Whereas T helper cell subsets differentiate from naive T cells via specific transcription factors, evidence for NK cell diversification is limited. In this report, we characterized intestinal lymphocytes expressing the NK cell natural cytotoxicity receptor NKp46. Gut NKp46+ cells were distinguished from classical NK cells by limited IFN-gamma production and absence of perforin, whereas several subsets expressed the nuclear hormone receptor retinoic acid receptor-related orphan receptor t (RORgammat) and interleukin-22 (IL-22). Intestinal NKp46+IL-22+ cells were generated via a local process that was conditioned by commensal bacteria and required RORgammat. Mice lacking IL-22-producing NKp46+ cells showed heightened susceptibility to the pathogen Citrobacter rodentium, consistent with a role for intestinal NKp46+ cells in immune protection. RORgammat-driven diversification of intestinal NKp46+ cells thereby specifies an innate cellular defense mechanism that operates at mucosal surfaces.

Conditional ablation of NKp46+ cells using a novel Ncr1(greenCre) mouse strain: NK cells are essential for protection against pulmonary B16 metastases.

To study gene functions specifically in NKp46+ cells we developed novel Cre mice allowing for conditional gene targeting in cells expressing Ncr1 (encoding NKp46). We generated transgenic Ncr1(greenCre) mice carrying an EGFPcre fusion under the control of a proximal Ncr1 promoter that faithfully directed EGFPcre expression to NKp46+ cells from lymphoid and nonlymphoid tissues. This approach allowed for direct detection of Cre-expressing NKp46+ cells via their GFP signature by flow cytometry and histology. Cre was functional as evidenced by the NKp46+ cell-specific expression of RFP in Ncr1(greenCre) Rosa-dtRFP reporter mice. We generated Ncr1(greenCre) Il2rg(fl/fl) mice that lack NKp46+ cells in an otherwise intact hematopoietic environment. Il2rg encodes the common gamma chain (γc ), which is an essential receptor subunit for cytokines (IL-2, -4, -7, -9, -15, and -21) that stimulate lymphocyte development and function. In Ncr1(greenCre) Il2rg(fl/fl) mice, NK cells are severely reduced and the few remaining NKp46+ cells escaping γc deletion failed to express GFP. Using this new NK-cell-deficient model, we demonstrate that the homeostasis of NKp46+ cells from all tissues (including the recently described intraepithelial ILC1 subset) requires Il2rg. Finally, Ncr1(greenCre) Il2rg(fl/fl) mice are unable to reject B16 lung metastases demonstrating the essential role of NKp46+ cells in antimelanoma immune responses.

Disease pathogenesis and barrier functions regulated by group 3 innate lymphoid cells.

The mucosal surface is in constant contact with foreign antigens and is regulated by unique mechanisms that are different from immune responses in the peripheral organs. For the last several decades, only adaptive immune cells such as helper T (Th) cells, Th1, Th2, or Th17 were targeted to study a wide variety of immune responses in the mucosal tissues. However, since their discovery, innate lymphoid cells (ILCs) have been attracting attention as a unique subset of immune cells that provide border defense with various functions and tissue specificity. ILCs are classified into different groups based on cell differentiation and functions. Group 3 innate lymphoid cells (ILC3s) are particularly in close proximity to mucosal surfaces and therefore have the opportunity to be exposed to a variety of bacteria including pathogenic bacteria. In recent years, studies have also provided much evidence that ILC3s contribute to disease pathogenesis as well as the defense of mucosal surfaces by rapidly responding to pathogens and coordinating other immune cells. As the counterpart of helper T cells, ILC3s together with other ILC subsets establish the immune balance between adaptive and innate immunity in protecting us from invasion or encounter with non-self-antigens for maintaining a complex homeostasis. In this review, we summarize recent advances in our understanding of ILCs, with a particular focus on the function of ILC3s in their involvement in bacterial infection and disease pathogenesis.

A lipidome landscape of aging in mice.

Understanding the molecular mechanisms of aging is crucial for enhancing healthy longevity. We conducted untargeted lipidomics across 13 biological samples from mice at various life stages (2, 12, 19 and 24 months) to explore the potential link between aging and lipid metabolism, considering sex (male or female) and microbiome (specific pathogen-free or germ-free) dependencies. By analyzing 2,704 molecules from 109 lipid subclasses, we characterized common and tissue-specific lipidome alterations associated with aging. For example, the levels of bis(monoacylglycero)phosphate containing polyunsaturated fatty acids increased in various organs during aging, whereas the levels of other phospholipids containing saturated and monounsaturated fatty acids decreased. In addition, we discovered age-dependent sulfonolipid accumulation, absent in germ-free mice, correlating with Alistipes abundance determined by 16S ribosomal RNA gene amplicon sequencing. In the male kidney, glycolipids such as galactosylceramides, galabiosylceramides (Gal2Cer), trihexosylceramides (Hex3Cer), and mono- and digalactosyldiacylglycerols were detected, with two lipid classes-Gal2Cer and Hex3Cer-being significantly enriched in aged mice. Integrated analysis of the kidney transcriptome revealed uridine diphosphate galactosyltransferase 8A (UGT8a), alkylglycerone phosphate synthase and fatty acyl-coenzyme A reductase 1 as potential enzymes responsible for the male-specific glycolipid biosynthesis in vivo, which would be relevant to sex dependency in kidney diseases. Inhibiting UGT8 reduced the levels of these glycolipids and the expression of inflammatory cytokines in the kidney. Our study provides a valuable resource for clarifying potential links between lipid metabolism and aging.

