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Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naïve limb progenitors and show that they differentiate progressively to form the skeleton in a complex nonconsecutive three-dimensional pattern.Single-cell RNA sequencing of the developing mouse forelimb revealed three progenitor states: naïve, proximal and autopodial, as well as Msx1 as a marker for the naïve progenitors. In vivo lineage tracing confirmed this role and localized the naïve progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naïve progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments.Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest a new model for limb skeleton development.

Tumors are supported by cancer-associated fibroblasts (CAFs). CAFs are heterogeneous and carry out distinct cancer-associated functions. Understanding the full repertoire of CAFs and their dynamic changes as tumors evolve could improve the precision of cancer treatment. Here we comprehensively analyze CAFs using index and transcriptional single-cell sorting at several time points along breast tumor progression in mice, uncovering distinct subpopulations. Notably, the transcriptional programs of these subpopulations change over time and in metastases, transitioning from an immunoregulatory program to wound-healing and antigen-presentation programs, indicating that CAFs and their functions are dynamic. Two main CAF subpopulations are also found in human breast tumors, where their ratio is associated with disease outcome across subtypes and is particularly correlated with BRCA mutations in triple-negative breast cancer. These findings indicate that the repertoire of CAF changes over time in breast cancer progression, with direct clinical implications. Scherz-Shouval and colleagues characterize the dynamic changes in cancer-associated fibroblast subpopulations during breast cancer progression. They find that the ratio of S100A4+ and PDPN+ CAF subsets is associated with clinical outcome.

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+ and Saa3+ monocytic subsets with a pathogenic potential. Transfer experiments with different monocyte and precursor subsets indicated that these Cxcl10+ and Saa3+ pathogenic cells were not derived from Ly6C+ monocytes but from early myeloid cell progenitors. These results suggest that blocking specific pathogenic monocytic subsets, including Cxcl10+ and Saa3+ monocytes, could be used for targeted therapeutic interventions.

Background: The clinical presentation of coronavirus disease 19 (COVID-19) is the result of intricate interactions between the novel coronavirus and the immune system. In patients with hematologic malignancies (HM), these interactions dramatically change the clinical course and outcomes of COVID-19. Summary: Patients with HM and COVID-19 are at an increased risk for prolonged viral shedding, more protracted and severe presentation, and death, even when compared to other immunocompromised hosts. HM (e.g., multiple myeloma, chronic lymphocytic leukemia) and anticancer treatments (e.g., anti-CD20 agents) that impair humoral immunity markedly increase the risk of severe COVID-19 as well as protracted viral shedding and possibly longer infectivity. Cytokine release syndrome (CRS) is an important player in the pathophysiology of severe and fatal COVID-19. Treatments targeting specific cytokines involved in CRS such as interleukin-6 and Janus kinase have proven beneficial in COVID-19 patients but were not assessed specifically in HM patients. Although neutropenia (as well as neutrophilia) was associated with increased COVID-19 mortality, granulocyte colony-stimulating factors were not beneficial in patients with COVID-19 and may have been associated with worse outcomes. Decreased levels of T lymphocytes and especially decreased CD4+ counts, and depletion of CD8+ lymphocytes, are a hallmark of severe COVID-19, and even more so among patients with HM, underlying the important role of T-helper dysfunction in severe COVID-19. In HM patients with intact cellular immunity, robust T-cell responses may compensate for an impaired humoral immune system. Further prospective studies are needed to evaluate the mechanisms of severe COVID-19 among patients with HM and assess the efficacy of new immunomodulating COVID-19 treatments in this population. Key Messages: Understanding the immunopathology of COVID-19 has greatly benefited from the previous research in patients with HM. So far, no COVID-19 treatments were properly evaluated in patients with HM. Patients with HM should be included in future RCTs assessing treatments for COVID-19.

