Stem-like intestinal Th17 cells give rise to pathogenic effector T cells during autoimmunity.

While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.

IFNγ and GM-CSF control complementary differentiation programs in the monocyte-to-phagocyte transition during neuroinflammation.

During inflammation, Ly6Chi monocytes are rapidly mobilized from the bone marrow (BM) and are recruited into inflamed tissues, where they undergo monocyte-to-phagocyte transition (MTPT). The in vivo developmental trajectories of the MTPT and the contribution of individual cytokines to this process remain unclear. Here, we used a murine model of neuroinflammation to investigate how granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-γ (IFNγ), two type 1 cytokines, controlled MTPT. Using genetic fate mapping, gene targeting and high-dimensional single-cell multiomics analyses, we found that IFNγ was essential for the gradual acquisition of a mature inflammatory phagocyte phenotype in Ly6Chi monocytes, while GM-CSF was required to license interleukin-1ß (IL-1ß) production, phagocytosis and oxidative burst. These results suggest that the proinflammatory cytokine environment guided MTPT trajectories in the inflamed central nervous system (CNS) and indicated that GM-CSF was the most prominent target for the disarming of monocyte progenies during neuroinflammation.

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity.

Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of ß-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1ß and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery.

Lung Single-Cell Signaling Interaction Map Reveals Basophil Role in Macrophage Imprinting.

Lung development and function arises from the interactions between diverse cell types and lineages. Using single-cell RNA sequencing (RNA-seq), we characterize the cellular composition of the lung during development and identify vast dynamics in cell composition and their molecular characteristics. Analyzing 818 ligand-receptor interaction pairs within and between cell lineages, we identify broadly interacting cells, including AT2, innate lymphocytes (ILCs), and basophils. Using interleukin (IL)-33 receptor knockout mice and in vitro experiments, we show that basophils establish a lung-specific function imprinted by IL-33 and granulocyte-macrophage colony-stimulating factor (GM-CSF), characterized by unique signaling of cytokines and growth factors important for stromal, epithelial, and myeloid cell fates. Antibody-depletion strategies, diphtheria toxin-mediated selective depletion of basophils, and co-culture studies show that lung resident basophils are important regulators of alveolar macrophage development and function. Together, our study demonstrates how whole-tissue signaling interaction map on the single-cell level can broaden our understanding of cellular networks in health and disease.

Migration-induced cell shattering due to DOCK8 deficiency causes a type 2-biased helper T cell response.

Mutations that impact immune cell migration and result in immune deficiency illustrate the importance of cell movement in host defense. In humans, loss-of-function mutations in DOCK8, a guanine exchange factor involved in hematopoietic cell migration, lead to immunodeficiency and, paradoxically, allergic disease. Here, we demonstrate that, like humans, Dock8-/- mice have a profound type 2 CD4+ helper T (TH2) cell bias upon pulmonary infection with Cryptococcus neoformans and other non-TH2 stimuli. We found that recruited Dock8-/-CX3CR1+ mononuclear phagocytes are exquisitely sensitive to migration-induced cell shattering, releasing interleukin (IL)-1ß that drives granulocyte-macrophage colony-stimulating factor (GM-CSF) production by CD4+ T cells. Blocking IL-1ß, GM-CSF or caspase activation eliminated the type-2 skew in mice lacking Dock8. Notably, treatment of infected wild-type mice with apoptotic cells significantly increased GM-CSF production and TH2 cell differentiation. This reveals an important role for cell death in driving type 2 signals during infection, which may have implications for understanding the etiology of type 2 CD4+ T cell responses in allergic disease.

Structure of the interleukin-5 receptor complex exemplifies the organizing principle of common beta cytokine signaling.

