The ATR inhibitor ceralasertib potentiates cancer checkpoint immunotherapy by regulating the tumor microenvironment.

The Ataxia telangiectasia and Rad3-related (ATR) inhibitor ceralasertib in combination with the PD-L1 antibody durvalumab demonstrated encouraging clinical benefit in melanoma and lung cancer patients who progressed on immunotherapy. Here we show that modelling of intermittent ceralasertib treatment in mouse tumor models reveals CD8+ T-cell dependent antitumor activity, which is separate from the effects on tumor cells. Ceralasertib suppresses proliferating CD8+ T-cells on treatment which is rapidly reversed off-treatment. Ceralasertib causes up-regulation of type I interferon (IFNI) pathway in cancer patients and in tumor-bearing mice. IFNI is experimentally found to be a major mediator of antitumor activity of ceralasertib in combination with PD-L1 antibody. Improvement of T-cell function after ceralasertib treatment is linked to changes in myeloid cells in the tumor microenvironment. IFNI also promotes anti-proliferative effects of ceralasertib on tumor cells. Here, we report that broad immunomodulatory changes following intermittent ATR inhibition underpins the clinical therapeutic benefit and indicates its wider impact on antitumor immunity.

Cis inhibition of NOTCH1 through JAGGED1 sustains embryonic hematopoietic stem cell fate.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta.

Tracking early mammalian organogenesis - prediction and validation of differentiation trajectories at whole organism scale.

Early organogenesis represents a key step in animal development, during which pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single-cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-h intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled timecourse of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-seq to transcriptionally determine cell fates of grafted primitive streak regions after 24 h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.

Single-cell multi-omics analysis of COVID-19 patients with pre-existing autoimmune diseases shows aberrant immune responses to infection.

In COVID-19, hyperinflammatory and dysregulated immune responses contribute to severity. Patients with pre-existing autoimmune conditions can therefore be at increased risk of severe COVID-19 and/or associated sequelae, yet SARS-CoV-2 infection in this group has been little studied. Here, we performed single-cell analysis of peripheral blood mononuclear cells from patients with three major autoimmune diseases (rheumatoid arthritis, psoriasis, or multiple sclerosis) during SARS-CoV-2 infection. We observed compositional differences between the autoimmune disease groups coupled with altered patterns of gene expression, transcription factor activity, and cell-cell communication that substantially shape the immune response under SARS-CoV-2 infection. While enrichment of HLA-DRlow CD14+ monocytes was observed in all three autoimmune disease groups, type-I interferon signaling as well as inflammatory T cell and monocyte responses varied widely between the three groups of patients. Our results reveal disturbed immune responses to SARS-CoV-2 in patients with pre-existing autoimmunity, highlighting important considerations for disease treatment and follow-up.

Autophagy counters inflammation-driven glycolytic impairment in aging hematopoietic stem cells.

Aging of the hematopoietic system promotes various blood, immune and systemic disorders and is largely driven by hematopoietic stem cell (HSC) dysfunction ( 1 ). Autophagy is central for the benefits associated with activation of longevity signaling programs ( 2 ), and for HSC function and response to nutrient stress ( 3,4 ). With age, a subset of HSCs increases autophagy flux and preserves some regenerative capacity, while the rest fail to engage autophagy and become metabolically overactivated and dysfunctional ( 4 ). However, the signals that promote autophagy in old HSCs and the mechanisms responsible for the increased regenerative potential of autophagy-activated old HSCs remain unknown. Here, we demonstrate that autophagy activation is an adaptive survival response to chronic inflammation in the aging bone marrow (BM) niche ( 5 ). We find that inflammation impairs glucose metabolism and suppresses glycolysis in aged HSCs through Socs3-mediated impairment of AKT/FoxO-dependent signaling. In this context, we show that inflammation-mediated autophagy engagement preserves functional quiescence by enabling metabolic adaptation to glycolytic impairment. Moreover, we demonstrate that transient autophagy induction via a short-term fasting/refeeding paradigm normalizes glucose uptake and glycolytic flux and significantly improves old HSC regenerative potential. Our results identify inflammation-driven glucose hypometabolism as a key driver of HSC dysfunction with age and establish autophagy as a targetable node to reset old HSC glycolytic and regenerative capacity. One-Sentence Summary: Autophagy compensates for chronic inflammation-induced metabolic deregulation in old HSCs, and its transient modulation can reset old HSC glycolytic and regenerative capacity.

