Evidence of innate lymphoid cell redundancy in humans.

Innate lymphoid cells (ILCs) have potent immunological functions in experimental conditions in mice, but their contributions to immunity in natural conditions in humans have remained unclear. We investigated the presence of ILCs in a cohort of patients with severe combined immunodeficiency (SCID). All ILC subsets were absent in patients with SCID who had mutation of the gene encoding the common γ-chain cytokine receptor subunit IL-2Rγ or the gene encoding the tyrosine kinase JAK3. T cell reconstitution was observed in patients with SCID after hematopoietic stem cell transplantation (HSCT), but the patients still had considerably fewer ILCs in the absence of myeloablation than did healthy control subjects, with the exception of rare cases of reconstitution of the ILC1 subset of ILCs. Notably, the ILC deficiencies observed were not associated with any particular susceptibility to disease, with follow-up extending from 7 years to 39 years after HSCT. We thus report here selective ILC deficiency in humans and show that ILCs might be dispensable in natural conditions, if T cells are present and B cell function is preserved.

The Heterogeneity of Ly6Chi Monocytes Controls Their Differentiation into iNOS+ Macrophages or Monocyte-Derived Dendritic Cells.

Inflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c-MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3-CD11c-MHCII-PU.1lo monocytes differentiated into FcγRIII+PD-L2-CD209a-iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/- mice had reduced Flt3+CD11c-MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs.

A wave of bipotent T/ILC-restricted progenitors shapes the embryonic thymus microenvironment in a time-dependent manner.

During embryonic development, multiple waves of hematopoietic progenitors with distinct lineage potential are differentially regulated in time and space. Two different waves of thymic progenitors colonize the fetal thymus where they contribute to thymic organogenesis and homeostasis. The origin, the lineage differentiation potential of the first wave, and their relative contribution in shaping the thymus architecture, remained, however, unclear. Here, we show that the first wave of thymic progenitors comprises a unique population of bipotent T and innatel lymphoid cells (T/ILC), generating a lymphoid tissue inducer cells (LTi's), in addition to invariant Vγ5+ T cells. Transcriptional analysis revealed that innate lymphoid gene signatures and, more precisely, the LTi-associated transcripts were expressed in the first, but not in the second, wave of thymic progenitors. Depletion of early thymic progenitors in a temporally controlled manner showed that the progeny of the first wave is indispensable for the differentiation of autoimmune regulator-expressing medullary thymic epithelial cells (mTECs). We further show that these progenitors are of strict hematopoietic stem cell origin, despite the overlap between lymphopoiesis initiation and the transient expression of lymphoid-associated transcripts in yolk sac (YS) erythromyeloid-restricted precursors. Our work highlights the relevance of the developmental timing on the emergence of different lymphoid subsets, required for the establishment of a functionally diverse immune system.

Tbet promotes CXCR6 expression in immature natural killer cells and natural killer cell egress from the bone marrow.

Tbet-deficient mice have reduced natural killer (NK) cells in blood and spleen, but increased NK cells in bone marrow and lymph nodes, a phenotype that is thought to be the result of defective migration. Here, we revisit the role of Tbet in NK cell bone marrow egress. We definitively show that the accumulation of NK cells in the bone marrow of Tbet-deficient Tbx21-/- animals occurs because of a migration defect and identify a module of genes, co-ordinated by Tbet, which affects the localization of NK cells in the bone marrow. Cxcr6 is approximately 125-fold underexpressed in Tbx21-/- , compared with wild-type, immature NK cells. Immature NK cells accumulate in the bone marrow of CXCR6-deficient mice, and CXCR6-deficient progenitors are less able to reconstitute the peripheral NK cell compartment than their wild-type counterparts. However, the CXCR6 phenotype is largely confined to immature NK cells, whereas the Tbet phenotype is present in both immature and mature NK cells, suggesting that genes identified as being more differentially expressed in mature NK cells, such as S1pr5, Cx3cr1, Sell and Cd69, may be the major drivers of the phenotype.

CCR10+ ILC2s with ILC1-like properties exhibit a protective function in severe allergic asthma.