Cytokines regulate immune responses by binding to cell surface receptors, including the common subunit beta (ßc), which mediates signaling for GM-CSF, IL-3, and IL-5. Despite known roles in inflammation, the structural basis of IL-5 receptor activation remains unclear. We present the cryo-EM structure of the human IL-5 ternary receptor complex, revealing architectural principles for IL-5, GM-CSF, and IL-3. In mammalian cell culture, single-molecule imaging confirms hexameric IL-5 complex formation on cell surfaces. Engineered chimeric receptors show that IL-5 signaling, as well as IL-3 and GM-CSF, can occur through receptor heterodimerization, obviating the need for higher-order assemblies of ßc dimers. These findings provide insights into IL-5 and ßc receptor family signaling mechanisms, aiding in the development of therapies for diseases involving deranged ßc signaling.

Amplification of autoimmune organ damage by NKp46-activated ILC1s.

In systemic lupus erythematosus, loss of immune tolerance, autoantibody production and immune complex deposition are required but not sufficient for organ damage1. How inflammatory signals are initiated and amplified in the setting of autoimmunity remains elusive. Here we set out to dissect layers and hierarchies of autoimmune kidney inflammation to identify tissue-specific cellular hubs that amplify autoinflammatory responses. Using high-resolution single-cell profiling of kidney immune and parenchymal cells, in combination with antibody blockade and genetic deficiency, we show that tissue-resident NKp46+ innate lymphoid cells (ILCs) are crucial signal amplifiers of disease-associated macrophage expansion and epithelial cell injury in lupus nephritis, downstream of autoantibody production. NKp46 signalling in a distinct subset of group 1 ILCs (ILC1s) instructed an unconventional immune-regulatory transcriptional program, which included the expression of the myeloid cell growth factor CSF2. CSF2 production by NKp46+ ILCs promoted the population expansion of monocyte-derived macrophages. Blockade of the NKp46 receptor (using the antibody clone mNCR1.15; ref. 2) or genetic deficiency of NKp46 abrogated epithelial cell injury. The same cellular and molecular patterns were operative in human lupus nephritis. Our data provide support for the idea that NKp46+ ILC1s promote parenchymal cell injury by granting monocyte-derived macrophages access to epithelial cell niches. NKp46 activation in ILC1s therefore constitutes a previously unrecognized, crucial tissue rheostat that amplifies organ damage in autoimmune hosts, with broad implications for inflammatory pathologies and therapies.

Tuberculosis in otherwise healthy adults with inherited TNF deficiency.

Severe defects in human IFNγ immunity predispose individuals to both Bacillus Calmette-Guérin disease and tuberculosis, whereas milder defects predispose only to tuberculosis1. Here we report two adults with recurrent pulmonary tuberculosis who are homozygous for a private loss-of-function TNF variant. Neither has any other clinical phenotype and both mount normal clinical and biological inflammatory responses. Their leukocytes, including monocytes and monocyte-derived macrophages (MDMs) do not produce TNF, even after stimulation with IFNγ. Blood leukocyte subset development is normal in these patients. However, an impairment in the respiratory burst was observed in granulocyte-macrophage colony-stimulating factor (GM-CSF)-matured MDMs and alveolar macrophage-like (AML) cells2 from both patients with TNF deficiency, TNF- or TNFR1-deficient induced pluripotent stem (iPS)-cell-derived GM-CSF-matured macrophages, and healthy control MDMs and AML cells differentiated with TNF blockers in vitro, and in lung macrophages treated with TNF blockers ex vivo. The stimulation of TNF-deficient iPS-cell-derived macrophages with TNF rescued the respiratory burst. These findings contrast with those for patients with inherited complete deficiency of the respiratory burst across all phagocytes, who are prone to multiple infections, including both Bacillus Calmette-Guérin disease and tuberculosis3. Human TNF is required for respiratory-burst-dependent immunity to Mycobacterium tuberculosis in macrophages but is surprisingly redundant otherwise, including for inflammation and immunity to weakly virulent mycobacteria and many other infectious agents.

The Cytokine IL-17A Limits Th17 Pathogenicity via a Negative Feedback Loop Driven by Autocrine Induction of IL-24.

