Divergent roles for STAT4 in shaping differentiation of cytotoxic ILC1 and NK cells during gut inflammation.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) require signal transducer and activator of transcription 4 (STAT4) to elicit rapid effector responses and protect against pathogens. By combining genetic and transcriptomic approaches, we uncovered divergent roles for STAT4 in regulating effector differentiation of these functionally related cell types. Stat4 deletion in Ncr1-expressing cells led to impaired NK cell terminal differentiation as well as to an unexpected increased generation of cytotoxic ILC1 during intestinal inflammation. Mechanistically, Stat4-deficient ILC1 exhibited upregulation of gene modules regulated by STAT5 in vivo and an aberrant effector differentiation upon in vitro stimulation with IL-2, used as a prototypical STAT5 activator. Moreover, STAT4 expression in NCR+ innate lymphocytes restrained gut inflammation in the dextran sulfate sodium-induced colitis model limiting pathogenic production of IL-13 from adaptive CD4+ T cells in the large intestine. Collectively, our data shed light on shared and distinctive mechanisms of STAT4-regulated transcriptional control in NK cells and ILC1 required for intestinal inflammatory responses.

Host genetic control of natural killer cell diversity revealed in the Collaborative Cross.

Natural killer (NK) cells are innate effectors armed with cytotoxic and cytokine-secreting capacities whose spontaneous antitumor activity is key to numerous immunotherapeutic strategies. However, current mouse models fail to mirror the extensive immune system variation that exists in the human population which may impact on NK cell-based therapies. We performed a comprehensive profiling of NK cells in the Collaborative Cross (CC), a collection of novel recombinant inbred mouse strains whose genetic diversity matches that of humans, thereby providing a unique and highly diverse small animal model for the study of immune variation. We demonstrate that NK cells from CC strains displayed a breadth of phenotypic and functional variation reminiscent of that reported for humans with regards to cell numbers, key marker expression, and functional capacities. We took advantage of the vast genetic diversity of the CC and identified nine genomic loci through quantitative trait locus mapping driving these phenotypic variations. SNP haplotype patterns and variant effect analyses identified candidate genes associated with lung NK cell numbers, frequencies of CD94+ NK cells, and expression levels of NKp46. Thus, we demonstrate that the CC represents an outstanding resource to study NK cell diversity and its regulation by host genetics.

Conditional Genetic Ablation Mouse Models as a Tool to Study Cancer Immunosurveillance In Vivo.

Over the last decades, it has been established that the immune system is crucial for the impediment of cancer development by recognizing and destroying transformed cells. This process has been termed cancer immunosurveillance. Small animal models have significantly facilitated our understanding of it. Dissecting the contribution of any specific immune cell type participating in this process requires the ability to specifically target it while leaving the other immune components as well as the cancer model system unperturbed in vivo. Here, we provide a simple and rapid protocol for the generation of transgenic mice expressing Cre recombinase in a cell type-specific manner-in our example we chose cells expressing Ncr1, which encodes for the surface protein NKp46-and the use of those mice to ablate NKp46+ cells in order to study their role in a model of cancer immunosurveillance against experimental pulmonary metastases. This protocol can easily be adapted to target other cell types and other cancer models.

Notch signaling in group 3 innate lymphoid cells modulates their plasticity.

The Notch signaling pathway is conserved throughout evolution, and it controls various processes, including cell fate determination, differentiation, and proliferation. Innate lymphoid cells (ILCs) are lymphoid cells lacking antigen receptors that fulfill effector and regulatory functions in innate immunity and tissue remodeling. Type 3 ILCs (ILC3s) reinforce the epithelial barrier and maintain homeostasis with intestinal microbiota. We demonstrated that the population of natural cytotoxicity receptor-positive (NCR(+)) ILC3s in mice is composed of two subsets that have distinct developmental requirements. A major subset depended on the activation of Notch2 in NCR(-) ILC3 precursors in the lamina propria of the small intestine to stimulate expression of the genes encoding the transcription factors T-bet, RORγt, and aryl hydrocarbon receptor (AhR). Notch signaling contributed to the transition of NCR(-) cells into NCR(+) cells, the more proinflammatory subset, in a cell-autonomous manner. In the absence of Notch signaling, this subset of NCR(-) ILC3s did not acquire the gene expression profile of NCR(+) ILC3s. A second subset of NCR(+) ILC3s did not depend on Notch for their development or for increased transcription factor abundance; however, their production of cytokines and cell surface abundance of NCRs were decreased in the absence of Notch signaling. Together, our data suggest that Notch is a regulator of the plasticity of ILC3s by controlling NCR(+) cell fate.

