Resting natural killer cell homeostasis relies on tryptophan/NAD+ metabolism and HIF-1α.

Natural killer (NK) cells are forced to cope with different oxygen environments even under resting conditions. The adaptation to low oxygen is regulated by oxygen-sensitive transcription factors, the hypoxia-inducible factors (HIFs). The function of HIFs for NK cell activation and metabolic rewiring remains controversial. Activated NK cells are predominantly glycolytic, but the metabolic programs that ensure the maintenance of resting NK cells are enigmatic. By combining in situ metabolomic and transcriptomic analyses in resting murine NK cells, our study defines HIF-1α as a regulator of tryptophan metabolism and cellular nicotinamide adenine dinucleotide (NAD+ ) levels. The HIF-1α/NAD+ axis prevents ROS production during oxidative phosphorylation (OxPhos) and thereby blocks DNA damage and NK cell apoptosis under steady-state conditions. In contrast, in activated NK cells under hypoxia, HIF-1α is required for glycolysis, and forced HIF-1α expression boosts glycolysis and NK cell performance in vitro and in vivo. Our data highlight two distinct pathways by which HIF-1α interferes with NK cell metabolism. While HIF-1α-driven glycolysis is essential for NK cell activation, resting NK cell homeostasis relies on HIF-1α-dependent tryptophan/NAD+ metabolism.

Brain endothelial CXCL12 attracts protective natural killer cells during ischemic stroke.

BACKGROUND: The innate lymphoid cell (ILC) family consists of NK cells, ILC type 1, 2, 3 and lymphoid tissue inducer cells. They have been shown to play important roles in homeostasis and immune responses and are generally considered tissue resident. Not much is known about the presence of ILC members within the central nervous system and whether they are tissue resident in this organ too. Therefore, we studied the presence of all ILC members within the central nervous system and after ischemic brain insult. METHODS: We used the photothrombotic ischemic lesion method to induce ischemic lesions within the mouse brain. Using whole-mount immunofluorescence imaging, we established that the ILCs were present at the rim of the lesion. We quantified the increase of all ILC members at different time-points after the ischemic lesion induction by flow cytometry. Their migration route via chemokine CXCL12 was studied by using different genetic mouse models, in which we induced deletion of Cxcl12 within the blood-brain barrier endothelium, or its receptor, Cxcr4, in the ILCs. The functional role of the ILCs was subsequently established using the beam-walk sensorimotor test. RESULTS: Here, we report that ILCs are not resident within the mouse brain parenchyma during steady-state conditions, but are attracted towards the ischemic stroke. Specifically, we identify NK cells, ILC1s, ILC2s and ILC3s within the lesion, the highest influx being observed for NK cells and ILC1s. We further show that CXCL12 expressed at the blood-brain barrier is essential for NK cells and NKp46+ ILC3s to migrate toward the lesion. Complementary, Cxcr4-deficiency in NK cells prevents NK cells from entering the infarct area. Lack of NK cell migration results in a higher neurological deficit in the beam-walk sensorimotor test. CONCLUSIONS: This study establishes the lack of ILCs in the mouse central nervous system at steady-state and their migration towards an ischemic brain lesion. Our data show a role for blood-brain barrier-derived CXCL12 in attracting protective NK cells to ischemic brain lesions and identifies a new CXCL12/CXCR4-mediated component of the innate immune response to stroke.

Tissue-specific transcriptional profiles and heterogeneity of natural killer cells and group 1 innate lymphoid cells.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are populations of non-T, non-B lymphocytes in peripheral tissues. Although NK and ILC1 subsets have been described, their identification and characteristics remain unclear. We performed single-cell RNA sequencing and CITE-seq to explore NK and ILC1 heterogeneity between tissues. We observed that although NK1 and NK2 subsets are conserved in spleen and liver, ILC1s are heterogeneous across tissues. We identified sets of genes expressed by related subsets or characterizing unique ILC1 populations in each organ. The syndecan-4 appeared as a marker discriminating murine ILC1 from NK cells across organs. Finally, we revealed that the expressions of EOMES, GZMA, IRF8, JAK1, NKG7, PLEK, PRF1, and ZEB2 define NK cells and that IL7R, LTB, and RGS1 differentiate ILC1s from NK cells in mice and humans. Our data constitute an important resource to improve our understanding of NK-ILC1 origin, phenotype, and biology.

