Yann M. Kerdiles

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.

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.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are populations of non-T, non-B lymphocytes found in peripheral tissues. Although several NK and ILC1 subsets have been described their identification and tissue specific characteristics remains unclear. We performed single-cell RNA sequencing and used CITE-seq technology to explore NK and ILC1 heterogeneity between tissues. We observed that the two main NK cell subsets are conserved in spleen and liver. In contrast, ILC1 are highly heterogeneous across tissues, and this diversity stems in part from tissue-restricted differentiation pathways. Using individual intra-tissues integrated analysis, we identified sets of genes expressed by related subsets or characterizing unique ILC1 populations in each organ. The transmembrane heparan sulfate proteoglycan syndecan-4 (SDC4) appeared as a cell surface protein discriminating ILC1 from NK cells across organs in the mouse. Finally, we revealed that the expression of EOMES, GZMA, IRF8, JAK1, NKG7, PLEK, PRF1 and ZEB2 define NK cells in mice and humans, and that IL7R, LTB and RGS1 differentiate ILC1 from NK cells in these species. Our data highlight the tissue-specific identities of ILC1s and precise the phenotypic and genetic bases of this heterogeneity, improving our understanding of NK cell and ILC1 origin, phenotype and biology. Funding Information: We thank the animal and flow cytometry facilities at the Centre d’Immunologie de Marseille-Luminy for technical support. The E.V. laboratory at CIML and Assistance-Publique des Hôpitaux de Marseille is supported by funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (TILC, grant agreement No. 694502 and MInfla-TILC, grant agreement No. 875102 - MInfla-Tilc), the Agence Nationale de la Recherche including the PIONEER Project (ANR-17-RHUS-0007), MSDAvenir, Innate Pharma and institutional grants awarded to the CIML (INSERM, CNRS, and Aix-Marseille University) and Marseille Immunopole. Declaration of Interests: N.L., B.E. and Y.K. have no competiting interests J.G. and S.B. are employee of Innate Pharma E.V is a cofounder and employee of Innate Pharma. Ethics Approval Statement: All experiments were performed in accordance with national and European regulations and were approved by the Ethics Committee for Animal Experimentation of Marseille (Comité National de Réflexion Ethique sur l’Expérimentation Animale no.014).

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.

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.

An IFN-γ feedback loop: Innate lymphoid cells (ILCs) play important roles in tissue homeostasis and host defense. Type 1 ILCs (ILC1s) produce interferon-γ (IFN-γ) and require the transcriptional master regulator T-bet. The pathways underlying how these cells develop and differentiate have remained poorly understood. Bai et al. found that the adult mouse liver contains a population of Lin–Sca-1 ⁺ Mac-1 ⁺ hematopoietic stem cells (LSM HSCs) that preferentially differentiate into tissue-resident liver ILC1s. They further show that IFN-γ produced by mature ILC1s promotes the expansion and differentiation of LSM HSCs into ILC1s but not natural killer cells. This work expands our understanding of extramedullary hematopoiesis and underscores the unique immune status of the liver. Science , this issue p. eaba4177

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.