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.

Transcriptional regulation of innate lymphoid cell fate.

Innate lymphoid cells (ILCs) are a recently described family of lymphoid effector cells that have important roles in immune defence, inflammation and tissue remodelling. It has been proposed that ILCs represent 'innate' homologues of differentiated effector T cells, and they have been categorized into three groups ­ namely, ILC1s, ILC2s and ILC3s ­ on the basis of their expression of cytokines and transcription factors that are typically associated with T helper 1 (T(H)1)-, T(H)2- and T(H)17-type immune responses, respectively. Indeed, remarkable similarity is seen between the specific transcription factors required for the development and diversification of different ILC groups and those that drive effector T cell differentiation. The recent identification of dedicated ILC precursors has provided a view of the mechanisms that control this first essential stage of ILC development. Here, we discuss the transcriptional mechanisms that regulate ILC development and diversification into distinct effector subsets with key roles in immunity and tissue homeostasis. We further caution against the current distinction between 'helper' versus 'killer' subsets in the evolving area of ILC nomenclature.

A novel immunoregulatory role for NK-cell cytotoxicity in protection from HLH-like immunopathology in mice.

The impairment of cytotoxic activity of lymphocytes disturbs immune surveillance and leads to the development of hemophagocytic lymphohistiocytic syndrome (HLH). Although cytotoxic T lymphocyte (CTL) control of HLH development is well documented, the role for natural killer (NK)-cell effector functions in the pathogenesis of this immune disorder remains unclear. In this study, we specifically targeted a defect in cytotoxicity to either CTL or NK cells in mice so as to dissect the contribution of these lymphocyte subsets to HLH-like disease severity after lymphocytic choriomeningitis virus (LCMV) infection. We found that NK-cell cytotoxicity was sufficient to protect mice from the fatal outcome that characterizes HLH-like disease and was also sufficient to reduce HLH-like manifestations. Mechanistically, NK-cell cytotoxicity reduced tissue infiltration by inflammatory macrophages and downmodulated LCMV-specific T-cell responses by limiting hyperactivation of CTL. Interestingly, the critical protective effect of NK cells on HLH was independent of interferon-γ secretion and changes in viral load. Therefore our findings identify a crucial role of NK-cell cytotoxicity in limiting HLH-like immunopathology, highlighting the important role of NK cytotoxic activity in immune homeostasis.

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.

Gata3 drives development of RORγt+ group 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3) include IL-22-producing NKp46(+) cells and IL-17A/IL-22-producing CD4(+) lymphoid tissue inducerlike cells that express RORγt and are implicated in protective immunity at mucosal surfaces. Whereas the transcription factor Gata3 is essential for T cell and ILC2 development from hematopoietic stem cells (HSCs) and for IL-5 and IL-13 production by T cells and ILC2, the role for Gata3 in the generation or function of other ILC subsets is not known. We found that abundant GATA-3 protein is expressed in mucosa-associated ILC3 subsets with levels intermediate between mature B cells and ILC2. Chimeric mice generated with Gata3-deficient fetal liver hematopoietic precursors lack all intestinal RORγt(+) ILC3 subsets, and these mice show defective production of IL-22 early after infection with the intestinal pathogen Citrobacter rodentium, leading to impaired survival. Further analyses demonstrated that ILC3 development requires cell-intrinsic Gata3 expression in fetal liver hematopoietic precursors. Our results demonstrate that Gata3 plays a generalized role in ILC lineage determination and is critical for the development of gut RORγt(+) ILC3 subsets that maintain mucosal barrier homeostasis. These results further extend the paradigm of Gata3-dependent regulation of diversified innate ILC and adaptive T cell subsets.

