Cell function and activity are regulated through integration of signaling, epigenetic, transcriptional, and metabolic pathways. Here, we introduce INs-seq, an integrated technology for massively ...parallel recording of single-cell RNA sequencing (scRNA-seq) and intracellular protein activity. We demonstrate the broad utility of INs-seq for discovering new immune subsets by profiling different intracellular signatures of immune signaling, transcription factor combinations, and metabolic activity. Comprehensive mapping of Arginase 1-expressing cells within tumor models, a metabolic immune signature of suppressive activity, discovers novel Arg1+ Trem2+ regulatory myeloid (Mreg) cells and identifies markers, metabolic activity, and pathways associated with these cells. Genetic ablation of Trem2 in mice inhibits accumulation of intra-tumoral Mreg cells, leading to a marked decrease in dysfunctional CD8+ T cells and reduced tumor growth. This study establishes INs-seq as a broadly applicable technology for elucidating integrated transcriptional and intra-cellular maps and identifies the molecular signature of myeloid suppressive cells in tumors.
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•INs-seq: a new technology for recording scRNA-seq and intracellular protein activity•INs-seq defines new immune subsets by TF combinations and metabolic activity•Mapping Arg1+ cells within tumors reveals novel Trem2+ suppressive myeloid cells•Genetic ablation of Trem2 decreases Mreg, exhausted CD8+ T cells and tumor growth
INs-seq, an integrated technology for scRNA-seq and intracellular protein activity uncovers a novel Arg1+ Trem2+ regulatory myeloid cells (Mreg), genetic ablation of Trem2 inhibits the accumulation of intra-tumoral Mreg, leading to immune reactivation and reduced tumor growth.
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.
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•Transcriptionally primed single-cell subpopulations in early myeloid progenitors•Transcription factors and epigenetic landscapes that regulate myeloid priming•Mixed lineage states are not observed but appear when regulation is perturbed•New reference model for studying hematopoiesis at single-cell resolution
Single-cell transcriptomic analysis of bone marrow myeloid progenitor populations reveals early transcriptional priming toward seven different fates and absence of progenitors of mixed lineages, challenging the current models of hematopoiesis based on progressive loss of differentiation potential.
Histone modifications play an important role in chromatin organization and transcriptional regulation, but despite the large amount of genome-wide histone modification data collected in different ...cells and tissues, little is known about co-occurrence of modifications on the same nucleosome. Here we present a genome-wide quantitative method for combinatorial indexed chromatin immunoprecipitation (co-ChIP) to characterize co-occurrence of histone modifications on nucleosomes. Using co-ChIP, we study the genome-wide co-occurrence of 14 chromatin marks (70 pairwise combinations), and find previously undescribed co-occurrence patterns, including the co-occurrence of H3K9me1 and H3K27ac in super-enhancers. Finally, we apply co-ChIP to measure the distribution of the bivalent H3K4me3-H3K27me3 domains in two distinct mouse embryonic stem cell (mESC) states and in four adult tissues. We observe dynamic changes in 5,786 regions and discover both loss and de novo gain of bivalency in key tissue-specific regulatory genes, suggesting a functional role for bivalent domains during different stages of development. These results show that co-ChIP can reveal the complex interactions between histone modifications.
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
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.
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.
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•Population-scale scRNA-seq of skin and blood of scleroderma (SSc) disease spectrum•Immune cell composition changes are confined to a specific subtype of dSSc patients•SSc is defined by global perturbation of a novel scleroderma-associated fibroblast (ScAF)•ScAF coordinates the signaling pathways implicated in key processes driving SSc
Analysis of skin and blood samples from patients with scleroderma, a severe autoimmune disease, reveals how molecular changes in the stroma rather than immune dysfunction predominantly explain disease severity and clinical features.
Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, ...we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene-expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.
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► High-throughput approach for in vivo mapping of protein binding to DNA ► A comprehensive view of transcriptional network dynamics during immune response ► Hierarchical layered division of labor among transcriptional regulators ► Organizational principles of transcription regulatory networks in mammalian cells
Glioblastoma is the most aggressive primary brain tumor with an unmet need for more effective therapies. Here, we investigated combination therapies based on L19TNF, an antibody-cytokine fusion ...protein based on tumor necrosis factor that selectively localizes to cancer neovasculature. Using immunocompetent orthotopic glioma mouse models, we identified strong anti-glioma activity of L19TNF in combination with the alkylating agent CCNU, which cured the majority of tumor-bearing mice, whereas monotherapies only had limited efficacy. In situ and ex vivo immunophenotypic and molecular profiling in the mouse models revealed that L19TNF and CCNU induced tumor DNA damage and treatment-associated tumor necrosis. In addition, this combination also up-regulated tumor endothelial cell adhesion molecules, promoted the infiltration of immune cells into the tumor, induced immunostimulatory pathways, and decreased immunosuppression pathways. MHC immunopeptidomics demonstrated that L19TNF and CCNU increased antigen presentation on MHC class I molecules. The antitumor activity was T cell dependent and completely abrogated in immunodeficient mouse models. On the basis of these encouraging results, we translated this treatment combination to patients with glioblastoma. The clinical translation is ongoing but already shows objective responses in three of five patients in the first recurrent glioblastoma patient cohort treated with L19TNF in combination with CCNU (NCT04573192).