The transcription factor HIF-1α mediates plasticity of NKp46+ innate lymphoid cells in the gut.

Gut innate lymphoid cells (ILCs) show remarkable phenotypic diversity, yet microenvironmental factors that drive this plasticity are incompletely understood. The balance between NKp46+, IL-22-producing, group 3 ILCs (ILC3s) and interferon (IFN)-γ-producing group 1 ILCs (ILC1s) contributes to gut homeostasis. The gut mucosa is characterized by physiological hypoxia, and adaptation to low oxygen is mediated by hypoxia-inducible transcription factors (HIFs). However, the impact of HIFs on ILC phenotype and gut homeostasis is not well understood. Mice lacking the HIF-1α isoform in NKp46+ ILCs show a decrease in IFN-γ-expressing, T-bet+, NKp46+ ILC1s and a concomitant increase in IL-22-expressing, RORγt+, NKp46+ ILC3s in the gut mucosa. Single-cell RNA sequencing revealed HIF-1α as a driver of ILC phenotypes, where HIF-1α promotes the ILC1 phenotype by direct up-regulation of T-bet. Loss of HIF-1α in NKp46+ cells prevents ILC3-to-ILC1 conversion, increases the expression of IL-22-inducible genes, and confers protection against intestinal damage. Taken together, our results suggest that HIF-1α shapes the ILC phenotype in the gut.

Type 1 Innate Lymphoid Cells Limit the Antitumoral Immune Response.

Natural killer (NK) cells are known to be able to kill established tumor cell lines, but important caveats remain regarding their roles in the detection and elimination of developing primary tumors. Using a genetic model of selective ILC1 and NK cell deficiency, we showed that these cells were dispensable for tumor immunosurveillance and immunoediting in the MCA-induced carcinogenesis model. However, we were able to generate primary cell lines derived from MCA-induced tumors with graded sensitivity to NK1.1+ cells (including NK cells and ILC1). This differential sensitivity was associated neither with a modulation of intratumoral NK cell frequency, nor the capacity of tumor cells to activate NK cells. Instead, ILC1 infiltration into the tumor was found to be a critical determinant of NK1.1+ cell-dependent tumor growth. Finally, bulk tumor RNAseq analysis identified a gene expression signature associated with tumor sensitivity to NK1.1+ cells. ILC1 therefore appear to play an active role in inhibiting the antitumoral immune response, prompting to evaluate the differential tumor infiltration of ILC1 and NK cells in patients to optimize the harnessing of immunity in cancer therapies.

NK cells in hypoxic skin mediate a trade-off between wound healing and antibacterial defence.

During skin injury, immune response and repair mechanisms have to be coordinated for rapid skin regeneration and the prevention of microbial infections. Natural Killer (NK) cells infiltrate hypoxic skin lesions and Hypoxia-inducible transcription factors (HIFs) mediate adaptation to low oxygen. We demonstrate that mice lacking the Hypoxia-inducible factor (HIF)-1α isoform in NK cells show impaired release of the cytokines Interferon (IFN)-γ and Granulocyte Macrophage - Colony Stimulating Factor (GM-CSF) as part of a blunted immune response. This accelerates skin angiogenesis and wound healing. Despite rapid wound closure, bactericidal activity and the ability to restrict systemic bacterial infection are impaired. Conversely, forced activation of the HIF pathway supports cytokine release and NK cell-mediated antibacterial defence including direct killing of bacteria by NK cells despite delayed wound closure. Our results identify, HIF-1α in NK cells as a nexus that balances antimicrobial defence versus global repair in the skin.