Comment in: DePICting T cell-APC crosstalk in cancer. Zhang T, Dong C. Nat Cancer. 2022 Mar;3(3):265-267. doi: 10.1038/s43018-022-00345-6. PMID: 35352059 No abstract available.; International audience; Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4+PD-1+CXCL13+ T cells are a major interacting hub with antigen-presenting cells in the tumor microenvironment of human non-small cell lung carcinoma. We define this clonally expanded, tumor-specific and conserved T-cell subset as T-helper tumor (Tht) cells. Reconstitution of Tht cells in vitro and in an ovalbumin-specific αβ TCR CD4+ T-cell mouse model, shows that the Tht program is primed in tumor-draining lymph nodes by dendritic cells presenting tumor antigens, and that their function is important for harnessing the antitumor response of anti-PD-1 treatment. Our molecular and functional findings support the modulation of Tht-dendritic cell interaction checkpoints as a major interventional strategy in immunotherapy.

The endocrine pancreas is able to regenerate in response to insult, including by driving beta-cells into the cell division cycle. Until recently, communication between neighboring cells in islets of Langerhans was overlooked by single-cell genomic technologies, which require rigorous tissue dissociation into single cells. Here, we utilize sorting of physically interacting cells (PICs) with single-cell RNA-sequencing to systematically map cellular interactions in the regenerating endocrine pancreas. The cellular landscape of the regenerated pancreas features regeneration-associated endocrine populations.We explore the unexpected heterogeneity of beta-cells in regeneration, including an interaction-specific program between paired beta and delta-cells. Our analysis suggests that the particular cluster of beta-cells that pair with delta-cells benefits from stress protection, implying that the interaction between beta and delta-cells safeguards against regeneration-associated challenges.

Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα+ common lymphoid progenitor population and identified Ly6D, IL-7Rα, CD81 and CD2 as key markers of pDC differentiation, which distinguish pDC precursors from cDC precursors. In conclusion, pDCs developed in the bone marrow from a Ly6DhiCD2hi lymphoid progenitor cell and differentiated independently of the myeloid cDC lineage.

International audience; 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 XCR1DTA mice or using anti-XCL1-blocking antibody attenuated liver pathology in NASH mouse models. Overall, our study provides a comprehensive characterization of cDC biology in NASH and identifies XCR1+ cDC1 as an important driver of liver pathology.

Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4

We found that acute insult to the central nervous system induces the formation of lymphocyte aggregates reminiscent of tertiary lymphoid structures within the spinal cord meninges. Unlike draining CNS-cervical lymph nodes, meningeal lymphocytes are locally activated during neuro-inflammtion and neurodegeneration., Tertiary lymphoid structures (TLS) are organized aggregates of B and T cells formed ectopically during different stages of life in response to inflammation, infection, or cancer. Here, we describe formation of structures reminiscent of TLS in the spinal cord meninges under several central nervous system (CNS) pathologies. After acute spinal cord injury, B and T lymphocytes locally aggregate within the meninges to form TLS-like structures, and continue to accumulate during the late phase of the response to the injury, with a negative impact on subsequent pathological conditions, such as experimental autoimmune encephalomyelitis. Using a chronic model of spinal cord pathology, the mSOD1 mouse model of amyotrophic lateral sclerosis, we further showed by single-cell RNA-sequencing that a meningeal lymphocyte niche forms, with a unique organization and activation state, including accumulation of pre-B cells in the spinal cord meninges. Such a response was not found in the CNS-draining cervical lymph nodes. The present findings suggest that a special immune response develops in the meninges during various neurological pathologies in the CNS, a possible reflection of its immune privileged nature.

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

Tumors are supported by cancer-associated fibroblasts (CAFs). CAFs are heterogeneous and carry out distinct cancer-associated functions. Understanding the full repertoire of CAFs and their dynamic changes could improve the precision of cancer treatment. CAFs are usually analyzed at a single time-point using specific markers, and it is therefore unclear whether CAFs display plasticity as tumors evolve. Here, we analyze thousands of CAFs using index and transcriptional single-cell sorting, at several time-points along breast tumor progression in mice, uncovering distinct subpopulations. Strikingly, the transcriptional programs of these subpopulations change over time and in metastases, transitioning from an immune-regulatory program to wound healing and antigen-presentation programs, indicating that CAFs and their functions are dynamic. Two main CAF subpopulations are also found in human breast tumors, where their ratio is associated with disease outcome across subtypes, and is particularly correlated with BRCA mutations in triple-negative breast cancer. These findings indicate that the repertoire of CAFs changes over time in breast cancer progression, with direct clinical implications.

Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies. Iannacone and colleagues show that the spatiotemporal regulation of type I interferon expression shapes the differentiation of antiviral CD4+ T cells into TFH or TH1 cells.

Spatially resolved single-cell RNA sequencing (scRNAseq) is a powerful approach for inferring connections between a cell's identity and its position in a tissue. We recently combined scRNAseq with spatially mapped landmark genes to infer the expression zonation of hepatocytes. However, determining zonation of small cells with low mRNA content, or without highly expressed landmark genes, remains challenging. Here we used paired-cell sequencing, in which mRNA from pairs of attached mouse cells were sequenced and gene expression from one cell type was used to infer the pairs' tissue coordinates. We applied this method to pairs of hepatocytes and liver endothelial cells (LECs). Using the spatial information from hepatocytes, we reconstructed LEC zonation and extracted a landmark gene panel that we used to spatially map LEC scRNAseq data. Our approach revealed the expression of both Wnt ligands and the Dkk3 Wnt antagonist in distinct pericentral LEC sub-populations. This approach can be used to reconstruct spatial expression maps of non-parenchymal cells in other tissues.

BackgroundHepatocellular carcinoma (HCC) has a dismal prognosis, and though checkpoint blocking antibodies have significantly improved patient outcome, many patients remain left out, highlighting the need to identify additional immune target to enhance therapeutic immunity. Macrophages (MF) are an abundant and heterogeneous population in the tumor microenvironment (TME), and are associated with a poor prognosis in multiple tumor types, including HCC, however, their molecular and functional diversity is still poorly understood.MethodsWe analyzed the molecular and spatial organization patterns of immune cells within the TME and adjacent tissue of 26 resected HCC lesions using single-cell RNA sequencing and multiplex immunohistochemistry (IHC).ResultsWe found that Kupffer cells, the self-renewing tissue-resident macrophages in liver tissue, are lacking from the TME, which is dominated by monocyte-derived macrophages. ScRNAseq followed by high-resolution clustering identified distinct MF molecular programs within the monocyte-derived macrophage compartment. One MF subset expressed a shared signature with monocytes including FCN1, S100A8 and T cell activation genes like CXCL9 and IL32. Conversely, one subset of MF expressed FOLR2, SEPP1 and genes of the complement (C1Qs), a program shared with KC in the adjacent tissue, and include another intratumoral subset enriched for the expression of TREM2 and GPNMB. Guided by these results, we are developing an IHC antibody panel that allows to visualize distinct MF localization in the TME. Intratumoral MF interface with, and potentially regulate, the T cell compartment within the TME. We are analyzing HCC tumor lesions in our treatment-naïve cohort and in patients treated with neoadjuvant anti-PD-1 therapy (NCT03916627) to study co-localization and direct interaction of MF and T cells using physically-interacting cell sequencing (PICseq). This analysis enables us to identify MF with direct cell-cell contact with T cells, and our preliminary analysis demonstrates an enrichment in MF with an immunosuppressive phenotype. We are also using spatial transcriptomic to map molecular programs of MF in the TME, that we will corroborated with additional patients.ConclusionsTaken together, our data provide a new understanding of intratumoral MF diversity and highlight the presence of specific immunoregulatory MF programs unique to tumor lesions, with subsets of these MF found to be directly interacting with T cells, potentially modulating anti-tumor responsiveness. Our analysis of resected tumor from anti-PD-1 treated patients, will allow us to correlate MF programs, and direct T cell interaction, with clinical response, and will inform therapeutic trials targeting specific MF populations so as to improve clinical efficacy of cancer immunotherapy.Trial RegistrationNCT03916627Ethics ApprovalSamples of tumor and non-involved liver were obtained from surgical specimens of patients undergoing resection at Mount Sinai Hospital (New York, NY) after obtaining informed consent in accordance with a protocol reviewed and approved by the Institutional Review Board at the Icahn School of Medicine at Mount Sinai (RUTH Human Subjects Electronic Submission System 18–00407 and 20–04150) and in collaboration with the Biorepository and Department of Pathology.