Deciphering the cell-state transitions underlying immune adaptation across time is fundamental for advancing biology. Empirical in vivo genomic technologies that capture cellular dynamics are ...currently lacking. We present Zman-seq, a single-cell technology recording transcriptomic dynamics across time by introducing time stamps into circulating immune cells, tracking them in tissues for days. Applying Zman-seq resolved cell-state and molecular trajectories of the dysfunctional immune microenvironment in glioblastoma. Within 24 hours of tumor infiltration, cytotoxic natural killer cells transitioned to a dysfunctional program regulated by TGFB1 signaling. Infiltrating monocytes differentiated into immunosuppressive macrophages, characterized by the upregulation of suppressive myeloid checkpoints Trem2, Il18bp, and Arg1, over 36 to 48 hours. Treatment with an antagonistic anti-TREM2 antibody reshaped the tumor microenvironment by redirecting the monocyte trajectory toward pro-inflammatory macrophages. Zman-seq is a broadly applicable technology, enabling empirical measurements of differentiation trajectories, which can enhance the development of more efficacious immunotherapies.
Introduction Substantial progress in the treatment of Multiple Myeloma (MM) extends survival for many patients (Pts), even after multiple relapses, owing to a broad and rapidly extending therapeutic ...arsenal, with major advances in the arena of bispecific antibodies and CART therapy. However, this also creates an increasingly complex challenge in selecting optimal therapeutic modalities for each patient, as current guidelines rely on empirical considerations. To address this, our study employs a population-scale single cell genomics database of over 250 patient samples covering all disease stages and cycles of treatment with complete patient medical records and machine learning approaches to analyze molecular signatures of myeloma cells and bone marrow immune microenvironment. Methods This study constitutes a meta-trial of RRMM patients who underwent single-cell RNA sequencing (scRNA-seq) between 6.2017 and 6.2023 in our lab. We gathered comprehensive demographic and myeloma clinical data, with a particular focus on therapeutic history and drug refractoriness, defined based on IMWG criteria (progression on or within 60 days from treatment). Our clinical scRNA-seq pipeline, includes a tailored protocol involving index sorting of CD38+ CD138+ positive cells for myeloma plasma cells (PCs) (Ledergor et al, Nat Med 2018; Cohen et al Nat Med 2020), together with CD45+ sorting and scRNA-seq cells from the tumor microenvironment for to identify tumor -immune molecular signatures associated with drug resistance. Results A total of 243 patient samples were included in the study, comprising RRMM (N=128), NDMM (N=21), SMM (N=9), and MGUS (N=4) patients with clinical annotation. Among RRMM patients, the median age was 72 (range 40-89), and 59% exhibited high-risk FISH cytogenetics (t(4:14), del17p, t(14:16), t(14:20), +1q21), while 17% had double-hit myeloma. Refractoriness to specific drugs was observed, such as bortezomib (49%), carfilzomib (27%), lenalidomide (49%), pomalidomide (33%), daratumumab (49). In total, we sequenced 158,444 myeloma/plasma cells, with an average of 880 cells per patient, along with 800 CD45+ cells. By using plasma cells from 11 healthy donors as a reference, we calculated transcriptional changes for each patient, determining the upregulated and downregulated genes profile. Clustering the patient's tumor profiles revealed 7 main tumor archetypes TA defined by the expression of 8 gene modules. Refractoriness score, defined as the average number of drugs to which myeloma is refractory, exhibited variations across different tumor archetypes. Notably, the expression levels of several tumor drivers and crucial targets of novel immunotherapies also showed diversity among the tumor archetypes. For instance, TA#6, primarily comprising newly diagnosed patients, displayed lower expression of GPRC5D. Conversely, in TA#1, which consisted of ultra-refractory patients with high MYC and CD47 expression, both GPRC5D and TNFRSF17 (BCMA) were downregulated. Examination of bone marrow CD45+ immune cells revealed a striking bimodal immune composition, characterized by one subtype with increased T-cell abundance and another enriched with myeloid cells. Each immune cell subtype contributed to the drug responsiveness score and provided additional insights complementary to the tumor cells' profile. Further clinical and transcriptional correlations involving both MM cells and immune cells will be presented at the meeting. Conclusions We constructed a clinically-annotated transcriptional matrix revealing the blueprint of relapsed refractory myeloma. This novel decision engine provides a basis for optimizing treatment selection by considering both tumor and tumor microenvironment characteristics. By incorporating critical information on drug resistance patterns and expression of key targets like GPRC5D, TNFRSF17 (BCMA) and FcRH5, we enable precision medicine approaches for improved management of relapsed refractory myeloma. These findings open avenues for tailored and more effective therapeutic strategies, promising better outcomes for patients with relapsed refractory myeloma.
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.
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