Liver type 1 innate lymphoid cells develop locally via an interferon-γ-dependent loop.

The pathways that lead to the development of tissue-resident lymphocytes, including liver type 1 innate lymphoid cells (ILC1s), remain unclear. We show here that the adult mouse liver contains Lin-Sca-1+Mac-1+ hematopoietic stem cells derived from the fetal liver. This population includes Lin-CD122+CD49a+ progenitors that can generate liver ILC1s but not conventional natural killer cells. Interferon-γ (IFN-γ) production by the liver ILC1s themselves promotes the development of these cells in situ, through effects on their IFN-γR+ liver progenitors. Thus, an IFN-γ-dependent loop drives liver ILC1 development in situ, highlighting the contribution of extramedullary hematopoiesis to regional immune composition within the liver.

Chronic IL-15 Stimulation and Impaired mTOR Signaling and Metabolism in Natural Killer Cells During Acute Myeloid Leukemia.

Natural Killer (NK) cells are potent anti-leukemic immune effectors. However, they display multiple defects in acute myeloid leukemia (AML) patients leading to reduced anti-tumor potential. Our limited understanding of the mechanisms underlying these defects hampers the development of strategies to restore NK cell potential. Here, we have used a mouse model of AML to gain insight into these mechanisms. We found that leukemia progression resulted in NK cell maturation defects and functional alterations. Next, we assessed NK cell cytokine signaling governing their behavior. We showed that NK cells from leukemic mice exhibit constitutive IL-15/mTOR signaling and type I IFN signaling. However, these cells failed to respond to IL-15 stimulation in vitro as illustrated by reduced activation of the mTOR pathway. Moreover, our data suggest that mTOR-mediated metabolic responses were reduced in NK cells from AML-bearing mice. Noteworthy, the reduction of mTOR-mediated activation of NK cells during AML development partially rescued NK cell metabolic and functional defects. Altogether, our data strongly suggest that NK cells from leukemic mice are metabolically and functionally exhausted as a result of a chronic cytokine activation, at least partially IL-15/mTOR signaling. NK cells from AML patients also displayed reduced IL-2/15Rß expression and showed cues of reduced metabolic response to IL-15 stimulation in vitro, suggesting that a similar mechanism might occur in AML patients. Our study pinpoints the dysregulation of cytokine stimulation pathways as a new mechanism leading to NK cell defects in AML.

Multidimensional molecular controls defining NK/ILC1 identity in cancers.

Innate Lymphoid Cells (ILCs) are a recently described heterogeneous population of non-T, non-B lymphocytes. They are highly abundant at mucosal interfaces and, unlike T and B cells, they do not express somatically rearranged antigen-specific receptors. ILCs may be seen as the innate counterparts of T cells, but, major ILC deficiencies in humans appear to be clinically silent in modern conditions of hygiene and medicine, provided that T and B functions are preserved. NK cells are the founder members of this family and were originally classified in group 1 ILCs with ILC1s, due to similarities in cytokine production and development between these two types of cell. The classification of the ILC subsets was subsequently reviewed and five groups were defined on the basis of cytokine production and the discovery of specific transcription factors determining the different lineages. ILCs include NK cells, lymphoid tissue-inducer (LTi) cells and three other main subsets: ILC1s, ILC2s and ILC3s. The nature of distinct ILC1 population in mice and human is not consensual due to the high degree of similarity between ILCs and NK cells and their plastic relationships in some context. In this review, we will discuss the characteristics currently used for the phenotyping of NK cells and ILC1s in mice and humans, in the context of cancers especially, in which inappropriate discrimination between these two cell types can lead to erroneous conclusions regarding the specific impact of their targeting on tumors. Here, we suggest that multidimensional molecular controls, with the co-ordination of ontogeny-related signals, tissue-specific and tumor microenvironment-derived signals, determine the identity of NK cells and ILC1s. All these molecular stratifications contribute to the construction of cell fate for NK cells and ILC1s and account for the difficulties distinguishing between these two groups of cells.

Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny.