Hepatocellular carcinoma (HCC) has a dismal prognosis, and though checkpoint blocking antibodies have significantly improved patient outcome, many patients remain left out highlighting the need to identify additional immune target to enhance therapeutic immunity. Macrophages (MΦ) are an abundant and heterogeneous population in the tumor microenvironment (TME), and are associated with a poor prognosis in multiple tumor types, including HCC, however, their molecular and functional diversity is still poorly understood. We analyzed the molecular and spatial organization patterns of immune cells within the TME and adjacent tissue of 23 resected HCC lesions using single-cell RNA sequencing and multiplex immunohistochemistry (IHC). Strikingly, we found that Kupffer cells (KC), the self-renewing tissue-resident macrophages in liver tissue, are lacking from the TME, which is dominated by monocyte-derived macrophages. ScRNAseq followed by high-resolution clustering identified distinct MΦ molecular programs within the monocyte-derived macrophage compartment. One MΦ subset expressed a shared signature with monocytes including FCN1 and S100A8 in addition to T cell activation genes like CXCL9 and IL32. Conversely, one subset of MΦ expressed FOLR2, SEPP1 and genes of the complement (C1Qs), a program shared with KC in the adjacent uninvolved tissue, and include another intratumoral subset enriched for the expression of TREM2 and GPNMB. Guided by these results, we are developing an IHC antibody panel that allows to visualize distinct MΦ localization in the TME. Intratumoral MΦ interface with, and potentially regulate, the T cell compartment within the TME. We are analyzing HCC tumor lesions in our treatment-naïve cohort and in patients treated with neoadjuvant anti-PD-1 therapy (NCT03916627) to study direct interaction of MΦ and T cells using an innovative technology of physically-interacting cell sequencing (PICseq). This analysis enables us to identify MΦ with direct cell-cell contact with T cells, and our preliminary analysis demonstrates an enrichment in MΦ with a highly immunosuppressive phenotype. We are also using PICseq to map the molecular program of MΦ that are interacting with T cells and that we will corroborated with additional patients. Taken together, our data provide a new understanding of intratumoral MΦ diversity and highlight the presence of specific immunoregulatory MΦ programs unique to tumor lesions, with subsets of these MΦ found to be directly interacting with T cells, potentially modulating anti-tumor responsiveness. Our analysis of resected tumor from anti-PD-1 treated patients, will allow us to correlate MΦ programs, and direct T cell interaction, with clinical response, and will inform therapeutic trials targeting specific MΦ populations so as to improve clinical efficacy of cancer immunotherapy. Citation Format: Pauline Hamon, Assaf Magen, Merav Cohen, Alexandra Tabachnikova, Christie Chang, Mark Buckup, Leanna Troncoso, Zhen Zhao, Joel Kim, Amir Giladi, Nausicaa Malissen, Fiona Desland, Jessica Le Berichel, Ido Amit, Ephraim Kenigsberg, Myron Schwartz, Thomas Marron, Miriam Merad. Characterization of molecular and spatial diversity of macrophages in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 64.

Cancer associated fibroblasts (CAFs) are prevalent in carcinomas. CAFs are also heterogeneous and perform various tumor-promoting tasks. Understanding whether distinct CAF-subsets exert specific functions, and how the composition of CAFs changes as tumors evolve could improve the accuracy of cancer treatment. Here, we analyzed thousands of CAFs by single-cell RNA-sequencing and index-sorting at several timepoints along breast tumor progression in mice, revealing distinct CAF-subsets. We discovered that the transcriptional programs of these subsets change over time, shifting from an immune-regulatory program at earlier timepoints to wound-healing and antigen-presenting programs at later timepoints, indicating that the composition and functions of CAFs are dynamic. We also found the two main CAF subsets in human breast tumors, wherein their ratio was associated with disease outcome. This association was particularly correlated with BRCA mutations in triple-negative breast cancer. Our findings indicate that the diverse composition of CAFs in breast cancer changes over time as tumors progress, and that these changes are linked to disease outcome. Citation Format: Gil Friedman, Oshrat Levi-Galibov, Eyal David, Chamutal Bornstein, Amir Giladi, Maya Dadiani, Avi Mayo, Coral Halperin, Meirav Pevsner-Fischer, Hagar Lavon, Shimrit Mayer, Reinat Nevo, Yaniv Stein, Nora Balint-Lahat, Iris Barshack, H. Raza Ali, Carlos Caldas, Einav Nili Gal-Yam, Uri Alon, Ido Amit, Ruth Scherz-Shouval. Dynamic changes in the compositions of cancer associated-fibroblasts correlate with clinical outcome in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB009.