BACKGROUND: We previously showed that patients with severe allergic asthma have high numbers of circulating ILC2s expressing CCR10. METHOD: Herein, CCR10+ ILC2s were further analyzed in the blood of healthy individuals or patients with allergic and non-allergic asthma. Characteristics of human CCR10+ and CCR10- ILC2s were assessed by flow cytometry as well as single-cell multiplex RT-qPCR. The role of CCR10+ ILC2s in asthma pathophysiology was studied in allergen-treated mice. RESULTS: When compared to healthy controls, CCR10+ ILC2s are enriched in the blood of both allergic and non-allergic severe asthmatic patients, and these cells are recruited to the lungs. Plasma concentrations of the CCR10 ligand CCL27 are significantly increased in severe asthmatics when compared to non-asthmatic patients. CCR10+ ILC2s secrete little TH 2 cytokines, but exhibit ILC1-like properties, including a capacity to produce IFN-γ. Also, single-cell analysis reveals that the CCR10+ ILC2 subset is enriched in cells expressing amphiregulin. CCR10+ ILC2 depletion, as well as blocking of IFN-γ activity, exacerbates airway hyperreactivity in allergen-challenged mice, providing evidence for a protective role of these cells in allergic inflammation. CONCLUSIONS: Frequencies of circulating CCR10+ ILC2s and CCL27 plasma concentrations represent candidate markers of asthma severity. The characterization of CCR10+ ILC2s in human samples and in mouse asthma models suggests that these cells downregulate allergic inflammation through IFN-γ production.

Maintenance of Type 2 Response by CXCR6-Deficient ILC2 in Papain-Induced Lung Inflammation.

Innate lymphoid cells (ILC) are important players of early immune defenses in situations like lymphoid organogenesis or in case of immune response to inflammation, infection and cancer. Th1 and Th2 antagonism is crucial for the regulation of immune responses, however mechanisms are still unclear for ILC functions. ILC2 and NK cells were reported to be both involved in allergic airway diseases and were shown to be able to interplay in the regulation of the immune response. CXCR6 is a common chemokine receptor expressed by all ILC, and its deficiency affects ILC2 and ILC1/NK cell numbers and functions in lungs in both steady-state and inflammatory conditions. We determined that the absence of a specific ILC2 KLRG1+ST2- subset in CXCR6-deficient mice is probably dependent on CXCR6 for its recruitment to the lung under inflammation. We show that despite their decreased numbers, lung CXCR6-deficient ILC2 are even more activated cells producing large amount of type 2 cytokines that could drive eosinophilia. This is strongly associated to the decrease of the lung Th1 response in CXCR6-deficient mice.

CXCR6 Expression Is Important for Retention and Circulation of ILC Precursors.

Innate lymphoid cells are present at mucosal sites and represent the first immune barrier against infections, but what contributes to their circulation and homing is still unclear. Using Rag2(-/-) Cxcr6(Gfp/+) reporter mice, we assessed the expression and role of CXCR6 in the circulation of ILC precursors and their progeny. We identify CXCR6 expressing ILC precursors in the bone marrow and characterize their significant increase in CXCR6-deficient mice at steady state, indicating their partial retention in the bone marrow after CXCR6 ablation. Circulation was also impaired during embryonic life as fetal liver from CXCR6-deficient embryos displayed decreased numbers of ILC3 precursors. When injected, fetal CXCR6-deficient ILC3 precursors also fail to home and reconstitute ILC compartments in vivo. We show that adult intestinal ILC subsets have heterogeneous expression pattern of CXCR6, integrin α 4 ß 7, CD62L, CD69, and CD44, with ILC1 and ILC3 being more likely tissue resident lymphocytes. Intestinal ILC subsets were unchanged in percentages and numbers in both mice. We demonstrate that the ILC frequency is maintained due to a significant increase of ILC peripheral proliferation, as well as an increased proliferation of the in situ ILC precursors to compensate their retention in the bone marrow.

Bone marrow monocytes sustain NK cell-poiesis during non-alcoholic steatohepatitis.

Natural killer (NK) cells are the predominant lymphocyte population in the liver. At the onset of non-alcoholic steatohepatitis (NASH), an accumulation of activated NK cells is observed in the liver in parallel with inflammatory monocyte recruitment and an increased systemic inflammation. Using in vivo and in vitro experiments, we unveil a specific stimulation of NK cell-poiesis during NASH by medullary monocytes that trans-present interleukin-15 (IL-15) and secrete osteopontin, a biomarker for patients with NASH. This cellular dialogue leads to increased survival and maturation of NK precursors that are recruited to the liver, where they dampen the inflammatory monocyte infiltration. The increase in the production of both osteopontin and the IL-15/IL-15Rα complex by bone marrow monocytes is induced by endotoxemia. We propose a tripartite gut-liver-bone marrow axis regulating the immune population dynamics and effector functions during liver inflammation.

