LGR5 expressing skin fibroblasts define a major cellular hub perturbed in scleroderma.

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.

Coupled scRNA-Seq and Intracellular Protein Activity Reveal an Immunosuppressive Role of TREM2 in Cancer.

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.

Transcriptional programs that control expression of the autoimmune regulator gene Aire.

Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire. In mTECs, however, Aire expression was facilitated by concurrent eviction of CTCF, specific demethylation of exon 2 and the proximal promoter, and the coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which acted on mTEC-specific accessible regions in the Aire locus.

Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies.

Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN-γ and VEGFα, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation.

Single-cell mapping of the thymic stroma identifies IL-25-producing tuft epithelial cells.

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.

A negative feedback loop of transcription factors specifies alternative dendritic cell chromatin States.

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.

A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals.

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.

CD74 is a novel transcription regulator.

CD74 is a cell-surface receptor for the cytokine macrophage migration inhibitory factor. Macrophage migration inhibitory factor binding to CD74 induces its intramembrane cleavage and the release of its cytosolic intracellular domain (CD74-ICD), which regulates cell survival. In the present study, we characterized the transcriptional activity of CD74-ICD in chronic lymphocytic B cells. We show that following CD74 activation, CD74-ICD interacts with the transcription factors RUNX (Runt related transcription factor) and NF-κB and binds to proximal and distal regulatory sites enriched for genes involved in apoptosis, immune response, and cell migration. This process leads to regulation of expression of these genes. Our results suggest that identifying targets of CD74 will help in understanding of essential pathways regulating B-cell survival in health and disease.

Identification of resistance pathways and therapeutic targets in relapsed multiple myeloma patients through single-cell sequencing.

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.

Cancer-associated fibroblast compositions change with breast cancer progression linking the ratio of S100A4+ and PDPN+ CAFs to clinical outcome.

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.

ETS Proteins Bind with Glucocorticoid Receptors: Relevance for Treatment of Ewing Sarcoma.

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.

Induction of CD4 T cell memory by local cellular collectivity.

Cell differentiation is directed by signals driving progenitors into specialized cell types. This process can involve collective decision-making, when differentiating cells determine their lineage choice by interacting with each other. We used live-cell imaging in microwell arrays to study collective processes affecting differentiation of naïve CD4+ T cells into memory precursors. We found that differentiation of precursor memory T cells sharply increases above a threshold number of locally interacting cells. These homotypic interactions involve the cytokines interleukin-2 (IL-2) and IL-6, which affect memory differentiation orthogonal to their effect on proliferation and survival. Mathematical modeling suggests that the differentiation rate is continuously modulated by the instantaneous number of locally interacting cells. This cellular collectivity can prioritize allocation of immune memory to stronger responses.

Single cell dissection of plasma cell heterogeneity in symptomatic and asymptomatic myeloma.

Multiple myeloma, a plasma cell malignancy, is the second most common blood cancer. Despite extensive research, disease heterogeneity is poorly characterized, hampering efforts for early diagnosis and improved treatments. Here, we apply single cell RNA sequencing to study the heterogeneity of 40 individuals along the multiple myeloma progression spectrum, including 11 healthy controls, demonstrating high interindividual variability that can be explained by expression of known multiple myeloma drivers and additional putative factors. We identify extensive subclonal structures for 10 of 29 individuals with multiple myeloma. In asymptomatic individuals with early disease and in those with minimal residual disease post-treatment, we detect rare tumor plasma cells with molecular characteristics similar to those of active myeloma, with possible implications for personalized therapies. Single cell analysis of rare circulating tumor cells allows for accurate liquid biopsy and detection of malignant plasma cells, which reflect bone marrow disease. Our work establishes single cell RNA sequencing for dissecting blood malignancies and devising detailed molecular characterization of tumor cells in symptomatic and asymptomatic patients.

SPATA18, a spermatogenesis-associated gene, is a novel transcriptional target of p53 and p63.

The transcription factor p53 functions not only to suppress tumorigenesis but also to maintain normal development and homeostasis. Although p53 was implicated in different aspects of fertility, including spermatogenesis and implantation, the mechanism underlying p53 involvement in spermatogenesis is poorly resolved. In this study we describe the identification of a spermatogenesis-associated gene, SPATA18, as a novel p53 transcriptional target and show that SPATA18 transcription is induced by p53 in a variety of cell types of both human and mouse origin. p53 binds a consensus DNA motif that resides within the first intron of SPATA18. We describe the spatiotemporal expression patterns of SPATA18 in mouse seminiferous tubules and suggest that SPATA18 transcription is regulated in vivo by p53. We also demonstrate the induction of SPATA18 by p63 and suggest that p63 can compensate for the loss of p53 activity in vivo. Our data not only enrich the known collection of p53 targets but may also provide insights on spermatogenesis defects that are associated with p53 deficiency.

Mutant p53 facilitates somatic cell reprogramming and augments the malignant potential of reprogrammed cells.

p53 deficiency enhances the efficiency of somatic cell reprogramming to a pluripotent state. As p53 is usually mutated in human tumors and many mutated forms of p53 gain novel activities, we studied the influence of mutant p53 (mut-p53) on somatic cell reprogramming. Our data indicate a novel gain of function (GOF) property for mut-p53, which markedly enhanced the efficiency of the reprogramming process compared with p53 deficiency. Importantly, this novel activity of mut-p53 induced alterations in the characteristics of the reprogrammed cells. Although p53 knockout (KO) cells reprogrammed with only Oct4 and Sox2 maintained their pluripotent capacity in vivo, reprogrammed cells expressing mutant p53 lost this capability and gave rise to malignant tumors. This novel GOF of mut-p53 is not attributed to its effect on proliferation, as both p53 KO and mut-p53 cells displayed similar proliferation rates. In addition, we demonstrate an oncogenic activity of Klf4, as its overexpression in either p53 KO or mut-p53 cells induced aggressive tumors. Overall, our data show that reprogrammed cells with the capacity to differentiate into the three germ layers in vitro can form malignant tumors, suggesting that in genetically unstable cells, such as those in which p53 is mutated, reprogramming may result in the generation of cells with malignant tumor-forming potential.

p53-dependent transcriptional regulation of EDA2R and its involvement in chemotherapy-induced hair loss.

The p53 tumor suppressor coordinates a multitude of cellular and organismal processes and exerts its activities mainly by activation of gene transcription. Here we describe the transcriptional activation of ectodysplasin A2 receptor (EDA2R) by p53 in a variety of cell types and tissues. We demonstrate that treatment of cancer cells with the ligand EDA-A2, known to specifically activate EDA2R, results in p53-dependent cell death. Moreover, we show that EDA2R is transactivated by p53 during chemotherapy-induced hair-loss, although its presence is not necessary for this process. These data shed new light on the role of EDA2R in exerting p53 function.