A Bcl11bN797K variant isolated from an immunodeficient patient inhibits early thymocyte development in mice.

BCL11B is a transcription factor with six C2H2-type zinc-finger domains. Studies in mice have shown that Bcl11b plays essential roles in T cell development. Several germline heterozygous BCL11B variants have been identified in human patients with inborn errors of immunity (IEI) patients. Among these, two de novo mis-sense variants cause asparagine (N) to lysine (K) replacement in distinct zinc-finger domains, BCL11BN441K and BCL11BN807K. To elucidate the pathogenesis of the BCL11BN807K variant, we generated a mouse model of BCL11BN807K by inserting the corresponding mutation, Bcl11bN797K, into the mouse genome. In Bcl11b+/N797K mice, the proportion of immature CD4-CD8+ single-positive thymocytes was increased, and the development of invariant natural killer cells was severely inhibited in a T-cell-intrinsic manner. Under competitive conditions, γδT cell development was outcompeted by control cells. Bcl11bN797K/N797K mice died within one day of birth. Recipient mice reconstituted with Bcl11bN797K/N797K fetal liver cells nearly lacked CD4+CD8+ double-positive thymocytes, which was consistent with the lack of their emergence in culture from Bcl11bN797K/N797K fetal liver progenitors. Interestingly, Bcl11bN797K/N797K progenitors gave rise to aberrant c-Kit+ and CD44+ cells both in vivo and in vitro. The increase in the proportion of immature CD8 single-positive thymocytes in the Bcl11bN797K mutants is caused, in part, by the inefficient activation of the Cd4 gene due to the attenuated function of the two Cd4 enhancers via distinct mechanisms. Therefore, we conclude that immunodeficient patient-derived Bcl11bN797K mutant mice elucidated a novel role for Bcl11b in driving the appropriate transition of CD4-CD8- into CD4+CD8+ thymocytes.

Lymphotoxin-ß receptor-independent development of intestinal IL-22-producing NKp46+ innate lymphoid cells.

The natural cytotoxicity receptor NKp46 is an activating receptor expressed by several distinct innate lymphoid cell (ILC) subsets, including NK cells, some γδ T cells and intestinal RORγt(+) IL-22(+) cells (NCR22 cells, IL-22-producing NKp46(+) cell). NCR22 cells may play a role in mucosal barrier function through IL-22-mediated production of anti-bacterial peptides from intestinal epithelial cells. Previous studies identified a predominant proportion of NCR22 cells in gut cryptopatches (CP), lymphoid structures that are strategically positioned to collect and integrate signals from luminal microbes; however, whether CP or other lymphoid structures condition NCR22 cell differentiation is not known. Programmed and inducible lymphoid tissue development requires cell-surface-expressed lymphotoxin (LT)α(1) ß(2) heterotrimers (provided by lymphoid tissue inducer (LTi) cells) to signal lymphotoxin-ß receptor (LTR)(+) stromal cells. Here, we analyzed NCR22 cells in LTßR-deficient Ncr1(GFP/+) mice that lack organized secondary lymphoid tissues. We found that NCR22 cells develop in the absence of LTßR, become functionally competent and localize to the lamina propria under steady-state conditions. Following infection of LTßR(-/-) mice with the Gram-negative pathogen Citrobacter rodentium, IL-22 production from NCR22 cells was not affected. These results indicate that organized lymphoid tissue structures are not critical for the generation of an intact and fully functional intestinal NCR22 cell compartment.

IL-22 is produced by γC-independent CD25+ CCR6+ innate murine spleen cells upon inflammatory stimuli and contributes to LPS-induced lethality.

