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Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors.

Multiple myeloma (MM) is a neoplastic plasma-cell disorder characterized by clonal proliferation of malignant plasma cells. Despite extensive research, disease heterogeneity within and between treatment-resistant patients is poorly characterized. In the present study, we conduct a prospective, multicenter, single-arm clinical trial (NCT04065789), combined with longitudinal single-cell RNA-sequencing (scRNA-seq) to study the molecular dynamics of MM resistance mechanisms. Newly diagnosed MM patients (41), who either failed to respond or experienced early relapse after a bortezomib-containing induction regimen, were enrolled to evaluate the safety and efficacy of a daratumumab, carfilzomib, lenalidomide and dexamethasone combination. The primary clinical endpoint was safety and tolerability. Secondary endpoints included overall response rate, progression-free survival and overall survival. Treatment was safe and well tolerated; deep and durable responses were achieved. In prespecified exploratory analyses, comparison of 41 primary refractory and early relapsed patients, with 11 healthy subjects and 15 newly diagnosed MM patients, revealed new MM molecular pathways of resistance, including hypoxia tolerance, protein folding and mitochondria respiration, which generalized to larger clinical cohorts (CoMMpass). We found peptidylprolyl isomerase A (PPIA), a central enzyme in the protein-folding response pathway, as a potential new target for resistant MM. CRISPR–Cas9 deletion of PPIA or inhibition of PPIA with a small molecule inhibitor (ciclosporin) significantly sensitizes MM tumor cells to proteasome inhibitors. Together, our study defines a roadmap for integrating scRNA-seq in clinical trials, identifies a signature of highly resistant MM patients and discovers PPIA as a potent therapeutic target for these tumors. Integration of longitudinal single-cell analysis of relapsed and refractory multiple myeloma patients in a prospective clinical trial uncovers new pathways of drug resistance and identifies potential actionable targets.

Brain macrophage populations include parenchymal microglia, border-associated macrophages, and recruited monocyte-derived cells; together, they control brain development and homeostasis but are also implicated in aging pathogenesis and neurodegeneration. The phenotypes, localization, and functions of each population in different contexts have yet to be resolved. We generated a murine brain myeloid scRNA-seq integration to systematically delineate brain macrophage populations. We show that the previously identified disease-associated microglia (DAM) population detected in murine Alzheimer's disease models actually comprises two ontogenetically and functionally distinct cell lineages: embryonically derived triggering receptor expressed on myeloid cells 2 (TREM2)-dependent DAM expressing a neuroprotective signature and monocyte-derived TREM2-expressing disease inflammatory macrophages (DIMs) accumulating in the brain during aging. These two distinct populations appear to also be conserved in the human brain. Herein, we generate an ontogeny-resolved model of brain myeloid cell heterogeneity in development, homeostasis, and disease and identify cellular targets for the treatment of neurodegeneration.

Significance Oral cancer is often diagnosed at late stages, resulting in a poor prognosis of this disease. Understanding early carcinogenic events taking place during the development of oral cancer is, therefore, an important unmet clinical need. This study identifies an early anticancer protective role of oral Langerhans cells (LCs), the principal mononuclear phagocyte involved in immunosurveillance of the oral epithelium. Continuous exposure to the carcinogen 4NQO hampers the local differentiation of LCs, while other mononuclear phagocytes and T cells with immunosuppressive characteristics populate the epithelium, leading to the development of oral cancer. These findings reveal an initial step by which immunosurveillance is compromised by a carcinogen, which could facilitate novel diagnostic approaches to better detection of oral cancer., Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αβT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

The functional activity and differentiation potential of cells are determined by their interactions with surrounding cells. Approaches that allow unbiased characterization of cell states while at the same time providing spatial information are of major value to assess this environmental influence. However, most current techniques are hampered by a tradeoff between spatial resolution and cell profiling depth. Here, we develop a photocage-based technology that allows isolation and in-depth analysis of live cells from regions of interest in complex ex vivo systems, including primary human tissues. The use of a highly sensitive 4-nitrophenyl(benzofuran) cage coupled to a set of nanobodies allows high-resolution photo-uncaging of different cell types in areas of interest. Single-cell RNA-sequencing of spatially defined CD8+ T cells is used to exemplify the feasibility of identifying location-dependent cell states. The technology described here provides a valuable tool for the analysis of spatially defined cells in diverse biological systems, including clinical samples. The development of a photocage-nanobody based technology enabled in-depth analysis of live cells from tissues while retaining their spatial information.