Dysregulated Th17 cell responses underlie multiple inflammatory and autoimmune diseases, including autoimmune uveitis and its animal model, EAU. However, clinical trials targeting IL-17A in uveitis were not successful. Here, we report that Th17 cells were regulated by their own signature cytokine, IL-17A. Loss of IL-17A in autopathogenic Th17 cells did not reduce their pathogenicity and instead elevated their expression of the Th17 cytokines GM-CSF and IL-17F. Mechanistic in vitro studies revealed a Th17 cell-intrinsic autocrine loop triggered by binding of IL-17A to its receptor, leading to activation of the transcription factor NF-κB and induction of IL-24, which repressed the Th17 cytokine program. In vivo, IL-24 treatment ameliorated Th17-induced EAU, whereas silencing of IL-24 in Th17 cells enhanced disease. This regulatory pathway also operated in human Th17 cells. Thus, IL-17A limits pathogenicity of Th17 cells by inducing IL-24. These findings may explain the disappointing therapeutic effect of targeting IL-17A in uveitis.

Establishment and Maintenance of the Macrophage Niche.

Self-maintaining resident macrophages populate all mammalian organs. In addition to their role as immune sentinels, macrophages also perform day-to-day functions essential to tissue homeostasis. The homeostatic functions of macrophages are regulated by so-called tissular "niches" that control the size of the macrophage population and imprint tissue-specific identity. Here, we review the mechanisms underlying self-maintenance of distinct macrophage populations and outline the organizing principles of the macrophage niche. We examine recent studies that uncovered mutually beneficial cell-cell circuits established between macrophages and their niche and propose a modular view of tissues that integrates the resident macrophage as an essential component of each individual module. Manipulating macrophage niche cells to control the function of resident macrophages in vivo might have therapeutic value in various disease settings.

Transcriptional and Functional Analysis of CD1c+ Human Dendritic Cells Identifies a CD163+ Subset Priming CD8+CD103+ T Cells.

Dendritic cells (DCs) are antigen-presenting cells controlling T cell activation. In humans, the diversity, ontogeny, and functional capabilities of DC subsets are not fully understood. Here, we identified circulating CD88-CD1c+CD163+ DCs (called DC3s) as immediate precursors of inflammatory CD88-CD14+CD1c+CD163+FcεRI+ DCs. DC3s develop via a specific pathway activated by GM-CSF, independent of cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors. Like classical DCs but unlike monocytes, DC3s drove activation of naive T cells. In vitro, DC3s displayed a distinctive ability to prime CD8+ T cells expressing a tissue homing signature and the epithelial homing alpha-E integrin (CD103) through transforming growth factor ß (TGF-ß) signaling. In vivo, DC3s infiltrated luminal breast cancer primary tumors, and DC3 infiltration correlated positively with CD8+CD103+CD69+ tissue-resident memory T cells. Together, these findings define DC3s as a lineage of inflammatory DCs endowed with a strong potential to regulate tumor immunity.

Epithelial IFNγ signalling and compartmentalized antigen presentation orchestrate gut immunity.

All nucleated cells express major histocompatibility complex I and interferon-γ (IFNγ) receptor1, but an epithelial cell-specific function of IFNγ signalling or antigen presentation by means of major histocompatibility complex I has not been explored. We show here that on sensing IFNγ, colonic epithelial cells productively present pathogen and self-derived antigens to cognate intra-epithelial T cells, which are critically located at the epithelial barrier. Antigen presentation by the epithelial cells confers extracellular ATPase expression in cognate intra-epithelial T cells, which limits the accumulation of extracellular adenosine triphosphate and consequent activation of the NLRP3 inflammasome in tissue macrophages. By contrast, antigen presentation by the tissue macrophages alongside inflammasome-associated interleukin-1α and interleukin-1ß production promotes a pathogenic transformation of CD4+ T cells into granulocyte-macrophage colony-stimulating-factor (GM-CSF)-producing T cells in vivo, which promotes colitis and colorectal cancer. Taken together, our study unravels critical checkpoints requiring IFNγ sensing and antigen presentation by epithelial cells that control the development of pathogenic CD4+ T cell responses in vivo.

