Publications by authors named "Cynthia J Guidos"

44 Publications

Biological and therapeutic implications of a unique subtype of NPM1 mutated AML.

Nat Commun 2021 02 16;12(1):1054. Epub 2021 Feb 16.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.

In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit.
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http://dx.doi.org/10.1038/s41467-021-21233-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886883PMC
February 2021

Early innate and adaptive immune perturbations determine long-term severity of chronic virus and Mycobacterium tuberculosis coinfection.

Immunity 2021 Mar 29;54(3):526-541.e7. Epub 2021 Jan 29.

Princess Margaret Cancer Center, University Health Network, Toronto, ON M5G 2M9, Canada; Department of Immunology, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address:

Chronic viral infections increase severity of Mycobacterium tuberculosis (Mtb) coinfection. Here, we examined how chronic viral infections alter the pulmonary microenvironment to foster coinfection and worsen disease severity. We developed a coordinated system of chronic virus and Mtb infection that induced central clinical manifestations of coinfection, including increased Mtb burden, extra-pulmonary dissemination, and heightened mortality. These disease states were not due to chronic virus-induced immunosuppression or exhaustion; rather, increased amounts of the cytokine TNFα initially arrested pulmonary Mtb growth, impeding dendritic cell mediated antigen transportation to the lymph node and subverting immune-surveillance, allowing bacterial sanctuary. The cryptic Mtb replication delayed CD4 T cell priming, redirecting T helper (Th) 1 toward Th17 differentiation and increasing pulmonary neutrophilia, which diminished long-term survival. Temporally restoring CD4 T cell induction overcame these diverse disease sequelae to enhance Mtb control. Thus, Mtb co-opts TNFα from the chronic inflammatory environment to subvert immune-surveillance, avert early immune function, and foster long-term coinfection.
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http://dx.doi.org/10.1016/j.immuni.2021.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946746PMC
March 2021

B cell acute lymphoblastic leukemia cells mediate RANK-RANKL-dependent bone destruction.

Sci Transl Med 2020 09;12(561)

Program in Genetics and Genome Biology, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada.

Although most children survive B cell acute lymphoblastic leukemia (B-ALL), they frequently experience long-term, treatment-related health problems, including osteopenia and osteonecrosis. Because some children present with fractures at ALL diagnosis, we considered the possibility that leukemic B cells contribute directly to bone pathology. To identify potential mechanisms of B-ALL-driven bone destruction, we examined the ; ; triple mutant (TM) mice and ; double mutant (DM) mouse models of spontaneous B-ALL. In contrast to DM animals, leukemic TM mice displayed brittle bones, and the TM leukemic cells overexpressed , encoding receptor activator of nuclear factor κB ligand. RANKL is a key regulator of osteoclast differentiation and bone loss. Transfer of TM leukemic cells into immunodeficient recipient mice caused trabecular bone loss. To determine whether human B-ALL can exert similar effects, we evaluated primary human B-ALL blasts isolated at diagnosis for RANKL expression and their impact on bone pathology after their transplantation into NOD. /SzJ (NSG) recipient mice. Primary B-ALL cells conferred bone destruction evident in increased multinucleated osteoclasts, trabecular bone loss, destruction of the metaphyseal growth plate, and reduction in adipocyte mass in these patient-derived xenografts (PDXs). Treating PDX mice with the RANKL antagonist recombinant osteoprotegerin-Fc (rOPG-Fc) protected the bone from B-ALL-induced destruction even under conditions of heavy tumor burden. Our data demonstrate a critical role of the RANK-RANKL axis in causing B-ALL-mediated bone pathology and provide preclinical support for RANKL-targeted therapy trials to reduce acute and long-term bone destruction in these patients.
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http://dx.doi.org/10.1126/scitranslmed.aba5942DOI Listing
September 2020

Cancers from Novel -Mutant Mouse Models Provide Insights into Polymerase-Mediated Hypermutagenesis and Immune Checkpoint Blockade.

Cancer Res 2020 12 16;80(24):5606-5618. Epub 2020 Sep 16.

Division of Haematology/Oncology, The Hospital for Sick Children, Toronto, Ontario, Canada.

mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype-phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation , independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, -driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of -driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients. SIGNIFICANCE: Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy..
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0624DOI Listing
December 2020

A network of immune and microbial modifications underlies viral persistence in the gastrointestinal tract.

J Exp Med 2020 12;217(12)

Princess Margaret Cancer Center, University Health Network, Toronto, Ontario, Canada.

Many pathogens subvert intestinal immunity to persist within the gastrointestinal tract (GIT); yet, the underlying mechanisms that enable sanctuary specifically in this reservoir are unclear. Using mass cytometry and network analysis, we demonstrate that chronic LCMV infection of the GIT leads to dysregulated microbial composition, a cascade of metabolic alterations, increased susceptibility to GI disease, and a system-wide recalibration of immune composition that defines viral persistence. Chronic infection led to outgrowth of activated Tbet-expressing T reg cell populations unique to the GIT and the rapid erosion of pathogen-specific CD8 tissue-resident memory T cells. Mechanistically, T reg cells and coinhibitory receptors maintained long-term viral sanctuary within the GIT, and their targeting reactivated T cells and eliminated this viral reservoir. Thus, our data provide a high-dimensional definition of the mechanisms of immune regulation that chronic viruses implement to exploit the unique microenvironment of the GIT and identify T reg cells as key modulators of viral persistence in the intestinal tract.
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http://dx.doi.org/10.1084/jem.20191473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953734PMC
December 2020

Relapse-Fated Latent Diagnosis Subclones in Acute B Lineage Leukemia Are Drug Tolerant and Possess Distinct Metabolic Programs.

Cancer Discov 2020 04 21;10(4):568-587. Epub 2020 Feb 21.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Disease recurrence causes significant mortality in B-progenitor acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples shows relapse often arising from minor diagnosis subclones. However, why therapy eradicates some subclones while others survive and progress to relapse remains obscure. Elucidation of mechanisms underlying these differing fates requires functional analysis of isolated subclones. Here, large-scale limiting dilution xenografting of diagnosis and relapse samples, combined with targeted sequencing, identified and isolated minor diagnosis subclones that initiate an evolutionary trajectory toward relapse [termed diagnosis Relapse Initiating clones (dRI)]. Compared with other diagnosis subclones, dRIs were drug-tolerant with distinct engraftment and metabolic properties. Transcriptionally, dRIs displayed enrichment for chromatin remodeling, mitochondrial metabolism, proteostasis programs, and an increase in stemness pathways. The isolation and characterization of dRI subclones reveals new avenues for eradicating dRI cells by targeting their distinct metabolic and transcriptional pathways before further evolution renders them fully therapy-resistant. SIGNIFICANCE: Isolation and characterization of subclones from diagnosis samples of patients with B-ALL who relapsed showed that relapse-fated subclones had increased drug tolerance and distinct metabolic and survival transcriptional programs compared with other diagnosis subclones. This study provides strategies to identify and target clinically relevant subclones before further evolution toward relapse.
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http://dx.doi.org/10.1158/2159-8290.CD-19-1059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122013PMC
April 2020

Validation of CyTOF Against Flow Cytometry for Immunological Studies and Monitoring of Human Cancer Clinical Trials.

Front Oncol 2019 17;9:415. Epub 2019 May 17.

Tumor Immunology Program, Princess Margaret Cancer Center, University Health Network, Toronto, ON, Canada.

Flow cytometry is a widely applied approach for exploratory immune profiling and biomarker discovery in cancer and other diseases. However, flow cytometry is limited by the number of parameters that can be simultaneously analyzed, severely restricting its utility. Recently, the advent of mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system, allowing simultaneous interrogation of a large number of parameters. This is important for deep interrogation of immune responses and particularly when sample sizes are limited (such as in tumors). Our goal was to compare the accuracy and reproducibility of CyTOF against flow cytometry as a reliable analytic tool for human PBMC and tumor tissues for cancer clinical trials. We developed a 40+ parameter CyTOF panel and demonstrate that compared to flow cytometry, CyTOF yields analogous quantification of cell lineages in conjunction with markers of cell differentiation, function, activation, and exhaustion for use with fresh and viably frozen PBMC or tumor tissues. Further, we provide a protocol that enables reliable quantification by CyTOF down to low numbers of input human cells, an approach that is particularly important when cell numbers are limiting. Thus, we validate CyTOF as an accurate approach to perform high dimensional analysis in human tumor tissue and to utilize low cell numbers for subsequent immunologic studies and cancer clinical trials.
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http://dx.doi.org/10.3389/fonc.2019.00415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534060PMC
May 2019

Method for Tagging Antibodies with Metals for Mass Cytometry Experiments.

Methods Mol Biol 2019 ;1989:47-54

The Hospital for Sick Children Research Institute, Toronto, ON, Canada.

Mass cytometers are time-of-flight (TOF) mass spectrometer-coupled flow cytometers (known as CyTOFs) that quantify the abundance of metal-tagged antibodies (Abs) or other cellular probes within single cell suspensions or laser-ablated tissue sections. While many strategies exist for covalently crosslinking to proteins, the Fluidigm MaxPar process is currently the most widely used and involves first loading a metal-chelating polymer with an elementally and isotopically enriched metal. Once the chelation sites have been filled, a maleimide moiety on the polymer is reacted with the free thiol groups on the partially reduced monoclonal immunoglobulin G (IgG) Ab to form an irreversible covalent bond. Here we describe modifications to the Fluidigm MaxPar protocol that increase the quantity of Ab per reaction up to 150 μg, introduce an initial Ab quality control step, utilize metal labels not included in the Fluidigm catalog, and provide an option to perform two reactions in one centrifugal filter.
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http://dx.doi.org/10.1007/978-1-4939-9454-0_4DOI Listing
March 2020

Cdh1 and Pik3ca Mutations Cooperate to Induce Immune-Related Invasive Lobular Carcinoma of the Breast.

Cell Rep 2018 10;25(3):702-714.e6

Program in Cell Biology, The Peter Gilgan Center for Research and Learning, The Hospital for Sick Children, Toronto, ON M5G-0A4, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. Electronic address:

CDH1 and PIK3CA are the two most frequently mutated genes in invasive lobular carcinoma (ILC) of the breast. Transcription profiling has identified molecular subtypes for ILC, one of which, immune-related (IR), is associated with gene expression linked to lymphocyte and macrophage infiltration. Here, we report that deletion of Cdh1, together with activation of Pik3ca in mammary epithelium of genetically modified mice, leads to formation of IR-ILC-like tumors with immune cell infiltration, as well as gene expression linked to T-regulatory (Treg) cell signaling and activation of targetable immune checkpoint pathways. Interestingly, these tumors show enhanced Rac1- and Yap-dependent transcription and signaling, as well as sensitivity to PI3K, Rac1, and Yap inhibitors in culture. Finally, high-dimensional immunophenotyping in control mouse mammary gland and IR-ILC tumors by mass cytometry shows dramatic alterations in myeloid and lymphoid populations associated with immune suppression and exhaustion, highlighting the potential for therapeutic intervention via immune checkpoint regulators.
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http://dx.doi.org/10.1016/j.celrep.2018.09.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276789PMC
October 2018

CD8 T Cell Priming in Established Chronic Viral Infection Preferentially Directs Differentiation of Memory-like Cells for Sustained Immunity.

Immunity 2018 10 9;49(4):678-694.e5. Epub 2018 Oct 9.

Princess Margaret Cancer Center, University Health Network, Toronto, ON, M5G 2M9 Canada; Department of Immunology, University of Toronto, Toronto, ON, M5S 1A8 Canada. Electronic address:

CD8 T cell exhaustion impedes control of chronic viral infection; yet how new T cell responses are mounted during chronic infection is unclear. Unlike T cells primed at the onset of infection that rapidly differentiate into effectors and exhaust, we demonstrate that virus-specific CD8 T cells primed after establishment of chronic LCMV infection preferentially generate memory-like transcription factor TCF1 cells that were transcriptionally and proteomically distinct, less exhausted, and more responsive to immunotherapy. Mechanistically, adaptations of antigen-presenting cells and diminished T cell signaling intensity promoted differentiation of the memory-like subset at the expense of rapid effector cell differentiation, which was now highly dependent on IL-21-mediated CD4 T cell help for its functional generation. Chronic viral infection similarly redirected de novo differentiation of tumor-specific CD8 T cells, ultimately preventing cancer control. Thus, targeting these T cell stimulatory pathways could enable strategies to control chronic infection, tumors, and enhance immunotherapeutic efficacy.
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http://dx.doi.org/10.1016/j.immuni.2018.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060917PMC
October 2018

Regulatory T Cells in Ovarian Cancer Are Characterized by a Highly Activated Phenotype Distinct from that in Melanoma.

Clin Cancer Res 2018 11 31;24(22):5685-5696. Epub 2018 Jul 31.

The Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada.

Regulatory T (Treg) cells expressing the transcription factor FOXP3 are essential for the maintenance of immunologic self-tolerance but play a detrimental role in most cancers due to their ability to suppress antitumor immunity. The phenotype of human circulating Treg cells has been extensively studied, but less is known about tumor-infiltrating Treg cells. We studied the phenotype and function of tumor-infiltrating Treg cells in ovarian cancer and melanoma to identify potential Treg cell-associated molecules that can be targeted by tumor immunotherapies. The phenotype of intratumoral and circulating Treg cells was analyzed by multicolor flow cytometry, mass cytometry, RNA-seq, and functional assays. Treg cells isolated from ovarian tumors displayed a distinct cell surface phenotype with increased expression of a number of receptors associated with TCR engagement, including PD-1, 4-1BB, and ICOS. Higher PD-1 and 4-1BB expression was associated with increased responsiveness to further TCR stimulation and increased suppressive capacity, respectively. Transcriptomic and mass cytometry analyses revealed the presence of Treg cell subpopulations and further supported a highly activated state specifically in ovarian tumors. In comparison, Treg cells infiltrating melanomas displayed lower FOXP3, PD-1, 4-1BB, and ICOS expression and were less potent suppressors of CD8 T-cell proliferation. The highly activated phenotype of ovarian tumor-infiltrating Treg cells may be a key component of an immunosuppressive tumor microenvironment. Receptors that are expressed by tumor-infiltrating Treg cells could be exploited for the design of novel combination tumor immunotherapies. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0554DOI Listing
November 2018

Functional screening of FGFR4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma.

Oncogene 2018 05 28;37(20):2630-2644. Epub 2018 Feb 28.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4 overexpression as murine myoblasts expressing FGFR4 undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4 were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4 tumor-derived cell and human RMS cell viability. FGFR4-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.
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http://dx.doi.org/10.1038/s41388-017-0122-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054765PMC
May 2018

A key role for IL-7R in the generation of microenvironments required for thymic dendritic cells.

Immunol Cell Biol 2017 11 11;95(10):933-942. Epub 2017 Sep 11.

Biological Sciences, Sunnybrook Research Institute, Toronto, Ontario, Canada.

Interleukin-7 receptor (IL-7R) signaling is critical for multiple stages of T-cell development, but a role in the establishment of the mature thymic architecture needed for T-cell development and thymocyte selection has not been established. Crosstalk signals between developing thymocytes and thymic epithelial cell (TEC) precursors are critical for their differentiation into cortical TECs (cTECs) and medullary TECs (mTECs). In addition, mTEC-derived factors have been implicated in the recruitment of thymic dendritic cells (DCs) and intrathymic DC development. We therefore examined corticomedullary structure and DC populations in the thymus of Il7r mice. Analysis of TEC phenotype and spatial organization revealed a striking shift in the mTEC to cTEC ratio, accompanied by disorganized corticomedullary structure. Several of the thymic subsets known to have DC potential were nearly absent, accompanied by reductions in DC cell numbers. We also examined chemokine expression in the Il7r thymus, and found a significant decrease in mTEC-derived CCR7 ligand expression, and high levels of cTEC-derived chemokines, including CCL25 and CXCL12. Although splenic DCs were similarly affected, bone marrow (BM) precursors capable of giving rise to DCs were unperturbed. Finally, BM chimeras showed that there was no intrinsic need for IL-7R signaling in the development or recruitment of thymic DCs, but that the provision of wild-type progenitors enhanced reconstitution of thymic DCs from Il7r progenitors. Our results are therefore supportive of a model in which Il7r-dependent cells are required to set up the microenvironments that allow accumulation of thymic DCs.
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http://dx.doi.org/10.1038/icb.2017.74DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698111PMC
November 2017

Chemo-genomic interrogation of CEBPA mutated AML reveals recurrent CSF3R mutations and subgroup sensitivity to JAK inhibitors.

Blood 2016 06 31;127(24):3054-61. Epub 2016 Mar 31.

The Leucegene Project at Institute for Research in Immunology and Cancer, Université de Montréal, Montréal, QC, Canada; Division of Hematology, Maisonneuve-Rosemont Hospital, Montréal, QC, Canada; Quebec Leukemia Cell Bank, Maisonneuve-Rosemont Hospital, Montréal, QC, Canada; and Department of Medicine, Faculty of Medicine, Université de Montréal, Montréal, QC, Canada.