Human tissues comprise trillions of cells that populate a complex space of molecular phenotypes and functions and that vary in abundance by 4-9 orders of magnitude. Relying solely on unbiased sampling to characterize cellular niches becomes infeasible, as the marginal utility of collecting more cells diminishes quickly. Furthermore, in many clinical samples, the relevant cell types are scarce and efficient processing is critical. We developed an integrated pipeline for index sorting and massively parallel single-cell RNA sequencing (MARS-seq2.0) that builds on our previously published MARS-seq approach. MARS-seq2.0 is based on >1 million cells sequenced with this pipeline and allows identification of unique cell types across different tissues and diseases, as well as unique model systems and organisms. Here, we present a detailed step-by-step procedure for applying the method. In the improved procedure, we combine sub-microliter reaction volumes, optimization of enzymatic mixtures and an enhanced analytical pipeline to substantially lower the cost, improve reproducibility and reduce well-to-well contamination. Data analysis combines multiple layers of quality assessment and error detection and correction, graphically presenting key statistics for library complexity, noise distribution and sequencing saturation. Importantly, our combined FACS and single-cell RNA sequencing (scRNA-seq) workflow enables intuitive approaches for depletion or enrichment of cell populations in a data-driven manner that is essential to efficient sampling of complex tissues. The experimental protocol, from cell sorting to a ready-to-sequence library, takes 2-3 d. Sequencing and processing the data through the analytical pipeline take another 1-2 d.

Alzheimer’s disease (AD) is a heterogeneous disorder with multiple etiologies. Harnessing the immune system by blocking the programmed cell death receptor (PD)-1 pathway in an amyloid beta mouse model was shown to evoke a sequence of immune responses that lead to disease modification. Here, blocking PD-L1, a PD-1 ligand, was found to have similar efficacy to that of PD-1 blocking in disease modification, in both animal models of AD and of tauopathy. Targeting PD-L1 in a tau-driven disease model resulted in increased immunomodulatory monocyte-derived macrophages within the brain parenchyma. Single cell RNA-seq revealed that the homing macrophages expressed unique scavenger molecules including macrophage scavenger receptor 1 (MSR1), which was shown here to be required for the effect of PD-L1 blockade in disease modification. Overall, our results demonstrate that immune checkpoint blockade targeting the PD-1/PD-L1 pathway leads to modification of common factors that go awry in AD and dementia, and thus can potentially provide an immunotherapy to help combat these diseases., Blocking the PD-1 pathway was shown to be effective in amyloid beta mouse models, yet little is known about its therapeutic potential in models of tauopathy. The authors show here that blocking PD-L1, a PD-1 ligand, is similarly effective, and that both treatments reversed cognitive deficiencies, and modified disease pathology not only in an animal model of AD, but also in the DM-hTAU mouse tauopathy model, through a mechanism that involves monocyte-derived macrophages.

Human organogenesis remains relatively unexplored for ethical and practical reasons. Here, we report the establishment of a single-cell transcriptome atlas of the human fetal pancreas between 7 and 10 post-conceptional weeks of development. To interrogate cell–cell interactions, we describe InterCom, an R-Package we developed for identifying receptor–ligand pairs and their downstream effects. We further report the establishment of a human pancreas culture system starting from fetal tissue or human pluripotent stem cells, enabling the long-term maintenance of pancreas progenitors in a minimal, defined medium in three-dimensions. Benchmarking the cells produced in 2-dimensions and those expanded in 3-dimensions to fetal tissue identifies that progenitors expanded in 3-dimensions are transcriptionally closer to the fetal pancreas. We further demonstrate the potential of this system as a screening platform and identify the importance of the EGF and FGF pathways controlling human pancreas progenitor expansion., From single-cell transcriptome analyses to defining culture media for spheroids, the authors provide a census of information to understand the development of human pancreatic progenitors. This approach identifies signalling pathways (EGF and FGF) regulating progenitor proliferation.

Controlling off-target editing activity is one of the central challenges in making CRISPR technology accurate and applicable in medical practice. Current algorithms for analyzing off-target activity do not provide statistical quantification, are not sufficiently sensitive in separating signal from noise in experiments with low editing rates, and do not address the detection of translocations. Here we present CRISPECTOR, a software tool that supports the detection and quantification of on- and off-target genome-editing activity from NGS data using paired treatment/control CRISPR experiments. In particular, CRISPECTOR facilitates the statistical analysis of NGS data from multiplex-PCR comparative experiments to detect and quantify adverse translocation events. We validate the observed results and show independent evidence of the occurrence of translocations in human cell lines, after genome editing. Our methodology is based on a statistical model comparison approach leading to better false-negative rates in sites with weak yet significant off-target activity., The control of off-target activity is a challenge for adapting CRISPR to therapeutic use. Here the authors present CRISPECTOR, a software tool to detect, evaluate and quantify editing activity, including translocations, from NGS data.