Fate-Mapping of GM-CSF Expression Identifies a Discrete Subset of Inflammation-Driving T Helper Cells Regulated by Cytokines IL-23 and IL-1ß.

Pathogenic lymphocytes initiate the development of chronic inflammatory diseases. The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) (encoded by Csf2) is a key communicator between pathogenic lymphocytes and tissue-invading inflammatory phagocytes. However, the molecular properties of GM-CSF-producing cells and the mode of Csf2 regulation in vivo remain unclear. To systematically study and manipulate GM-CSF+ cells and their progeny in vivo, we generated a fate-map and reporter of GM-CSF expression mouse strain (FROG). We mapped the phenotypic and functional profile of auto-aggressive T helper (Th) cells during neuroinflammation and identified the signature and pathogenic memory of a discrete encephalitogenic Th subset. These cells required interleukin-23 receptor (IL-23R) and IL-1R but not IL-6R signaling for their maintenance and pathogenicity. Specific ablation of this subset interrupted the inflammatory cascade, despite the unperturbed tissue accumulation of other Th subsets (e.g., Th1 and Th17), highlighting that GM-CSF expression not only marks pathogenic Th cells, but that this subset mediates immunopathology and tissue destruction.

NKp46+ ILC3s promote early neutrophil defense against Clostridioides difficile infection through GM-CSF secretion.

Clostridioides difficile infection (CDI) is a common cause of antibiotic-associated colitis. C. difficile proliferates and produces toxins that damage the colonic epithelium, leading to symptoms ranging from mild diarrhea to severe pseudomembranous colitis. The host's innate response to CDI occurs in two phases: an early phase in which neutrophils reduce the bacterial load and a late phase involving repair mechanisms to restore epithelial integrity. Group 3 innate lymphoid cells (ILC3s) are crucial in protecting the gut from CDI. Previous studies have shown that ILC3-derived IL-22 is essential in the late phase of CDI for epithelial repair and maintaining an intestinal microbiota that competes with C. difficile, preventing its expansion. Our study finds that ILC3s also protect during the early stages of CDI by sustaining neutrophils through GM-CSF. Less neutrophil production, accumulation, and activation was evident in ILC3-deficient mice than in wild-type (WT) mice, which led to exacerbated symptoms, impaired pathogen clearance, a compromised epithelial barrier, and increased mortality. The adoptive transfer of ILC3s into ILC3-deficient mice restored neutrophil responses and improved disease outcomes. Both in vitro and in vivo experiments revealed that GM-CSF production by ILC3s is crucial for neutrophil production and effective resistance during CDI. Using mice lacking NKp46+ ILC3s, we found that this subset significantly contributes to GM-CSF production in CDI. These findings highlight the critical role of the ILC3-neutrophil connection in early innate responses to CDI. Enhancing ILC3 production of GM-CSF could be a promising strategy for improving host defense against CDI and other enteric infections.

Induction of the nuclear receptor PPAR-γ by the cytokine GM-CSF is critical for the differentiation of fetal monocytes into alveolar macrophages.

Tissue-resident macrophages constitute heterogeneous populations with unique functions and distinct gene-expression signatures. While it has been established that they originate mostly from embryonic progenitor cells, the signals that induce a characteristic tissue-specific differentiation program remain unknown. We found that the nuclear receptor PPAR-γ determined the perinatal differentiation and identity of alveolar macrophages (AMs). In contrast, PPAR-γ was dispensable for the development of macrophages located in the peritoneum, liver, brain, heart, kidneys, intestine and fat. Transcriptome analysis of the precursors of AMs from newborn mice showed that PPAR-γ conferred a unique signature, including several transcription factors and genes associated with the differentiation and function of AMs. Expression of PPAR-γ in fetal lung monocytes was dependent on the cytokine GM-CSF. Therefore, GM-CSF has a lung-specific role in the perinatal development of AMs through the induction of PPAR-γ in fetal monocytes.

Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells.

Innate lymphoid cells (ILCs) regulate stromal cells, epithelial cells and cells of the immune system, but their effect on B cells remains unclear. Here we identified RORγt(+) ILCs near the marginal zone (MZ), a splenic compartment that contains innate-like B cells highly responsive to circulating T cell-independent (TI) antigens. Splenic ILCs established bidirectional crosstalk with MAdCAM-1(+) marginal reticular cells by providing tumor-necrosis factor (TNF) and lymphotoxin, and they stimulated MZ B cells via B cell-activation factor (BAFF), the ligand of the costimulatory receptor CD40 (CD40L) and the Notch ligand Delta-like 1 (DLL1). Splenic ILCs further helped MZ B cells and their plasma-cell progeny by coopting neutrophils through release of the cytokine GM-CSF. Consequently, depletion of ILCs impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune system and circulatory system.

Plasmodium yoelii Infection Enhances the Expansion of Myeloid-Derived Suppressor Cells via JAK/STAT3 Pathway.

Myeloid-derived suppressor cells (MDSCs), the negative immune regulators, have been demonstrated to be involved in immune responses to a variety of pathological conditions, such as tumors, chronic inflammation, and infectious diseases. However, the roles and mechanisms underlying the expansion of MDSCs in malaria remain unclear. In this study, the phenotypic and functional characteristics of splenic MDSCs during Plasmodium yoelii NSM infection are described. Furthermore, we provide compelling evidence that the sera from P. yoelii-infected C57BL/6 mice containing excess IL-6 and granulocyte-macrophage colony-stimulating factor promote the accumulation of MDSCs by inducing Bcl2 expression. Serum-induced MDSCs exert more potent suppressive effects on T cell responses than control MDSCs within both in vivo P. yoelii infection and in vitro serum-treated bone marrow cells experiments. Serum treatment increases the MDSC inhibitory effect, which is dependent on Arg1 expression. Moreover, mechanistic studies reveal that the serum effects are mediated by JAK/STAT3 signaling. By inhibiting STAT3 phosphorylation with the JAK inhibitor JSI-124, effects of serum on MDSCs are almost eliminated. In vivo depletion of MDSCs with anti-Gr-1 or 5-fluorouracil significantly reduces the parasitemia and promotes Th1 immune response in P. yoelii-infected C57BL/6 mice by upregulating IFN-γ expression. In summary, this study indicates that P. yoelii infection facilitates the accumulation and function of MDSCs by upregulating the expression of Bcl2 and Arg1 via JAK/STAT3 signaling pathway in vivo and in vitro. Manipulating the JAK/STAT3 signaling pathway or depleting MDSCs could be promising therapeutic interventions to treat malaria.

MAFG-driven astrocytes promote CNS inflammation.

Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.

Low-Voltage-Activated CaV3.1 Calcium Channels Shape T Helper Cell Cytokine Profiles.

Activation of T cells is mediated by the engagement of T cell receptors (TCRs) followed by calcium entry via store-operated calcium channels. Here we have shown an additional route for calcium entry into T cells-through the low-voltage-activated T-type CaV3.1 calcium channel. CaV3.1 mediated a substantial current at resting membrane potentials, and its deficiency had no effect on TCR-initiated calcium entry. Mice deficient for CaV3.1 were resistant to the induction of experimental autoimmune encephalomyelitis and had reduced productions of the granulocyte-macrophage colony-stimulating factor (GM-CSF) by central nervous system (CNS)-infiltrating T helper 1 (Th1) and Th17 cells. CaV3.1 deficiency led to decreased secretion of GM-CSF from in vitro polarized Th1 and Th17 cells. Nuclear translocation of the nuclear factor of activated T cell (NFAT) was also reduced in CaV3.1-deficient T cells. These data provide evidence for T-type channels in immune cells and their potential role in shaping the autoimmune response.