An atlas of rabbit development as a model for single-cell comparative genomics.

Traditionally, the mouse has been the favoured vertebrate model for biomedical research, due to its experimental and genetic tractability. However, non-rodent embryological studies highlight that many aspects of early mouse development, such as its egg-cylinder gastrulation and method of implantation, diverge from other mammals, thus complicating inferences about human development. Like the human embryo, rabbits develop as a flat-bilaminar disc. Here we constructed a morphological and molecular atlas of rabbit development. We report transcriptional and chromatin accessibility profiles for over 180,000 single cells and high-resolution histology sections from embryos spanning gastrulation, implantation, amniogenesis and early organogenesis. Using a neighbourhood comparison pipeline, we compare the transcriptional landscape of rabbit and mouse at the scale of the entire organism. We characterize the gene regulatory programmes underlying trophoblast differentiation and identify signalling interactions involving the yolk sac mesothelium during haematopoiesis. We demonstrate how the combination of both rabbit and mouse atlases can be leveraged to extract new biological insights from sparse macaque and human data. The datasets and computational pipelines reported here set a framework for a broader cross-species approach to decipher early mammalian development, and are readily adaptable to deploy single-cell comparative genomics more broadly across biomedical research.

Mapping interindividual dynamics of innate immune response at single-cell resolution.

Common genetic variants across individuals modulate the cellular response to pathogens and are implicated in diverse immune pathologies, yet how they dynamically alter the response upon infection is not well understood. Here, we triggered antiviral responses in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-sequencing. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), a statistical approach designed to identify nonlinear dynamic genetic effects across transcriptional trajectories of cells. This approach identified 1,275 expression quantitative trait loci (local false discovery rate 10%) that manifested during the responses, many of which were colocalized with susceptibility loci identified by genome-wide association studies of infectious and autoimmune diseases, including the OAS1 splicing quantitative trait locus in a COVID-19 susceptibility locus. In summary, our analytical approach provides a unique framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

Proteomic analysis of circulating immune cells identifies cellular phenotypes associated with COVID-19 severity.

Certain serum proteins, including C-reactive protein (CRP) and D-dimer, have prognostic value in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nonetheless, these factors are non-specific, providing limited mechanistic insight into the peripheral blood mononuclear cell (PBMC) populations that drive the pathogenesis of severe COVID-19. To identify cellular phenotypes associated with disease, we performed a comprehensive, unbiased analysis of total and plasma-membrane PBMC proteomes from 40 unvaccinated individuals with SARS-CoV-2, spanning the whole disease spectrum. Combined with RNA sequencing (RNA-seq) and flow cytometry from the same donors, we define a comprehensive multi-omic profile for each severity level, revealing that immune-cell dysregulation progresses with increasing disease. The cell-surface proteins CEACAMs1, 6, and 8, CD177, CD63, and CD89 are strongly associated with severe COVID-19, corresponding to the emergence of atypical CD3+CD4+CEACAM1/6/8+CD177+CD63+CD89+ and CD16+CEACAM1/6/8+ mononuclear cells. Utilization of these markers may facilitate real-time patient assessment by flow cytometry and identify immune populations that could be targeted to ameliorate immunopathology.

Stromal niche inflammation mediated by IL-1 signalling is a targetable driver of haematopoietic ageing.