During embryogenesis, lymphoid tissue inducer (LTi) cells are essential for lymph node organogenesis. These cells are part of the innate lymphoid cell (ILC) family. Although their earliest embryonic hematopoietic origin is unclear, other innate immune cells have been shown to be derived from early hemogenic endothelium in the yolk sac as well as the aorta-gonad-mesonephros. A proper model to discriminate between these locations was unavailable. In this study, using a Cxcr4-CreERT2 lineage tracing model, we identify a major contribution from embryonic hemogenic endothelium, but not the yolk sac, toward LTi progenitors. Conversely, embryonic LTi cells are replaced by hematopoietic stem cell-derived cells in adults. We further show that, in the fetal liver, common lymphoid progenitors differentiate into highly dynamic alpha-lymphoid precursor cells that, at this embryonic stage, preferentially mature into LTi precursors and establish their functional LTi cell identity only after reaching the periphery.

Loss of HIF-1α in natural killer cells inhibits tumour growth by stimulating non-productive angiogenesis.

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.

Natural killer cell immunotherapies against cancer: checkpoint inhibitors and more.

After many years of research, recent advances have shed new light on the role of the immune system in advanced-stage cancer. Various types of immune cells may be useful for therapeutic purposes, along with chemical molecules and engineered monoclonal antibodies. The immune effectors suitable for manipulation for adoptive transfer or drug targeting in vivo include natural killer (NK) cells. These cells are of particular interest because they are tightly regulated by an array of inhibitory and activating receptors, enabling them to kill tumor cells while sparing normal cells. New therapeutic antibodies blocking the interactions of inhibitory receptors (immune checkpoint inhibitors, ICI) with their ligands have been developed and can potentiate NK cell functions in vivo.

Complement factor P is a ligand for the natural killer cell-activating receptor NKp46.

Innate lymphoid cells (ILCs) are involved in immune responses to microbes and various stressed cells, such as tumor cells. They include group 1 [such as natural killer (NK) cells and ILC1], group 2, and group 3 ILCs. Besides their capacity to respond to cytokines, ILCs detect their targets through a series of cell surface-activating receptors recognizing microbial and nonmicrobial ligands. The nature of some of these ligands remains unclear, limiting our understanding of ILC biology. We focused on NKp46, which is highly conserved in mammals and expressed by all mature NK cells and subsets of ILC1 and ILC3. We show here that NKp46 binds to a soluble plasma glycoprotein, the complement factor P (CFP; properdin), the only known positive regulator of the alternative complement pathway. Consistent with the selective predisposition of patients lacking CFP to lethal Neisseria meningitidis (Nm) infections, NKp46 and group 1 ILCs bearing this receptor were found to be required for mice to survive Nm infection. Moreover, the beneficial effects of CFP treatment for Nm infection were dependent on NKp46 and group 1 NKp46+ ILCs. Thus, group 1 NKp46+ ILCs interact with the complement pathway, via NKp46, revealing a cross-talk between two partners of innate immunity in the response to an invasive bacterial infection.

Lessons from NK Cell Deficiencies in the Mouse.

Since their discovery in the late 1970s, in vivo studies on mouse natural killer (NK) cell almost entirely relied on the use of depleting antibodies and were associated with significant limitations. More recently, large-scale gene-expression analyses allowed the identification of NKp46 as one of the best markers of NK cells across mammalian species. Since then, NKp46 has been shown to be expressed on other subsets of innate lymphoid cells (ILCs) such as the closely related ILC1 and the mucosa-associated NCR(+) ILC3. Based on this marker, several mouse models specifically targeting NKp46-expressing cell have recently been produced. Here, we review recent advances in the generation of models of deficiency in NKp46-expressing cells and their use to address the role of NK cells in immunity, notably on the regulation of adaptive immune responses.

Complementarity and redundancy of IL-22-producing innate lymphoid cells.