The Rag2⁻Il2rb⁻Dmd⁻ mouse: a novel dystrophic and immunodeficient model to assess innovating therapeutic strategies for muscular dystrophies.

The development of innovative therapeutic strategies for muscular dystrophies, particularly cell-based approaches, is still a developing field. Although positive results have been obtained in animal models, they have rarely been confirmed in patients and resulted in very limited clinical improvements, suggesting some specificity in humans. These findings emphasized the need for an appropriate animal model (i.e., immunodeficient and dystrophic) to investigate in vivo the behavior of transplanted human myogenic stem cells. We report a new model, the Rag2(-)Il2rb(-)Dmd(-) mouse, which lacks T, B, and NK cells, and also carries a mutant Dmd allele that prevents the production of any dystrophin isoform. The dystrophic features of this new model are comparable with those of the classically used mdx mouse, but with the total absence of any revertant dystrophin positive fiber. We show that Rag2(-)Il2rb(-)Dmd(-) mice allow long-term xenografts of human myogenic cells. Altogether, our findings indicate that the Rag2(-)Il2rb(-)Dmd(-) mouse represents an ideal model to gain further insights into the behavior of human myogenic stem cells in a dystrophic context, and can be used to assess innovative therapeutic strategies for muscular dystrophies.

Essential, dose-dependent role for the transcription factor Gata3 in the development of IL-5+ and IL-13+ type 2 innate lymphoid cells.

Group 2 innate lymphoid cells (ILC2s; also called nuocytes, innate helper cells, or natural helper cells) provide protective immunity during helminth infection and play an important role in influenza-induced and allergic airway hyperreactivity. Whereas the transcription factor GATA binding protein 3 (Gata3) is important for the production of IL-5 and -13 by ILC2s in response to IL-33 or -25 stimulation, it is not known whether Gata3 is required for ILC2 development from hematopoietic stem cells. Here, we show that chimeric mice generated with Gata3-deficient fetal liver hematopoietic stem cells fail to develop systemically dispersed ILC2s. In these chimeric mice, in vivo administration of IL-33 or -25 fails to expand ILC2 numbers or to induce characteristic ILC2-dependent IL-5 or -13 production. Moreover, cell-intrinsic Gata3 expression is required for ILC2 development in vitro and in vivo. Using mutant and transgenic mice in which Gata3 gene copy number is altered, we show that ILC2 generation from common lymphoid progenitors, as well as ILC2 homeostasis and cytokine production, is regulated by Gata3 expression levels in a dose-dependent fashion. Collectively, these results identify Gata3 as a critical early regulator of ILC2 development, thereby extending the paradigm of Gata3-dependent control of type 2 immunity to include both innate and adaptive lymphocytes.

Developmental programming of natural killer and innate lymphoid cells.

In recent years we have witnessed a blooming interest in innate lymphoid cell (ILC) biology thanks to the discovery of novel lineages of ILC that are phenotypically and functionally distinct from NK cells. While the importance of these novel ILC subsets as essential functional components of the early immune responses are now clearly established, many questions remain as to how early ILC developmental fates are determined and how specific effector functions associated with individual ILC subsets are achieved. As the founding member of the ILC family, properties of NK cells have defining attributes that characterize this group of innate effectors. Analysing their developmental rules may provide clues to principles that guide ILC development in general.

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-1ß regulates a novel myeloid-derived suppressor cell subset that impairs NK cell development and function.