Inflammatory responses rapidly detect pathogen invasion and mount a regulated reaction. However, dysregulated anti-pathogen immune responses can provoke life-threatening inflammatory pathologies collectively known as cytokine release syndrome (CRS), exemplified by key clinical phenotypes unearthed during the SARS-Cov-2 pandemic. The underlying pathophysiology of CRS remains elusive. We found that FLIP, a protein that controls caspase-8 death pathways, was highly expressed in myeloid cells of COVID-19 lungs. FLIP controlled CRS by fueling a STAT3-dependent inflammatory program. Indeed, constitutive expression of a viral FLIP homologue in myeloid cells triggered a STAT3-linked, progressive and fatal inflammatory syndrome in mice, characterized by elevated cytokine output, lymphopenia, lung injury and multiple organ dysfunctions that mimicked human CRS. As STAT3-targeting approaches relieved inflammation, immune disorders, and organ failures in these mice, targeted intervention towards this pathway could suppress the lethal CRS inflammatory state.One sentence summaryFLIP-expressing myeloid cells are key drivers of CRS through aberrant overexpression of STAT3 pathway. STAT3-targeting is effective in mitigating CRS like severe COVID-19.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are heterogenous innate lymphocytes broadly defined in mice as Lin(−)NK1.1(+)NKp46(+) cells that express the transcription factor T-BET and produce interferon-γ. The ILC1 definition primarily stems from studies on liver and small intestinal populations. However, NK1.1(+)NKp46(+) cells in the salivary glands, uterus, adipose, and other tissues exhibit nonuniform programs that differ from those of liver or intestinal ILC1s or NK cells. Here, we performed single-cell RNA sequencing on murine NK1.1(+)NKp46(+) cells from blood, spleen, various tissues, and solid tumors. We identified gene expression programs of tissue-specific ILC1s, tissue-specific NK cells, and non-tissue-specific populations in blood, spleen, and other tissues largely corresponding to circulating cells. Moreover, we found that circulating NK cell programs were reshaped in tumor-bearing mice. Core programs of circulating and tumor NK cells paralleled conserved human NK cells signatures, advancing our understanding of the human NK-ILC1 spectrum.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1–5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH–HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH–HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH–HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment., In hepatocellular carcinoma driven by non-alcoholic steatohepatitis, aberrant T cell activation and impaired immune surveillance seem to make hepatocellular carcinoma less responsive to anti-PD1 or anti-PDL1 immunotherapy.

Therapeutic use of agonistic anti-CD40 antibodies is a potentially powerful approach for activation of the immune response to eradicate tumors. However, the translation of this approach to clinical practice has been substantially restricted due to the severe dose-limiting toxicities observed in multiple clinical trials. Here, we demonstrate that conventional type 1 dendritic cells are essential for triggering antitumor immunity but not the toxicity of CD40 agonists, while macrophages, platelets and monocytes lead to toxic events. Therefore, we designed bispecific antibodies that target CD40 activation preferentially to dendritic cells, by coupling the CD40 agonist arm with CD11c-, DEC-205- or CLEC9A-targeting arms. These bispecific reagents demonstrate a superior safety profile compared to their parental CD40 monospecific antibody while triggering potent antitumor activity. We suggest such cell-selective bispecific agonistic antibodies as a drug platform to bypass the dose-limiting toxicities of anti-CD40, and of additional types of agonistic antibodies used for cancer immunotherapy.

Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation–related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.

Group 2 innate lymphoid cells (ILC2s) represent innate homologs of type 2 helper T cells (TH2) that participate in immune defense and tissue homeostasis through production of type 2 cytokines. While T lymphocytes metabolically adapt to microenvironmental changes, knowledge of human ILC2 metabolism is limited, and its key regulators are unknown. Here, we show that circulating ‘naive’ ILC2s have an unexpected metabolic profile with a higher level of oxidative phosphorylation (OXPHOS) than natural killer (NK) cells. Accordingly, ILC2s are severely reduced in individuals with mitochondrial disease (MD) and impaired OXPHOS. Metabolomic and nutrient receptor analysis revealed ILC2 uptake of amino acids to sustain OXPHOS at steady state. Following activation with interleukin-33 (IL-33), ILC2s became highly proliferative, relying on glycolysis and mammalian target of rapamycin (mTOR) to produce IL-13 while continuing to fuel OXPHOS with amino acids to maintain cellular fitness and proliferation. Our results suggest that proliferation and function are metabolically uncoupled in human ILC2s, offering new strategies to target ILC2s in disease settings., ILC2 metabolism has been largely unexplored. Di Santo and colleagues examine metabolic profiles from naive and cytokine-activated ILC2s and find that IL-33-triggered ILC2s rely on distinct metabolic pathways to sustain proliferation and function.

Malignant cell growth is fueled by interactions between tumor cells and the stromal cells composing the tumor microenvironment. The human liver is a major site of tumors and metastases, but molecular identities and intercellular interactions of different cell types have not been resolved in these pathologies. Here, we apply single cell RNA‐sequencing and spatial analysis of malignant and adjacent non‐malignant liver tissues from five patients with cholangiocarcinoma or liver metastases. We find that stromal cells exhibit recurring, patient‐independent expression programs, and reconstruct a ligand–receptor map that highlights recurring tumor–stroma interactions. By combining transcriptomics of laser‐capture microdissected regions, we reconstruct a zonation atlas of hepatocytes in the non‐malignant sites and characterize the spatial distribution of each cell type across the tumor microenvironment. Our analysis provides a resource for understanding human liver malignancies and may expose potential points of interventions., Molecular Systems Biology, 16 (12), ISSN:1744-4292