Natural Killer Cells and Type 1 Innate Lymphoid Cells Are New Actors in Non-alcoholic Fatty Liver Disease.

Obesity and associated liver diseases (Non Alcoholic Fatty Liver Disease, NAFLD) are a major public health problem with increasing incidence in Western countries (25% of the affected population). These complications develop from a fatty liver (steatosis) to an inflammatory state (steatohepatitis) evolving toward fibrosis and hepatocellular carcinoma. Lipid accumulation in the liver contributes to hepatocyte cell death and promotes liver injury. Local immune cells are activated either by Danger Associated Molecular Patterns (DAMPS) released by dead hepatocytes or by bacterial products (PAMPS) reaching the liver due to increased intestinal permeability. The resulting low-grade inflammatory state promotes the progression of liver complications toward more severe grades. Innate lymphoid cells (ILC) are an heterogeneous family of five subsets including circulating Natural Killer (NK) cells, ILC1, ILC2, ILC3, and lymphocytes tissue-inducer cells (LTi). NK cells and tissue-resident ILCs, mainly located at epithelial surfaces, are prompt to rapidly react to environmental changes to mount appropriate immune responses. Recent works have demonstrated the interplay between ILCs subsets and the environment within metabolic active organs such as liver, adipose tissue and gut during diet-induced obesity leading or not to hepatic abnormalities. Here, we provide an overview of the newly roles of NK cells and ILC1 in metabolism focusing on their contribution to the development of NAFLD. We also discuss recent studies that demonstrate the ability of these two subsets to influence tissue-specific metabolism and how their function and homeostasis are affected during metabolic disorders.

The Notch Signaling Pathway Is Balancing Type 1 Innate Lymphoid Cell Immune Functions.

The Notch pathway is one of the canonical signaling pathways implicated in the development of various solid tumors. During carcinogenesis, the Notch pathway dysregulation induces tumor expression of Notch receptor ligands participating to escape the immune surveillance. The Notch pathway conditions both the development and the functional regulation of lymphoid subsets. Its importance on T cell subset polarization has been documented contrary to its action on innate lymphoid cells (ILC). We aim to analyze the effect of the Notch pathway on type 1 ILC polarization and functions after disruption of the RBPJk-dependent Notch signaling cascade. Indeed, type 1 ILC comprises conventional NK (cNK) cells and type 1 helper innate lymphoid cells (ILC1) that share Notch-related functional characteristics such as the IFNg secretion downstream of T-bet expression. cNK cells have strong antitumor properties. However, data are controversial concerning ILC1 functions during carcinogenesis with models showing antitumoral capacities and others reporting ILC1 inability to control tumor growth. Using various mouse models of Notch signaling pathway depletion, we analyze the effects of its absence on type 1 ILC differentiation and cytotoxic functions. We also provide clues into its role in the maintenance of immune homeostasis in tissues. We show that modulating the Notch pathway is not only acting on tumor-specific T cell activity but also on ILC immune subset functions. Hence, our study uncovers the intrinsic Notch signaling pathway in ILC1/cNK populations and their response in case of abnormal Notch ligand expression. This study help evaluating the possible side effects mediated by immune cells different from T cells, in case of multivalent forms of the Notch receptor ligand delta 1 treatments. In definitive, it should help determining the best novel combination of therapeutic strategies in case of solid tumors.

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli. Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis. Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture. This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

Single-Cell Gene Expression Analyses Reveal Heterogeneous Responsiveness of Fetal Innate Lymphoid Progenitors to Notch Signaling.

T and innate lymphoid cells (ILCs) share some aspects of their developmental programs. However, although Notch signaling is strictly required for T cell development, it is dispensable for fetal ILC development. Constitutive activation of Notch signaling, at the common lymphoid progenitor stage, drives T cell development and abrogates ILC development by preventing Id2 expression. By combining single-cell transcriptomics and clonal culture strategies, we characterize two heterogeneous α4ß7-expressing lymphoid progenitor compartments. αLP1 (Flt3(+)) still retains T cell potential and comprises the global ILC progenitor, while αLP2 (Flt3(-)) consists of ILC precursors that are primed toward the different ILC lineages. Only a subset of αLP2 precursors is sensitive to Notch signaling required for their proliferation. Our study identifies, in a refined manner, the diversity of transitional stages of ILC development, their transcriptional signatures, and their differential dependence on Notch signaling.

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.