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.

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.

scRNA-seq profiles each represent a highly partial sample of mRNA molecules from a unique cell that can never be resampled, and robust analysis must separate the sampling effect from biological variance. We describe a methodology for partitioning scRNA-seq datasets into metacells: disjoint and homogenous groups of profiles that could have been resampled from the same cell. Unlike clustering analysis, our algorithm specializes at obtaining granular as opposed to maximal groups. We show how to use metacells as building blocks for complex quantitative transcriptional maps while avoiding data smoothing. Our algorithms are implemented in the MetaCell R/C++ software package.

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

Differentiation of CD4

Dendritic cells (DC) are unrivaled in their potential to prime naive T cells by presenting antigen and providing costimulation. DC are furthermore believed to decode antigen context by virtue of pattern recognition receptors and to polarize T cells through cytokine secretion toward distinct effector functions. Diverse polarized T helper (TH) cells have been explored in great detail. In contrast, studies of instructing DC have to date largely been restricted to in vitro settings or adoptively transferred DC. Here we report efforts to unravel the DC response to cognate T cell encounter in antigen-challenged lymph nodes (LN). Mice engrafted with antigen-specific T cells were immunized with nanoparticles (NP) entrapping adjuvants and absorbed with antigen to study the immediate DC response to T cell encounter using bulk and single cell RNA-seq profiling. NP induced robust antigen-specific TH1 cell responses with minimal bystander activation. Fluorescent-labeled NP allowed identification of antigen-carrying DC and focus on transcriptional changes in DC that encounter T cells. Our results support the existence of a bi-directional crosstalk between DC and T cells that promotes TH1 responses, including involvement of the ubiquitin-like molecule Isg15 that merits further study.

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

Single cell RNA-seq (scRNA-seq) has become the method of choice for analyzing mRNA distributions in heterogeneous cell populations. scRNA-seq only partially samples the cells in a tissue and the RNA in each cell, resulting in sparse data that challenge analysis. We develop a methodology that addresses scRNA-seq’s sparsity through partitioning the data into metacells: disjoint, homogenous and highly compact groups of cells, each exhibiting only sampling variance. Metacells constitute local building blocks for clustering and quantitative analysis of gene expression, while not enforcing any global structure on the data, thereby maintaining statistical control and minimizing biases. We illustrate the MetaCell framework by re-analyzing cell type and transcriptional gradients in peripheral blood and whole organism scRNA-seq maps. Our algorithms are implemented in the new MetaCell R/C++ software package.

Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα

The dynamics of haematopoietic stem cell differentiation and the hierarchy of oligopotent stem cells in the bone marrow remain controversial. Here we dissect haematopoietic progenitor populations at single cell resolution, deriving an unbiased reference model of transcriptional states in normal and perturbed murine bone marrow. We define the signature of the naive haematopoietic stem cell and find a continuum of core progenitor states. Core cell populations mix transcription of pre-myeloid and pre-lymphoid programs, but do not mix erythroid or megakaryocyte programs with other fates. CRISP-seq perturbation analysis confirms our models and reveals that Cebpa regulates entry into all myeloid fates, while Irf8 and PU.1 deficiency block later differentiation towards monocyte or granulocyte fates. Our transcriptional map defines a reference network model for blood progenitors and their differentiation trajectories during normal and perturbed haematopoiesis. Using a multi-tier scRNA-seq and CRISP-seq approach, Giladi et al. define a transcriptional signature for the naive haematopoietic stem cell state, and follow progenitor plasticity and fate commitment under the influence of cytokines and growth factors.