Intestinal T cells and group 3 innate lymphoid cells (ILC3 cells) control the composition of the microbiota and gut immune responses. Within the gut, ILC3 subsets coexist that either express or lack the natural cytoxicity receptor (NCR) NKp46. We identified here the transcriptional signature associated with the transcription factor T-bet-dependent differentiation of NCR(-) ILC3 cells into NCR(+) ILC3 cells. Contrary to the prevailing view, we found by conditional deletion of the key ILC3 genes Stat3, Il22, Tbx21 and Mcl1 that NCR(+) ILC3 cells were redundant for the control of mouse colonic infection with Citrobacter rodentium in the presence of T cells. However, NCR(+) ILC3 cells were essential for cecal homeostasis. Our data show that interplay between intestinal ILC3 cells and adaptive lymphocytes results in robust complementary failsafe mechanisms that ensure gut homeostasis.

Transcription factor Foxo1 is a negative regulator of natural killer cell maturation and function.

Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes by upregulating CD62L expression and inhibited late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21(+/-) mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions.

ICOS coreceptor signaling inactivates the transcription factor FOXO1 to promote Tfh cell differentiation.

T follicular helper (Tfh) cells are essential in the induction of high-affinity, class-switched antibodies. The differentiation of Tfh cells is a multi-step process that depends upon the co-receptor ICOS and the activation of phosphoinositide-3 kinase leading to the expression of key Tfh cell genes. We report that ICOS signaling inactivates the transcription factor FOXO1, and a Foxo1 genetic deletion allowed for generation of Tfh cells with reduced dependence on ICOS ligand. Conversely, enforced nuclear localization of FOXO1 inhibited Tfh cell development even though ICOS was overexpressed. FOXO1 regulated Tfh cell differentiation through a broad program of gene expression exemplified by its negative regulation of Bcl6. Final differentiation to germinal center Tfh cells (GC-Tfh) was instead FOXO1 dependent as the Foxo1(-/-) GC-Tfh cell population was substantially reduced. We propose that ICOS signaling transiently inactivates FOXO1 to initiate a Tfh cell contingency that is completed in a FOXO1-dependent manner.

T cell regulation of natural killer cells.

In light of their role in the immune response against tumors and viruses, natural killer (NK) cells represent a promising target for immunotherapy. Before this target is reached, the various mechanisms that control NK cell activity must first be identified and understood. In the past decades, studies have identified two critical processes that prevent spontaneous NK cell-mediated autoimmune activation while maximizing the efficiency of these cells during an immune response. First is the education process, whereby NK cells adapt to their environment by sensing ligands for inhibitory and activating receptors. Second is the priming phase of NK cell activation, which arms NK cells with appropriate cytotoxic molecules during inflammation. New studies now indicate that NK cell proliferation, accumulation, and activation are also under the control of regulatory T cells that restrict availability of IL-2 released by activated CD4(+) T cells. Together with other recent studies, these data highlight the importance of the adaptive immune system in the regulation of NK cell activity.

Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor.

NKp46 is a cell surface receptor expressed on natural killer (NK) cells, on a minute subset of T cells, and on a population of innate lymphoid cells that produce IL-22 and express the transcription factor retinoid-related orphan receptor (ROR)-γt, referred to as NK cell receptor (NKR)(+)ROR-γt(+) cells. Here we describe Nkp46(iCre) knock-in mice in which the gene encoding the improved Cre (iCre) recombinase was inserted into the Nkp46 locus. This mouse was used to noninvasively trace cells expressing NKp46 in vivo. Fate mapping experiments demonstrated the stable expression of NKp46 on NK cells and allowed a reappraisal of the sequential steps of NK cell maturation. NKp46 genetic tracing also showed that gut NKR(+)ROR-γt(+) and NK cells represent two distinct lineages. In addition, the genetic heterogeneity of liver NK cells was evidenced. Finally, Nkp46(iCre) mice also represent a unique mouse model of conditional mutagenesis specifically in NKp46(+) cells, paving the way for further developments in the biology of NKp46(+) NK, T, and NKR(+)ROR-γt(+) cells.