Summary Blood myeloid cells are known to be dysregulated in the coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity, and whether markers of innate immunity discriminate high risk patients. Thus, we performed high dimensional flow cytometry and single cell RNA sequencing of COVID-19 patient peripheral blood cells and detected the disappearance of non-classical CD14LowCD16High monocytes, the accumulation of HLA-DRLow classical monocytes, and the release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10LowCD101-CXCR4+/- neutrophils with an immuno-suppressive profile accumulated as well in blood and lungs, suggesting emergency myelopoiesis. We finally showed that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe COVID-19 form, suggesting a predictive value that deserves prospective evaluation., Highlights • Severe patients accumulate HLA-DRLow monocytes and immature neutrophils in blood/lung. • Calprotectin level positively correlates with neutrophil count and disease severity. • Loss of non-classical monocytes could identify high risk of severe COVID-19.

Since the beginning of the SARS-CoV-2 pandemic, COVID-19 appeared as a unique disease with unconventional tissue and systemic immune features. Here we show a COVID-19 immune signature associated with severity by integrating single-cell RNA-seq analysis from blood samples and broncho-alveolar lavage fluids with clinical, immunological and functional ex vivo data. This signature is characterized by lung accumulation of naïve lymphoid cells associated with a systemic expansion and activation of myeloid cells. Myeloid-driven immune suppression is a hallmark of COVID-19 evolution, highlighting arginase-1 expression with immune regulatory features of monocytes. Monocyte-dependent and neutrophil-dependent immune suppression loss is associated with fatal clinical outcome in severe patients. Additionally, our analysis shows a lung CXCR6+ effector memory T cell subset is associated with better prognosis in patients with severe COVID-19. In summary, COVID-19-induced myeloid dysregulation and lymphoid impairment establish a condition of ‘immune silence’ in patients with critical COVID-19., Integrated studies of matched tissue sites and cell types in COVID-19 patients are important to define the immune mechanisms of pathology. Here, the authors describe an immune signature in fatal COVID-19 patients harmonizing single-cell RNA sequencing of blood and matched BAL cells with deep clinical, immunological and functional data.

Viruses are a constant threat to global health as highlighted by the current COVID-19 pandemic. Currently, lack of data underlying how the human host interacts with viruses, including the SARS-CoV-2 virus, limits effective therapeutic intervention. We introduce Viral-Track, a computational method that globally scans unmapped scRNA-seq data for the presence of viral RNA, enabling transcriptional cell sorting of infected versus bystander cells. We demonstrate the sensitivity and specificity of Viral-Track to systematically detect viruses from multiple models of infection, including hepatitis B virus in an unsupervised manner. Applying Viral-Track to Bronchoalveloar-Lavage samples from severe and mild COVID-19 patients reveals a dramatic impact of the virus on the immune system of severe patients compared to mild cases. Viral-Track detects an unexpected co-infection of the human MetaPneumoVirus, present mainly in monocytes perturbed in type-I IFN-signaling. Viral-Track provides a robust technology for dissecting the mechanisms of viral-infection and pathology., Highlights Viral-track: a computational framework to analyze host-viral infection maps Viral-track sorts infected from bystander cells and reveals virus-induced expression SARS-CoV-2 infects epithelial cells and alters immune landscape in severe patients Coinfection of SARS-Cov-2 and hMPV affects monocytes and dampen interferon response, A computational framework that allows for the identification and characterizaton of virus-infected cells as well as bystander cell responses reveals how SARS-CoV-2 alters the immune responses of patients.

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1

Respiratory immune characteristics associated with Coronavirus Disease 2019 (COVID-19) severity are currently unclear. We characterized bronchoalveolar lavage fluid immune cells from patients with varying severity of COVID-19 and from healthy people by using single-cell RNA sequencing. Proinflammatory monocyte-derived macrophages were abundant in the bronchoalveolar lavage fluid from patients with severe COVID-9. Moderate cases were characterized by the presence of highly clonally expanded CD8+ T cells. This atlas of the bronchoalveolar immune microenvironment suggests potential mechanisms underlying pathogenesis and recovery in COVID-19.

Alzheimer's disease, obesity-related metabolic syndrome, and cancer are the leading causes of death and among the most costly medical conditions in the Western world. In all three cases, recent discoveries establish the TREM2 receptor as a major pathology-induced immune signaling hub that senses tissue damage and activates robust immune remodeling in response to it. In this review, we summarize and question what is known and remains to be discovered about TREM2 signaling pathway, track the consequences of its activation in physiological niches and pathological contexts, and highlight the promising potential of therapeutic manipulation of TREM2 signaling.