Summary: Hematopoietic stem cells (HSCs) are considered a heterogeneous cell population. To further resolve the HSC compartment, we characterized a retinoic acid (RA) reporter mouse line. Sub-fractionation of the HSC compartment in RA-CFP reporter mice demonstrated that RA-CFP-dim HSCs were largely non-proliferative and displayed superior engraftment potential in comparison with RA-CFP-bright HSCs. Gene expression analysis demonstrated higher expression of RA-target genes in RA-CFP-dim HSCs, in contrast to the RA-CFP reporter expression, but both RA-CFP-dim and RA-CFP-bright HSCs responded efficiently to RA in vitro. Single-cell RNA sequencing (RNA-seq) of >1,200 HSCs showed that differences in cell cycle activity constituted the main driver of transcriptional heterogeneity in HSCs. Moreover, further analysis of the single-cell RNA-seq data revealed that stochastic low-level expression of distinct lineage-affiliated transcriptional programs is a common feature of HSCs. Collectively, this work demonstrates the utility of the RA-CFP reporter line as a tool for the isolation of superior HSCs. : HSCs are considered a functional heterogeneous population. Lauridsen et al. use scRNA-seq to demonstrate that most transcriptional heterogeneity within the HSC compartment is associated with differences in cell cycle status. They further use an RA-CFP reporter mouse line to isolate slow-cycling HSCs characterized by superior engraftment potential. Keywords: hematopoietic stem cells, single-cell RNA-sequencing, retinoic acid, transcriptional heterogeneity, hematopoiesis

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.

International audience; T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations1-3. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing4,5, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.

Spatial information from NICHE-seq Immune functions depend on the interactions of heterogeneous cells in a range of microenvironments in the body. Although information regarding immune cell function has been collected using single-cell RNA-sequencing methods, these techniques have traditionally lacked spatial information. Medaglia et al. describe NICHE-seq, a technique that allows the sorting and analysis of cells from within visually selected territories in transgenic mice that express photoactivatable green fluorescent protein. The method successfully identified T and B cell-specific niches in mouse lymph nodes and spleens after virus infection. The approach will allow us to bridge the gap between cellular and spatial information in studies of organs. Science , this issue p. 1622

Innate lymphoid cells (ILCs) are critical modulators of mucosal immunity, inflammation, and tissue homeostasis, but their full spectrum of cellular states and regulatory landscapes remains elusive. Here, we combine genome-wide RNA-seq, ChIP-seq, and ATAC-seq to compare the transcriptional and epigenetic identity of small intestinal ILCs, identifying thousands of distinct gene profiles and regulatory elements. Single-cell RNA-seq and flow and mass cytometry analyses reveal compartmentalization of cytokine expression and metabolic activity within the three classical ILC subtypes and highlight transcriptional states beyond the current canonical classification. In addition, using antibiotic intervention and germ-free mice, we characterize the effect of the microbiome on the ILC regulatory landscape and determine the response of ILCs to microbial colonization at the single-cell level. Together, our work characterizes the spectrum of transcriptional identities of small intestinal ILCs and describes how ILCs differentially integrate signals from the microbial microenvironment to generate phenotypic and functional plasticity.

Summary Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.

Monocytes are circulating, short-lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C+ monocytes display developmental plasticity and are recruited to complement tissue-resident macrophages and dendritic cells on demand. Murine vascular Ly6C- monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA-seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C+ into Ly6C- monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre-established (poised) enhancers. Generation of Ly6C- monocytes involved induction of the transcription factor C/EBPβ and C/EBPβ-deficient mice lacked Ly6C- monocytes. Mechanistically, C/EBPβ bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor.

Crosstalk between neighboring cells underlies many biological processes, including cell signaling, proliferation and differentiation. Current single-cell genomic technologies profile each cell separately after tissue dissociation, losing information on cell-cell interactions. In the present study, we present an approach for sequencing physically interacting cells (PIC-seq), which combines cell sorting of physically interacting cells (PICs) with single-cell RNA-sequencing. Using computational modeling, PIC-seq systematically maps in situ cellular interactions and characterizes their molecular crosstalk. We apply PIC-seq to interrogate diverse interactions including immune-epithelial PICs in neonatal murine lungs. Focusing on interactions between T cells and dendritic cells (DCs) in vitro and in vivo, we map T cell-DC interaction preferences, and discover regulatory T cells as a major T cell subtype interacting with DCs in mouse draining lymph nodes. Analysis of T cell-DC pairs reveals an interaction-specific program between pathogen-presenting migratory DCs and T cells. PIC-seq provides a direct and broadly applicable technology to characterize intercellular interaction-specific pathways at high resolution.

Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5(+)-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies.