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Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by... more
Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by various niche-confined factors. R-spondin 3 (RSPO3) is one of the most potent enhancers of Wnt signaling, and its expression is usually restricted to the stem cell niche where it provides localized enhancement of Wnt signaling to regulate stem cell expansion and differentiation. Disruption of this niche-confined expression can disturb proper tissue organization and lead to cancers. Here, we investigate the consequences of disrupting the niche-restricted expression of RSPO3 in various tissues, including the hematopoietic system. We show that normal Rspo3 expression is confined to the perivascular niche in the bone marrow. Induction of increased systemic levels of circulating RSPO3 outside of the niche results in prominent loss of early B-cell progenitors a...
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NOTCH1 gain-of-function mutations are recurrent in B-cell chronic lymphocytic leukemia (B-CLL), where they are associated with accelerated disease progression and refractoriness to chemotherapy. The specific role of NOTCH1 in the... more
NOTCH1 gain-of-function mutations are recurrent in B-cell chronic lymphocytic leukemia (B-CLL), where they are associated with accelerated disease progression and refractoriness to chemotherapy. The specific role of NOTCH1 in the development and progression of this malignancy is unclear. Here, we assess the impact of loss of Notch signaling and pathway hyperactivation in an in vivo mouse model of CLL (IgH.TEμ) that faithfully replicates many features of the human pathology. Ablation of canonical Notch signaling using conditional gene inactivation of RBP-J in immature hematopoietic or B-cell progenitors delayed CLL induction and reduced incidence of mice developing disease. In contrast, forced expression of a dominant active form of Notch resulted in more animals developing CLL with early disease onset. Comparative analysis of gene expression and epigenetic features of Notch gain-of-function and control CLL cells revealed direct and indirect regulation of cell cycle–associated genes,...
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Mammalian cells use cytosolic nucleic acid receptors to detect pathogens and other stress signals. In innate immune cells the presence of cytosolic DNA is sensed by the cGAS–STING signalling pathway, which initiates a gene expression... more
Mammalian cells use cytosolic nucleic acid receptors to detect pathogens and other stress signals. In innate immune cells the presence of cytosolic DNA is sensed by the cGAS–STING signalling pathway, which initiates a gene expression programme linked to cellular activation and cytokine production. Whether the outcome of the STING response varies between distinct cell types remains largely unknown. Here we show that T cells exhibit an intensified STING response, which leads to the expression of a distinct set of genes and results in the induction of apoptosis. Of note, this proapoptotic STING response is still functional in cancerous T cells and delivery of small molecule STING agonists prevents in vivo growth of T-cell-derived tumours independent of its adjuvant activity. Our results demonstrate how the magnitude of STING signalling can shape distinct effector responses, which may permit for cell type-adjusted behaviours towards endogenous or exogenous insults.
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Follicular helper T (TFH) cells are specialized in providing help for B cell differentiation and Ab secretion. Several positive and negative regulators of TFH cell differentiation have been described but their control is not fully... more
Follicular helper T (TFH) cells are specialized in providing help for B cell differentiation and Ab secretion. Several positive and negative regulators of TFH cell differentiation have been described but their control is not fully understood. In this study, we show that Notch signaling in T cells is a major player in the development and function of TFH cells. T cell–specific gene ablation of Notch1 and Notch2 impaired differentiation of TFH cells in draining lymph nodes of mice immunized with T-dependent Ags or infected with parasites. Impaired TFH cell differentiation correlated with deficient germinal center development and the absence of high-affinity Abs. The impact of loss of Notch on TFH cell differentiation was largely independent of its effect on IL-4. These results show a previously unknown role for Notch in the regulation of TFH cell differentiation and function with implications for the control of this T cell population.
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Significance The Notch signaling cascade is deregulated by oncogenic lesions in human cancers and has therefore become an attractive therapeutic target. Inhibitory monoclonal antibodies and small-molecule γ-secretase inhibitors have been... more
Significance The Notch signaling cascade is deregulated by oncogenic lesions in human cancers and has therefore become an attractive therapeutic target. Inhibitory monoclonal antibodies and small-molecule γ-secretase inhibitors have been developed to target the pathway at the most proximal point of the cascade. Major hurdles to the therapeutic application of these Notch inhibitors have been prevalent dose-limiting toxicities in the intestine. Here we report identification and preclinical validation of a small molecule (CB-103) that inhibits the pathway at the level of the Notch transcription complex without causing intestinal toxicity. Its properties and mechanism of action provide CB-103 with a more favorable therapeutic index than other types of Notch targeting agents, a feature that is currently being tested in cancer patients.
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In this issue of JEM, Varga et al. (https://doi.org/10.1084/jem.20191515) describe a mouse model of invasive and metastatic colorectal cancer (CRC) closely resembling the human consensus molecular subtype (CMS) 4 associated with the... more
In this issue of JEM, Varga et al. (https://doi.org/10.1084/jem.20191515) describe a mouse model of invasive and metastatic colorectal cancer (CRC) closely resembling the human consensus molecular subtype (CMS) 4 associated with the poorest overall survival of the four CMSs. Transcriptomic and bioinformatic analysis combined with pharmacological and genetic studies identified Notch3 as a promoter of tumor progression and metastasis. NOTCH3 expression was up-regulated in CMS4 CRC patients and associated with tumor staging, lymph node and distant metastasis. These findings feature NOTCH3 as putative therapeutic target for advanced CMS4 CRC patients.
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NOTCH1 is a well-established lineage specifier for T cells and among the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone... more
NOTCH1 is a well-established lineage specifier for T cells and among the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology, allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of preleu...
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The interaction between CD4 and major histocompatibility complex class II proteins provides a critical co-receptor function for the activation of CD4+ T cells implicated in the pathogenesis of a number of autoimmune diseases and... more
The interaction between CD4 and major histocompatibility complex class II proteins provides a critical co-receptor function for the activation of CD4+ T cells implicated in the pathogenesis of a number of autoimmune diseases and transplantation responses. A ...
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Computer Graphics, Chemistry, Biology, Cell Adhesion, Biological Chemistry, and 15 moreMedicine, Biological Sciences, Humans, Female, Animals, Male, Biological, Drug Design, CHEMICAL SCIENCES, Amino Acid Sequence, Lymphocytes, Graft Rejection, Epitopes, binding sites, and Medical and Health Sciences
Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T... more
Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). The mechanisms controlling cTEC and mTEC production from the common TEPC are not however understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH-independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPC at the expense of cTEC differentiation. Finally, we uncover a cross-regulatory relationship between NOTCH and FOXN1, a master regulator of TEC differentiation. These data establish NOTCH as ...
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Notch signals drive pathogenic T cell alloreactivity in mouse models of allogeneic bone marrow transplantation (allo-BMT) leading to lethal graft-versus-host disease (GVHD). In a mouse model of MHC-mismatched allo-BMT with high intensity... more
Notch signals drive pathogenic T cell alloreactivity in mouse models of allogeneic bone marrow transplantation (allo-BMT) leading to lethal graft-versus-host disease (GVHD). In a mouse model of MHC-mismatched allo-BMT with high intensity whole body conditioning (11 Gy), residual recipient or donor hematopoietic cells were dispensable sources of Notch signals to drive T cell alloreactivity. Instead, abrogating the expression of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands in Ccl19-Cre+ secondary lymphoid organ (SLO) fibroblastic stromal cells protected mice from GVHD (Chung et al., JCI 2017). In this study we sought to 1) understand the expression and regulation of Notch ligands in fibroblastic stromal cells during allo-BMT; 2) test whether fibroblastic stromal cells are still critical sources of Notch signals in models of allotransplantation with reduced or no conditioning (when other radiation-sensitive sources of Notch ligands are better preserved); and 3) assess whethe...
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Notch signaling is a critical regulator of T cell effector functions during acute graft-versus-host disease (GVHD). Pan-Notch inhibition in donor-derived T cells or systemic antibody-mediated blockade of Delta-like1 (Dll1) and Delta-like4... more
Notch signaling is a critical regulator of T cell effector functions during acute graft-versus-host disease (GVHD). Pan-Notch inhibition in donor-derived T cells or systemic antibody-mediated blockade of Delta-like1 (Dll1) and Delta-like4 (Dll4) Notch ligands results in near-complete protection from acute GVHD in mouse models of allogeneic bone marrow transplantation. Notch-deprived alloreactive T cells proliferate and accumulate in vivo, but produce dramatically reduced levels of the proinflammatory cytokines IFNγ, TNFα and interleukin-2 (IL-2) (Zhang et al., Blood 2011; Sandy et al., J Immunol 2013; Tran et al., J Clin Invest 2013). In this study, we sought to: 1) determine the kinetic requirements for Notch signaling in the pathogenesis of acute GVHD; 2) identify the essential cellular compartment that delivers Dll1 and/or Dll4 ligands to incoming alloreactive T cells. In the B6 anti-BALB/c major histocompatibility complex-mismatched model, a single dose of Dll1 and Dll4 blocking...
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Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T... more
Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). The mechanisms controlling cTEC and mTEC production from the common TEPC are not however understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH-independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPC at the expense of cTEC differentiation. Finally, we uncover a direct interaction between NOTCH and FOXN1, the master regulator of TEC differentiation. These data establish NOTCH as a potent ...
Although c-Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used themouse melanoma model to show that c-Myc is essential for tumor initiation,... more
Although c-Myc is essential for melanocyte development, its role in cutaneous melanoma, the most aggressive skin cancer, is only partly understood. Here we used themouse melanoma model to show that c-Myc is essential for tumor initiation, maintenance, and metastasis. c-Myc-expressing melanoma cells were preferentially found at metastatic sites, correlated with increased tumor aggressiveness and high tumor initiation potential. Abrogation of c-Myc caused apoptosis in primary murine and human melanoma cells. Mechanistically, c-Myc-positive melanoma cells activated and became dependent on the metabolic energy sensor AMP-activated protein kinase (AMPK), a metabolic checkpoint kinase that plays an important role in energy and redox homeostasis under stress conditions. AMPK pathway inhibition caused apoptosis of c-Myc-expressing melanoma cells, while AMPK activation protected against cell death of c-Myc-depleted melanoma cells through suppression of oxidative stress. Furthermore, TCGA dat...
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Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a... more
Alloimmune T cell responses induce graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (allo-BMT). Although Notch signaling mediated by Delta-like 1/4 (DLL1/4) Notch ligands has emerged as a major regulator of GVHD pathogenesis, little is known about the timing of essential Notch signals and the cellular source of Notch ligands after allo-BMT. Here, we have shown that critical DLL1/4-mediated Notch signals are delivered to donor T cells during a short 48-hour window after transplantation in a mouse allo-BMT model. Stromal, but not hematopoietic, cells were the essential source of Notch ligands during in vivo priming of alloreactive T cells. GVHD could be prevented by selective inactivation of Dll1 and Dll4 in subsets of fibroblastic stromal cells that were derived from chemokine Ccl19-expressing host cells, including fibroblastic reticular cells and follicular dendritic cells. However, neither T cell recruitment into secondary lymphoid ...
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Although Notch signaling plays important roles in lineage commitment and differentiation of multiple cell types including conventional T cells, nothing is currently known concerning Notch function in innate-like T cells. We have found... more
Although Notch signaling plays important roles in lineage commitment and differentiation of multiple cell types including conventional T cells, nothing is currently known concerning Notch function in innate-like T cells. We have found that the homeostasis of several well-characterized populations of innate-like T cells including invariant NKT cells (iNKT), CD8ααTCRαβ small intestinal intraepithelial lymphocytes, and innate memory phenotype CD8 T cells is controlled by Notch. Notch selectively regulates hepatic iNKT cell survival via tissue-restricted control of B cell lymphoma 2 and IL-7Rα expression. More generally, Notch regulation of innate-like T cell homeostasis involves both cell-intrinsic and -extrinsic mechanisms and relies upon context-dependent interactions with Notch ligand-expressing fibroblastic stromal cells. Collectively, using conditional ablation of Notch receptors on peripheral T cells or Notch ligands on putative fibroblastic stromal cells, we show that Notch sign...
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Nuocytes are essential in innate type 2 immunity and contribute to the exacerbation of asthma responses. Here we found that nuocytes arose in the bone marrow and differentiated from common lymphoid progenitors, which indicates they are... more
Nuocytes are essential in innate type 2 immunity and contribute to the exacerbation of asthma responses. Here we found that nuocytes arose in the bone marrow and differentiated from common lymphoid progenitors, which indicates they are distinct, previously unknown members of the lymphoid lineage. Nuocytes required interleukin 7 (IL-7), IL-33 and Notch signaling for development in vitro. Pro-T cell progenitors
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ABSTRACT
The modulatory function of individual miRNAs in Notch driven T-ALLs has recently been established. Although pro-tumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T cell leukemia,... more
The modulatory function of individual miRNAs in Notch driven T-ALLs has recently been established. Although pro-tumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T cell leukemia, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. Here we used conditional and inducible genetic loss of function approaches to test whether the development and maintenance of Notch-driven T-ALL was dependent on Dicer1 function. Mice with specific inactivation of both Dicer1 alleles in the T cell lineage did not develop Notch-driven T-ALL. In contrast, loss of one functional Dicer1 allele did not significantly perturb T-ALL onset and tumor progression. Inducible inactivation of Dicer1 in early stage polyclonal T-ALL cells was sufficient to abrogate T-ALL progression in leukemic mice whereas late stage monoclonal T-ALL cells were counter-selected against loss of Dicer1. Lineage tracing experiments revealed that D...
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In recent years a substantial body of evidence derived from not only preclinical but also clinical studies has accumulated in support of Notch signaling playing important oncogenic roles in several types of cancer. The finding that... more
In recent years a substantial body of evidence derived from not only preclinical but also clinical studies has accumulated in support of Notch signaling playing important oncogenic roles in several types of cancer. The finding that activating Notch mutations are frequently found in patients suffering from acute lymphoblastic leukemia is one of the best examples for a critical role of Notch signaling in cancer, a fact that motivated many researchers and clinicians to study the role of Notch also in solid tumors. Hence Notch signaling has gained increasing attention as a potential therapeutic target. In this book chapter we would like to discuss our current knowledge of Notch signaling within different types of solid cancers as well as advantages and disadvantages of potential new therapies that try to target the oncogenic properties of Notch signaling.
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The efficacy of a synthetic peptide analogue (rD-mPGPtide), mimicking the CDR3 region in the first domain of the CD4 surface molecule, was investigated in a murine model for CD4+ T cell-mediated skin allograft rejection. A single... more
The efficacy of a synthetic peptide analogue (rD-mPGPtide), mimicking the CDR3 region in the first domain of the CD4 surface molecule, was investigated in a murine model for CD4+ T cell-mediated skin allograft rejection. A single injection of rD-mPGPtide shortly before transplantation exhibited significantly prolonged graft survival in the B6 anti-B6.C-H2bm12 MHC class II-disparate strain combination. Long-term graft survival (>100 days) was achieved when thymectomized adult recipient mice were transplanted along with rD-mPGPtide treatment. The peptide also affected secondary rechallenge responses with MHC class II allografts. In addition, the inhibitory effect of the rD-mPGPtide in this transplantation model was directed against CD4+ T cells and was exclusively specific toward donor alloantigen. In vitro analysis of CD4+ T cells isolated from the draining lymph nodes of rD-mPGPtide-treated recipients indicated a 450-fold decrease in precursor frequency in response to donor allos...
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The efficacy of a synthetic peptide analog mimicking the CDR3-D1 domain of the CD4 molecule was investigated in murine models of allogeneic bone marrow engraftment after transplantation across major histocompatibility complex (MHC)... more
The efficacy of a synthetic peptide analog mimicking the CDR3-D1 domain of the CD4 molecule was investigated in murine models of allogeneic bone marrow engraftment after transplantation across major histocompatibility complex (MHC) barriers. A single dose of a CD4-CDR3 peptide analog was administered at the time of marrow transplantation to three different allogeneic mouse strain combinations after appropriate sublethal total body irradiation: (1) B10.BR --> C57BL/6J (B6), a full allogeneic MHC difference; (2) (B6xDBA/2)F1 --> (B6xCBA)F1, a haploidentical MHC combination; and (3) B6.C-H2bm12 --> B6-Ly5.2, involving only a MHC class II difference. Donor-host chimerism was assessed after 1 and 2 months posttransplantation by flow cytometric analysis of spleen and/or lymph node cells. Peptide-treated animals in all three strain combinations exhibited significantly enhanced donor lymphoid engraftment, which was similarly reflected in the total lymphocyte compartment and its T-c...
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Fibroblast-like cells of secondary lymphoid organs (SLO) are important for tissue architecture. In addition, they regulate lymphocyte compartmentalization through the secretion of chemokines, and participate in the orchestration of... more
Fibroblast-like cells of secondary lymphoid organs (SLO) are important for tissue architecture. In addition, they regulate lymphocyte compartmentalization through the secretion of chemokines, and participate in the orchestration of appropriate cell-cell interactions required for adaptive immunity. Here, we provide data demonstrating the functional importance of SLO fibroblasts during Notch-mediated lineage specification and immune response. Genetic ablation of the Notch ligand Delta-like (DL)1 identified splenic fibroblasts rather than hematopoietic or endothelial cells as niche cells, allowing Notch 2-driven differentiation of marginal zone B cells and of Esam(+) dendritic cells. Moreover, conditional inactivation of DL4 in lymph node fibroblasts resulted in impaired follicular helper T cell differentiation and, consequently, in reduced numbers of germinal center B cells and absence of high-affinity antibodies. Our data demonstrate previously unknown roles for DL ligand-expressing ...