The cytokine interferon (IFN)-γ produced by tumor-reactive T cells is a key effector molecule with pleiotropic effects during anti-tumor immune responses. Although IFN-γ production is targeted at the immunologic synapse, its spatiotemporal activity within the tumor remains elusive. In the present study, we report that, although IFN-γ secretion requires local antigen recognition, IFN-γ diffuses extensively to alter the tumor microenvironment in distant areas. Using intravital imaging and a reporter for STAT1 translocation, we provide evidence that T cells mediate sustained IFN-γ signaling in remote tumor cells. Furthermore, tumor phenotypic alterations required several hours of exposure to IFN-γ, a feature that disfavored local IFN-γ activity over diffusion and bystander activity. Finally, single-cell RNA-sequencing data from melanoma patients also suggested bystander IFN-γ activity in human tumors. Thus, tumor-reactive T cells act collectively to create large cytokine fields that profoundly modify the tumor microenvironment. Two papers by Schumacher and colleagues and Bousso and colleagues show that sensing of IFN-γ by a small number of tumor cells leads to propagation of IFN-γ sensing and response across a larger fraction of the tumor.

Summary Hematopoietic stem cells (HSCs) maintain life-long production of immune cells and can directly respond to infection, but sustained effects on the immune response remain unclear. We show that acute immune stimulation with lipopolysaccharide (LPS) induced only transient changes in HSC abundance, composition, progeny, and gene expression, but persistent alterations in accessibility of specific myeloid lineage enhancers occurred, which increased responsiveness of associated immune genes to secondary stimulation. Functionally, this was associated with increased myelopoiesis of pre-exposed HSCs and improved innate immunity against the gram-negative bacterium P. aeruginosa. The accessible myeloid enhancers were enriched for C/EBPβ targets, and C/EBPβ deletion erased the long-term inscription of LPS-induced epigenetic marks and gene expression. Thus, short-term immune signaling can induce C/EBPβ-dependent chromatin accessibility, resulting in HSC-trained immunity, during secondary infection. This establishes a mechanism for how infection history can be epigenetically inscribed in HSCs as an integral memory function of innate immunity.

Erratum inCross-Species Single-Cell Analysis Reveals Divergence of the Primate Microglia Program.Geirsdottir L, David E, Keren-Shaul H, Weiner A, Bohlen SC, Neuber J, Balic A, Giladi A, Sheban F, Dutertre CA, Pfeifle C, Peri F, Raffo-Romero A, Vizioli J, Matiasek K, Scheiwe C, Meckel S, Mätz-Rensing K, van der Meer F, Thormodsson FR, Stadelmann C, Zilkha N, Kimchi T, Ginhoux F, Ulitsky I, Erny D, Amit I, Prinz M.Cell. 2020 Apr 30;181(3):746. doi: 10.1016/j.cell.2020.04.002.PMID: 32359440; International audience; Microglia, the brain-resident immune cells, are critically involved in many physiological and pathological brain processes, including neurodegeneration. Here we characterize microglia morphology and transcriptional programs across ten species spanning more than 450 million years of evolution. We find that microglia express a conserved core gene program of orthologous genes from rodents to humans, including ligands and receptors associated with interactions between glia and neurons. In most species, microglia show a single dominant transcriptional state, whereas human microglia display significant heterogeneity. In addition, we observed notable differences in several gene modules of rodents compared with primate microglia, including complement, phagocytic, and susceptibility genes to neurodegeneration, such as Alzheimer's and Parkinson's disease. Our study provides an essential resource of conserved and divergent microglia pathways across evolution, with important implications for future development of microglia-based therapies in humans.

A major challenge in the field of neurodegenerative diseases and brain aging is to identify the body's intrinsic mechanism that could sense the central nervous system (CNS) damage early and protect the brain from neurodegeneration. Accumulating evidence suggests that disease-associated microglia (DAM), a recently identified subset of CNS resident macrophages found at sites of neurodegeneration, might play such a protective role. Here, we propose that microglia are endowed with a dedicated sensory mechanism, which includes the Trem2 signaling pathway, to detect damage within the CNS in the form of neurodegeneration-associated molecular patterns (NAMPs). Combining data from transcriptional analysis of DAM at single-cell level and from human genome-wide association studies (GWASs), we discuss potential function of different DAM pathways in the diseased brain and outline how manipulating DAM may create new therapeutic opportunities.

The extent and nature of epigenomic changes associated with melanoma progression is poorly understood. Through systematic epigenomic profiling of 35 epigenetic modifications and transcriptomic analysis, we define chromatin state changes associated with melanomagenesis by using a cell phenotypic model of non-tumorigenic and tumorigenic states. Computation of specific chromatin state transitions showed loss of histone acetylations and H3K4me2/3 on regulatory regions proximal to specific cancer-regulatory genes in important melanoma-driving cell signaling pathways. Importantly, such acetylation changes were also observed between benign nevi and malignant melanoma human tissues. Intriguingly, only a small fraction of chromatin state transitions correlated with expected changes in gene expression patterns. Restoration of acetylation levels on deacetylated loci by histone deacetylase (HDAC) inhibitors selectively blocked excessive proliferation in tumorigenic cells and human melanoma cells, suggesting functional roles of observed chromatin state transitions in driving hyperproliferative phenotype. Through these results, we define functionally relevant chromatin states associated with melanoma progression.