Chronic inflammation is associated with promotion of malignancy and tumor progression. Many tumors enhance the accumulation of myeloid-derived suppressor cells (MDSC), which contribute to tumor progression and growth by suppressing anti-tumor immune responses. Tumor-derived IL-1ß secreted into the tumor microenvironment has been shown to induce the accumulation of MDSC possessing an enhanced capacity to suppress T cells. In this study, we found that the enhanced suppressive potential of IL-1ß-induced MDSC was due to the activity of a novel subset of MDSC lacking Ly6C expression. This subset was present at low frequency in tumor-bearing mice in the absence of IL-1ß-induced inflammation; however, under inflammatory conditions, Ly6C(neg) MDSC were predominant. Ly6C(neg) MDSC impaired NK cell development and functions in vitro and in vivo. These results identify a novel IL-1ß-induced subset of MDSC with unique functional properties. Ly6C(neg) MDSC mediating NK cell suppression may thus represent useful targets for therapeutic interventions.

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.

The intrathymic crossroads of T and NK cell differentiation.

T lymphocytes depend on the thymic microenvironment for initiation of the T-cell developmental program. As the progenitors in the thymus have lost the capacity to self-renew, this process depends on the constant influx of hematopoietic progenitors that originate in the bone marrow. Nevertheless, thymic emigrants are heterogeneous and retain developmental plasticity for both the myeloid and lymphoid lineages. It is the role of the thymic microenvironment to steer these uncommitted progenitors toward a T-cell fate. Still, the thymus also generates a unique population of thymic NK cells, thus raising the question of how the T versus NK lymphoid cell fate is determined intrathymically. Many factors have been implicated in the developmental pathways in the thymus, and the processes are characterized by both subtle and not so subtle modifications in gene expression. In this review, we consider the crucial factors governing lineage determination of T cells versus NK cells from bi-potent thymic NK/T precursors. Recent reports have shed new light on the complex interactions of cytokines and transcription factors at different cell fate decision branch points in thymopoiesis. We discuss the implications of these findings and propose a model that may be applicable at this critical thymic NK/T juncture.

A 'natural' way to provide innate mucosal immunity.

The mucosal barrier comprises a layered defense system including physio-chemical and immunological strategies to contain commensal microflora while protecting the host against potential pathogens. In contrast to the clearly established and well-characterized role for the adaptive immune system in intestinal defense, our knowledge on innate immune mechanisms that operate in the gut is much less defined. The recent identification of novel innate lymphoid cells (ILC), including 'NK-like' cells that naturally produce IL-22 and appear to play a role in intestinal defense, demonstrates an unexpected and increasing complexity in mucosal innate immunity.

IL-7 and IL-15 independently program the differentiation of intestinal CD3-NKp46+ cell subsets from Id2-dependent precursors.

The natural cytotoxicity receptor NKp46 (encoded by Ncr1) was recently shown to identify a subset of noncytotoxic, Rag-independent gut lymphocytes that express the transcription factor Rorc, produce interleukin (IL)-22, and provide innate immune protection at the intestinal mucosa. Intestinal CD3(-)NKp46(+) cells are phenotypically heterogeneous, comprising a minority subset that resembles classical mature splenic natural killer (NK) cells (NK1.1(+), Ly49(+)) but also a large CD127(+)NK1.1(-) subset of lymphoid tissue inducer (LTi)-like Rorc(+) cells that has been proposed to include NK cell precursors. We investigated the developmental relationships between these intestinal CD3(-)NKp46(+) subsets. Gut CD3(-)NKp46(+) cells were related to LTi and NK cells in requiring the transcriptional inhibitor Id2 for normal development. Overexpression of IL-15 in intestinal epithelial cells expanded NK1.1(+) cells within the gut but had no effect on absolute numbers of the CD127(+)NK1.1(-)Rorc(+) subset of CD3(-)NKp46(+) cells. In contrast, IL-7 deficiency strongly reduced the overall numbers of CD3(-)NKp46(+)NK1.1(-) cells that express Rorc and produce IL-22 but failed to restrict homeostasis of classical intestinal NK1.1(+) cells. Finally, in vivo fate-mapping experiments demonstrated that intestinal NK1.1(+)CD127(-) cells are not the progeny of Rorc-expressing progenitors, indicating that CD127(+)NK1.1(-)Rorc(+) cells are not canonical NK cell precursors. These studies highlight the independent cytokine regulation of functionally diverse intestinal NKp46(+) cell subsets.

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.

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.