IL-22 is a Th17 cytokine that plays a key role in immune responses against extracellular bacteria. In mucosal lymphoid tissues, IL-22 production is mainly due to an IL-23-responsive NK-like cell subset that shares some markers with lymphoid tissue inducer (LTi) cells. Here, we identified a new spleen cell population responsible for IL-22 production upon either in vitro stimulation by anti-CD3 antibodies or in vivo stimulation by lipopolysaccharide (LPS) via IL-2- and an IL-23-dependent mechanisms, respectively. These cells represent 1% of spleen cells from recombination activating gene (Rag2)-deficient mice, and correspond to a discrete innate lymphoid cell population expressing CD25, CCR6 and IL-7R. This population comprises 60-70% CD4(+) cells, which produce IL-22, and are still present in common γ chain-deficient mice; the CD4(-) subset coexpresses IL-22 and IL-17, and is common γ chain-dependent. The importance of IL-22 production for the LPS-triggered response is highlighted by the fact that IL-22-deficient mice are more resistant to LPS-induced mortality.

Cutting edge: Thymic NK cells develop independently from T cell precursors.

Although NK cells in the mouse are thought to develop in the bone marrow, a small population of NK cells in the thymus has been shown to derive from a GATA3-dependent pathway. Characteristically, thymic NK cells express CD127 and few Ly49 molecules and lack CD11b. Because these NK cells develop in the thymus, the question of their relationship to the T cell lineage has been raised. Using several different mouse models, we find that unlike T cells, thymic NK cells are not the progeny of Rorc-expressing progenitors and do not express Rag2 or rearrange the TCRγ locus. We further demonstrate that thymic NK cells develop independently of the Notch signaling pathway, supporting the idea that thymic NK cells represent bona fide NK cells that can develop independently of all T cell precursors.

A Japanese Herbal Formula, Daikenchuto, Alleviates Experimental Colitis by Reshaping Microbial Profiles and Enhancing Group 3 Innate Lymphoid Cells.

Daikenchuto (DKT) is one of the most widely used Japanese herbal formulae for various gastrointestinal disorders. It consists of Zanthoxylum Fructus (Japanese pepper), Zingiberis Siccatum Rhizoma (processed ginger), Ginseng radix, and maltose powder. However, the use of DKT in clinical settings is still controversial due to the limited molecular evidence and largely unknown therapeutic effects. Here, we investigated the anti-inflammatory actions of DKT in the dextran sodium sulfate (DSS)-induced colitis model in mice. We observed that DKT remarkably attenuated the severity of experimental colitis while maintaining the members of the symbiotic microbiota such as family Lactobacillaceae and increasing levels of propionate, an immunomodulatory microbial metabolite, in the colon. DKT also protected colonic epithelial integrity by upregulating the fucosyltransferase gene Fut2 and the antimicrobial peptide gene Reg3g. More remarkably, DKT restored the reduced colonic group 3 innate lymphoid cells (ILC3s), mainly RORγthigh-ILC3s, in DSS-induced colitis. We further demonstrated that ILC3-deficient mice showed increased mortality during experimental colitis, suggesting that ILC3s play a protective function on colonic inflammation. These findings demonstrate that DKT possesses anti-inflammatory activity, partly via ILC3 function, to maintain the colonic microenvironment. Our study also provides insights into the molecular basis of herbal medicine effects, promotes more profound mechanistic studies towards herbal formulae and contributes to future drug development.

Possible Metastatic Stage-Dependent ILC2 Activation Induces Differential Functions of MDSCs through IL-13/IL-13Rα1 Signaling during the Progression of Breast Cancer Lung Metastasis.

Breast cancer is the most common cancer in women worldwide, and lung metastasis is one of the most frequent distant metastases. When breast cancer metastasizes to the lung, group 2 innate lymphoid cells (ILC2s) are thought to promote tumor growth via the activation of myeloid-derived suppressor cells (MDSCs), which are known to negatively regulate anticancer immune responses. However, it remains to be elucidated exactly how this ILC2-MDSC interaction is involved in tumor growth during metastases formation. Using a 4T1/LM4 breast cancer mouse model, we found that ILC2s were activated in both the micro- and macrometastatic regions, suggesting sustained activation throughout the metastatic cascades via IL-33/ST2 signaling. Consistent with IL-13 secretion from activated ILC2s, the frequencies of polymorphonuclear (PMN)- and monocytic (M)-MDSCs were also significantly elevated during the progression from micro- to macrometastatic cancer. However, the effects of ILC2-induced MDSC functionality on the microenvironment differed in a metastatic-stage-specific manner. Our findings indicate that ILC2s may induce the immunosuppressive functions of MDSCs during the later stages of metastasis. Concomitantly, ILC2 may instigate extracellular matrix remodeling by PMN-MDSC activation during the early stages of metastasis. These metastatic-stage-specific changes may contribute to metastatic tumor growth in the microenvironment of breast cancer lung metastasis.

The natural cytotoxicity receptor NKp46 is dispensable for IL-22-mediated innate intestinal immune defense against Citrobacter rodentium.

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-gamma, 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.