The mammalian liver consists of hexagon-shaped lobules that are radially polarized by blood flow and morphogens. Key liver genes have been shown to be differentially expressed along the lobule axis, a phenomenon termed zonation, but a detailed genome-wide reconstruction of this spatial division of labour has not been achieved. Here we measure the entire transcriptome of thousands of mouse liver cells and infer their lobule coordinates on the basis of a panel of zonated landmark genes, characterized with single-molecule fluorescence in situ hybridization. Using this approach, we obtain the zonation profiles of all liver genes with high spatial resolution. We find that around 50% of liver genes are significantly zonated and uncover abundant non-monotonic profiles that peak at the mid-lobule layers. These include a spatial order of bile acid biosynthesis enzymes that matches their position in the enzymatic cascade. Our approach can facilitate the reconstruction of similar spatial genomic blueprints for other mammalian organs.

Acute liver failure (ALF) is a fulminant complication of multiple etiologies, characterized by rapid hepatic destruction, multi-organ failure and mortality. ALF treatment is mainly limited to supportive care and liver transplantation. Here we utilize the acetaminophen (APAP) and thioacetamide (TAA) ALF models in characterizing 56,527 single-cell transcriptomes to define the mouse ALF cellular atlas. We demonstrate that unique, previously uncharacterized stellate cell, endothelial cell, Kupffer cell, monocyte and neutrophil subsets, and their intricate intercellular crosstalk, drive ALF. We unravel a common MYC-dependent transcriptional program orchestrating stellate, endothelial and Kupffer cell activation during ALF, which is regulated by the gut microbiome through Toll-like receptor (TLR) signaling. Pharmacological inhibition of MYC, upstream TLR signaling checkpoints or microbiome depletion suppress this cell-specific, MYC-dependent program, thereby attenuating ALF. In humans, we demonstrate upregulated hepatic MYC expression in ALF transplant recipients compared to healthy donors. Collectively we demonstrate that detailed cellular/genetic decoding may enable pathway-specific ALF therapeutic intervention.

Recent progress in single-cell genomics urges its application in drug development, particularly of cancer immunotherapies. Current immunotherapy pipelines are focused on functional outcome and simple cellular and molecular readouts. A thorough mechanistic understanding of the cells and pathways targeted by immunotherapy agents is lacking, which limits the success rate of clinical trials. A large leap forward can be made if the immunotherapy target cells and pathways are characterized at high resolution before and after treatment, in clinical cohorts and model systems. This will enable rapid development of effective immunotherapies and data-driven design of synergistic drug combinations. In this Perspective, we discuss how emerging single-cell genomic technologies can serve as an engine for target identification and drug development. Amit and colleagues discuss where single-cell genomic technologies can be applied both in trial design and in the clinical trial stage to improve the development of immunotherapies.

Multiple sclerosis (MS) is characterized by pathological inflammation that results from the recruitment of lymphoid and myeloid immune cells from the blood into the brain. Due to subset heterogeneity, defining the functional roles of the various cell subsets in acute and chronic stages of MS has been challenging. Here, we used index and transcriptional single-cell sorting to characterize the mononuclear phagocytes that infiltrate the central nervous system from the periphery in mice with experimentally induced autoimmune encephalomyelitis, a model of MS. We identified eight monocyte and three dendritic cell subsets at acute and chronic disease stages in which the defined transcriptional programs pointed toward distinct functions. Monocyte-specific cell ablation identified Cxcl10

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.

Immune cells residing in white adipose tissue have been highlighted as important factors contributing to the pathogenesis of metabolic diseases, but the molecular regulators that drive adipose tissue immune cell remodeling during obesity remain largely unknown. Using index and transcriptional single-cell sorting, we comprehensively map all adipose tissue immune populations in both mice and humans during obesity. We describe a novel and conserved Trem2(+) lipid-associated macrophage (LAM) subset and identify markers, spatial localization, origin, and functional pathways associated with these cells. Genetic ablation of Trem2 in mice globally inhibits the downstream molecular LAM program, leading to adipocyte hypertrophy as well as systemic hypercholesterolemia, body fat accumulation, and glucose intolerance. These findings identify Trem2 signaling as a major pathway by which macrophages respond to loss of tissue-level lipid homeostasis, highlighting Trem2 as a key sensor of metabolic pathologies across multiple tissues and a potential therapeutic target in metabolic diseases.

International audience; The immune system generates pathogen-tailored responses. The precise innate immune cell types and pathways that direct robust adaptive immune responses have not been fully characterized. By using fluorescent pathogens combined with massively parallel single-cell RNA-seq, we comprehensively characterized the initial 48 h of the innate immune response to diverse pathogens. We found that across all pathogens tested, most of the lymph node cell types and states showed little pathogen specificity. In contrast, the rare antigen-positive cells displayed pathogen-specific transcriptional programs as early as 24 h after immunization. In addition, mycobacteria activated a specific NK-driven IFNγ response. Depletion of NK cells and IFNγ showed that IFNγ initiated a monocyte-specific signaling cascade, leading to the production of major chemokines and cytokines that promote Th1 development. Our systems immunology approach sheds light on early events in innate immune responses and may help further development of safe and efficient vaccines.