Haematopoietic ageing is marked by a loss of regenerative capacity and skewed differentiation from haematopoietic stem cells (HSCs), leading to impaired blood production. Signals from the bone marrow niche tailor blood production, but the contribution of the old niche to haematopoietic ageing remains unclear. Here we characterize the inflammatory milieu that drives both niche and haematopoietic remodelling. We find decreased numbers and functionality of osteoprogenitors at the endosteum and expansion of central marrow LepR+ mesenchymal stromal cells associated with deterioration of the sinusoidal vasculature. Together, they create a degraded and inflamed old bone marrow niche. Niche inflammation in turn drives the chronic activation of emergency myelopoiesis pathways in old HSCs and multipotent progenitors, which promotes myeloid differentiation and hinders haematopoietic regeneration. Moreover, we show how production of interleukin-1ß (IL-1ß) by the damaged endosteum acts in trans to drive the proinflammatory nature of the central marrow, with damaging consequences for the old blood system. Notably, niche deterioration, HSC dysfunction and defective regeneration can all be ameliorated by blocking IL-1 signalling. Our results demonstrate that targeting IL-1 as a key mediator of niche inflammation is a tractable strategy to improve blood production during ageing.

Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19.

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

Local and systemic responses to SARS-CoV-2 infection in children and adults.

It is not fully understood why COVID-19 is typically milder in children1-3. Here, to examine the differences between children and adults in their response to SARS-CoV-2 infection, we analysed paediatric and adult patients with COVID-19 as well as healthy control individuals (total n = 93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In the airways of healthy paediatric individuals, we observed cells that were already in an interferon-activated state, which after SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon responses restrict viral replication and disease progression. The systemic response in children was characterized by increases in naive lymphocytes and a depletion of natural killer cells, whereas, in adults, cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signalling in early infection, and identify epithelial cell states associated with COVID-19 and age. Our matching nasal and blood data show a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were substantially reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children.

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

Single-Cell Analysis of Hematopoietic Stem Cells.

The study of hematopoiesis has been revolutionized in recent years by the application of single-cell RNA sequencing technologies. The technique coupled with rapidly developing bioinformatic analysis has provided great insight into the cell type compositions of many populations previously defined by their cell surface phenotype. Moreover, transcriptomic information enables the identification of individual molecules and pathways which define novel cell populations and their transitions including cell lineage decisions. Combining single-cell transcriptional profiling with molecular perturbations allows functional analysis of individual factors in gene regulatory networks and better understanding of the earliest stages of malignant transformation. In this chapter we describe a comprehensive protocol for scRNA-Seq analysis of the mouse bone marrow, using both plate-based (low throughput) and droplet-based (high throughput) methods. The protocol includes instructions for sample preparation, an antibody panel for flow cytometric purification of hematopoietic progenitors with index sorting for plate-based analysis or in bulk for droplet-based methods. The plate-based protocol described in this chapter is a combination of the Smart-Seq2 and mcSCRB-Seq protocols, optimized in our laboratory. It utilizes off-the-shelf reagents for cDNA preparation, is amenable to automation using a liquid handler, and takes 4 days from preparation of the cells for sorting to producing a sequencing-ready library. The droplet-based method (using for instance the 10× Genomics platform) relies on the manufacturer's user guide and commercial reagents, and takes 3 days from isolation of the cells to the production of a library ready for sequencing.

Single-cell multi-omics analysis of the immune response in COVID-19.

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy.

Single-cell transcriptome analysis of CAR T-cell products reveals subpopulations, stimulation, and exhaustion signatures.