Summary The glucocorticoid receptor (GR) acts as a ubiquitous cortisol-dependent transcription factor (TF). To identify co-factors, we used protein-fragment complementation assays and found that GR recognizes FLI1 and additional ETS family proteins, TFs relaying proliferation and/or migration signals. Following steroid-dependent translocation of FLI1 and GR to the nucleus, the FLI1-specific domain (FLS) binds with GR and strongly enhances GR’s transcriptional activity. This interaction has functional consequences in Ewing sarcoma (ES), childhood and adolescence bone malignancies driven by fusions between EWSR1 and FLI1. In vitro, GR knockdown inhibited the migration and proliferation of ES cells, and in animal models, antagonizing GR (or lowering cortisol) retarded both tumor growth and metastasis from bone to lung. Taken together, our findings offer mechanistic rationale for repurposing GR-targeting drugs for the treatment of patients with ES., Graphical Abstract, Highlights • Glucocorticoids activate GR, an inducible TF regulating growth and metabolism • Protein complementation assays identified FLI1 and ERG as GR binders and activators • EWS-FLI1, a fusion protein, drives Ewing sarcoma (ES) and binds with activated GR • Pharmacological inhibition of GR activation retards progression in ES animal models, The single oncogene of Ewing sarcoma (ES), a childhood cancer, encodes a FLI1 fusion protein. Srivastava et al. report physical interactions between the fusion protein and the glucocorticoid receptor. Drug-induced inhibition of these interactions retards the progression of ES in mouse models.

Single-cell expression profiling (scRNA-seq) is a rich resource of cellular heterogeneity. While profiling every sample under study would be advantageous, it is time-consuming and costly. Here we introduce Cell Population Mapping (CPM), a deconvolution algorithm in which the composition of cell types and states is inferred from the bulk transcriptome using reference scRNA-seq profiles ('scBio' CRAN R-package). Analysis of individual variations in lungs of influenza virus-infected mice, using CPM, revealed that the relationship between cell abundance and clinical symptoms is a cell-state-specific property that varies gradually along the continuum of cell-activation states. The gradual change was confirmed in subsequent experiments and was further explained by a mathematical model in which clinical outcomes relate to cell-state dynamics along the activation process. Our results demonstrate the power of CPM in reconstructing the continuous spectrum of cell states within heterogeneous tissues.

Tumor immune cell compositions play a major role in response to immunotherapy, but the heterogeneity and dynamics of immune infiltrates in human cancer lesions remain poorly characterized. Here, we identify conserved intratumoral CD4 and CD8 T cell behaviors in scRNA-seq data from 25 melanoma patients. We discover a large population of CD8 T cells showing continuous progression from an early effector "transitional" into a dysfunctional T cell state. CD8 T cells that express a complete cytotoxic gene set are rare, and TCR sharing data suggest their independence from the transitional and dysfunctional cell states. Notably, we demonstrate that dysfunctional T cells are the major intratumoral proliferating immune cell compartment and that the intensity of the dysfunctional signature is associated with tumor reactivity. Our data demonstrate that CD8 T cells previously defined as exhausted are in fact a highly proliferating, clonal, and dynamically differentiating cell population within the human tumor microenvironment.

Nature Biotechnology asks a selection of researchers about the most exciting frontier in their field and the most needed technologies for advancing knowledge and applications.

Analysing the DNA, RNA and protein of single cells is transforming our understanding of the immune system, say Amir Giladi and Ido Amit.

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Summary The influenza virus is a major cause of morbidity and mortality worldwide. Yet, both the impact of intracellular viral replication and the variation in host response across different cell types remain uncharacterized. Here we used single-cell RNA sequencing to investigate the heterogeneity in the response of lung tissue cells to in vivo influenza infection. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of bystander (exposed but uninfected) as compared with infected cells. We reveal that all major immune and non-immune cell types manifest substantial fractions of infected cells, albeit at low viral transcriptome loads relative to epithelial cells. We show that all cell types respond primarily with a robust generic transcriptional response, and we demonstrate novel markers specific for influenza-infected as opposed to bystander cells. These findings open new avenues for targeted therapy aimed exclusively at infected cells., Graphical Abstract, Highlights • Combined measurements of host-viral scRNA-seq during in vivo influenza infection • High prevalence of infection in a variety of immune and non-immune cell types • Extensive cellular heterogeneity exists within infected and bystander cells • Generic and cell-type-specific differences between infected and bystander cells, Simultaneous mapping of host and viral transcriptomes at the same single cell provides a framework to study host-viral interactions. Analysis of cells derived from lungs of in vivo influenza infection reveals both generic and cell-type-specific infection response. By analyzing the cellular heterogeneity of both bystander (exposed but uninfected) and infected cells, we characterize novel markers specific for influenza-infected as opposed to bystanders. These findings open new avenues for targeted therapy aimed exclusively at infected cells.

4sUDRB-seq separately measures, on a genomic scale, the distinct contributions of transcription elongation speed and rate of RNA polymerase II (Pol II) transition into active elongation (TAE) to the overall mRNA production rate. It uses reversible inhibition of transcription elongation with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), combined with a pulse of 4-thiouridine (4sU), to tag newly transcribed RNA. After DRB removal, cells are collected at several time points, and tagged RNA is biotinylated, captured on streptavidin beads and sequenced. 4sUDRB-seq enables the comparison of elongation speeds between different developmental stages or different cell types, and it allows the impact of specific transcription factors on transcription elongation speed versus TAE to be studied. RNA preparation takes ∼4 d to complete, with deep sequencing requiring an additional ∼4-11 d plus 1-3 d for bioinformatics analysis. The experimental protocol requires basic molecular biology skills, whereas data analysis requires knowledge in bioinformatics, particularly MATLAB and the Linux environment.

Hematopoiesis and immunity are mediated through complex interactions between multiple cell types and states. This complexity is currently addressed following a reductionist approach of characterizing cell types by a small number of cell surface molecular features and gross functions. While the introduction of global transcriptional profiling technologies enabled a more comprehensive view, heterogeneity within sampled populations remained unaddressed, obscuring the true picture of hematopoiesis and immune system function. A critical mass of technological advances in molecular biology and genomics has enabled genome-wide measurements of single cells - the fundamental unit of immunity. These new advances are expected to boost detection of less frequent cell types and fuzzy intermediate cell states, greatly expanding the resolution of current available classifications. This new era of single-cell genomics in immunology research holds great promise for further understanding of the mechanisms and circuits regulating hematopoiesis and immunity in both health and disease. In the near future, the accuracy of single-cell genomics will ultimately enable precise diagnostics and treatment of multiple hematopoietic and immune related diseases.

Summary Pluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage specific behavior of selected factors. In addition to the orchestrated remodeling of the chromatin landscape, we find that the binding of several transcription factors is strongly associated with specific loss of DNA methylation in one germ layer and in many cases a reciprocal gain in the other layers. Taken together, our work shows context-dependent rewiring of transcription factor binding, downstream signaling effectors, and the epigenome during human embryonic stem cell differentiation.

During hematopoiesis, cells originating from the same stem cell reservoir differentiate into distinct cell types. The mechanisms enabling common progenitors to differentiate into alternative cell fates are not fully understood. Here, we identify cell-fate-determining transcription factors (TFs) governing dendritic cell (DC) development by annotating the enhancer landscapes of the DC lineage. Combining these analyses with detailed overexpression, knockdown, and ChIP-Seq studies, we show that Irf8 functions as a plasmacytoid DC epigenetic and fate-determining TF, regulating massive, cell-specific chromatin changes in thousands of pDC enhancers. Importantly, Irf8 forms a negative feedback loop with Cebpb, a monocyte-derived DC epigenetic fate-determining TF. We show that using this circuit logic, a pulse of TF expression can stably define epigenetic and transcriptional states, regardless of the microenvironment. More broadly, our study proposes a general paradigm that allows closely related cells with a similar set of signal-dependent factors to generate differential and persistent enhancer landscapes.

Item does not contain fulltext The International Human Epigenome Consortium (IHEC) coordinates the generation of a catalog of high-resolution reference epigenomes of major primary human cell types. The studies now presented (see the Cell Press IHEC web portal at http://www.cell.com/consortium/IHEC) highlight the coordinated achievements of IHEC teams to gather and interpret comprehensive epigenomic datasets to gain insights in the epigenetic control of cell states relevant for human health and disease. PAPERCLIP.

Systemic immune suppression may curtail the ability to mount the protective, cell-mediated immune responses that are needed for brain repair. By using mouse models of Alzheimer's disease (AD), we show that immune checkpoint blockade directed against the programmed death-1 (PD-1) pathway evokes an interferon (IFN)-γ-dependent systemic immune response, which is followed by the recruitment of monocyte-derived macrophages to the brain. When induced in mice with established pathology, this immunological response leads to clearance of cerebral amyloid-β (Aβ) plaques and improved cognitive performance. Repeated treatment sessions were required to maintain a long-lasting beneficial effect on disease pathology. These findings suggest that immune checkpoints may be targeted therapeutically in AD.

The immunology field has invested great efforts and ingenuity to characterize the various immune cell types and elucidate their functions. However, accumulating evidence indicates that current technologies and classification schemes are limited in their ability to account for the functional heterogeneity of immune processes. Single-cell genomics hold the potential to revolutionize the way we characterize complex immune cell assemblies and study their spatial organization, dynamics, clonal distribution, pathways, function, and crosstalks. In this Perspective, we consider recent and forthcoming technological and analytical advances in single-cell genomics and the potential impact of those advances on the future of immunology research and immunotherapy.