Chimeric antigen receptor (CAR) T-cell adoptive therapy is set to transform the treatment of a rapidly expanding range of malignancies. Although the activation process of normal T cells is well characterized, comparatively little is known about the activation of cells via the CAR. Here we have used flow cytometry together with single-cell transcriptome profiling to characterize the starting material (peripheral blood mononuclear cells) and CAR therapeutic products of 3 healthy donors in the presence and absence of antigen-specific stimulation. Analysis of 53,191 single-cell transcriptomes showed APRIL-based CAR products to contain several subpopulations of cells, with cellular composition reproducible from donor to donor, and all major cellular subsets compatible with CAR expression. Only 50% of CAR-expressing cells displayed transcriptional changes upon CAR-specific antigen exposure. The resulting molecular signature for CAR T-cell activation provides a rich resource for future dissection of underlying mechanisms. Targeted data interrogation also revealed that a small proportion of antigen-responding CAR-expressing cells displayed an exhaustion signature, with both known markers and genes not previously associated with T-cell exhaustion. Comprehensive single-cell transcriptomic analysis thus represents a powerful way to guide the assessment and optimization of clinical-grade CAR-T-cells, and inform future research into the underlying molecular processes.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

Single-cell chromatin accessibility maps reveal regulatory programs driving early mouse organogenesis.

During mouse embryonic development, pluripotent cells rapidly divide and diversify, yet the regulatory programs that define the cell repertoire for each organ remain ill-defined. To delineate comprehensive chromatin landscapes during early organogenesis, we mapped chromatin accessibility in 19,453 single nuclei from mouse embryos at 8.25 days post-fertilization. Identification of cell-type-specific regions of open chromatin pinpointed two TAL1-bound endothelial enhancers, which we validated using transgenic mouse assays. Integrated gene expression and transcription factor motif enrichment analyses highlighted cell-type-specific transcriptional regulators. Subsequent in vivo experiments in zebrafish revealed a role for the ETS factor FEV in endothelial identity downstream of ETV2 (Etsrp in zebrafish). Concerted in vivo validation experiments in mouse and zebrafish thus illustrate how single-cell open chromatin maps, representative of a mammalian embryo, provide access to the regulatory blueprint for mammalian organogenesis.

Dissecting the early steps of MLL induced leukaemogenic transformation using a mouse model of AML.

Leukaemogenic mutations commonly disrupt cellular differentiation and/or enhance proliferation, thus perturbing the regulatory programs that control self-renewal and differentiation of stem and progenitor cells. Translocations involving the Mll1 (Kmt2a) gene generate powerful oncogenic fusion proteins, predominantly affecting infant and paediatric AML and ALL patients. The early stages of leukaemogenic transformation are typically inaccessible from human patients and conventional mouse models. Here, we take advantage of cells conditionally blocked at the multipotent haematopoietic progenitor stage to develop a MLL-r model capturing early cellular and molecular consequences of MLL-ENL expression based on a clear clonal relationship between parental and leukaemic cells. Through a combination of scRNA-seq, ATAC-seq and genome-scale CRISPR-Cas9 screening, we identify pathways and genes likely to drive the early phases of leukaemogenesis. Finally, we demonstrate the broad utility of using matched parental and transformed cells for small molecule inhibitor studies by validating both previously known and other potential therapeutic targets.

Notch ligand Dll4 impairs cell recruitment to aortic clusters and limits blood stem cell generation.

Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium in cluster structures that protrude into the embryonic aortic lumen. Although much is known about the molecular characteristics of the developing hematopoietic cells, we lack a complete understanding of their origin and the three-dimensional organization of the niche. Here, we use advanced live imaging techniques of organotypic slice cultures, clonal analysis, and mathematical modeling to show the two-step process of intra-aortic hematopoietic cluster (IACH) formation. First, a hemogenic progenitor buds up from the endothelium and undergoes division forming the monoclonal core of the IAHC. Next, surrounding hemogenic cells are recruited into the IAHC, increasing their size and heterogeneity. We identified the Notch ligand Dll4 as a negative regulator of the recruitment phase of IAHC. Blocking of Dll4 promotes the entrance of new hemogenic Gfi1+ cells into the IAHC and increases the number of cells that acquire HSC activity. Mathematical modeling based on our data provides estimation of the cluster lifetime and the average recruitment time of hemogenic cells to the cluster under physiologic and Dll4-inhibited conditions.

GATA2 Promotes Hematopoietic Development and Represses Cardiac Differentiation of Human Mesoderm.

In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.