In this study, we analyzed RNA-sequencing data of 14 samples characterized by biallelic CEBPA (CEBPA(bi)) mutations included in the Leucegene collection of 415 primary acute myeloid leukemia (AML) specimens, and describe for the first time high frequency recurrent mutations in the granulocyte colony-stimulating factor receptor gene CSF3R, which signals through JAK-STAT proteins. Chemical interrogation of these primary human specimens revealed a uniform and specific sensitivity to all JAK inhibitors tested irrespective of their CSF3R mutation status, indicating a general sensitization of JAK-STAT signaling in this leukemia subset. Altogether, these results identified the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors and paves the way to personalized clinical trials for this disease.
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http://dx.doi.org/10.1182/blood-2016-03-705053DOI Listing
June 2016

Notch signal strength controls cell fate in the haemogenic endothelium.

Nat Commun 2015 Oct 14;6:8510. Epub 2015 Oct 14.

Program in Cancer Research, Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Barcelona 08003, Spain.

Acquisition of the arterial and haemogenic endothelium fates concurrently occur in the aorta-gonad-mesonephros (AGM) region prior to haematopoietic stem cell (HSC) generation. The arterial programme depends on Dll4 and the haemogenic endothelium/HSC on Jag1-mediated Notch1 signalling. How Notch1 distinguishes and executes these different programmes in response to particular ligands is poorly understood. By using two Notch1 activation trap mouse models with different sensitivity, here we show that arterial endothelial cells and HSCs originate from distinct precursors, characterized by different Notch1 signal strengths. Microarray analysis on AGM subpopulations demonstrates that the Jag1 ligand stimulates low Notch strength, inhibits the endothelial programme and is permissive for HSC specification. In the absence of Jag1, endothelial cells experience high Dll4-induced Notch activity and select the endothelial programme, thus precluding HSC formation. Interference with the Dll4 signal by ligand-specific blocking antibodies is sufficient to inhibit the endothelial programme and favour specification of the haematopoietic lineage.
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http://dx.doi.org/10.1038/ncomms9510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634136PMC
October 2015

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

PLoS Genet 2015 Jun 9;11(6):e1005288. Epub 2015 Jun 9.

Program in Genetics & Genome Biology, Research Institute, The Hospital for Sick Children, Toronto, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Canada.

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to be p53-dependent. Our findings therefore point to cell/tissue-specific responses to p53-activation that include distinction between apoptosis and senescence pathways, in the context of translation disruption.
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http://dx.doi.org/10.1371/journal.pgen.1005288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4461263PMC
June 2015

IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection.

Nat Immunol 2015 Apr 2;16(4):397-405. Epub 2015 Mar 2.

1] Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, Canada. [2] Department of Immunology, University of Toronto, Toronto, Canada.

Signaling via the pre-T cell antigen receptor (pre-TCR) and the receptor Notch1 induces transient self-renewal (β-selection) of TCRβ(+) CD4(-)CD8(-) double-negative stage 3 (DN3) and DN4 progenitor cells that differentiate into CD4(+)CD8(+) double-positive (DP) thymocytes, which then rearrange the locus encoding the TCR α-chain (Tcra). Interleukin 7 (IL-7) promotes the survival of TCRβ(-) DN thymocytes by inducing expression of the pro-survival molecule Bcl-2, but the functions of IL-7 during β-selection have remained unclear. Here we found that IL-7 signaled TCRβ(+) DN3 and DN4 thymocytes to upregulate genes encoding molecules involved in cell growth and repressed the gene encoding the transcriptional repressor Bcl-6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate but rearranged Tcra prematurely and differentiated rapidly. Deletion of Bcl6 partially restored the self-renewal of DN4 cells in the absence of IL-7, but overexpression of BCL2 did not. Thus, IL-7 critically acts cooperatively with signaling via the pre-TCR and Notch1 to coordinate proliferation, differentiation and Tcra recombination during β-selection.
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http://dx.doi.org/10.1038/ni.3122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4368453PMC
April 2015

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

PLoS One 2014 29;9(8):e105602. Epub 2014 Aug 29.

Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada; Dept. of Medical Biophysics, University of Toronto, Toronto, ON, Canada.

Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105602PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149490PMC
June 2015

MuLV-related endogenous retroviral elements and Flt3 participate in aberrant end-joining events that promote B-cell leukemogenesis.

Genes Dev 2014 Jun;28(11):1179-90

Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, Toronto, Ontario M5G 0A4, Canada; Department of Immunology.

During V(D)J recombination of immunoglobulin genes, p53 and nonhomologous end-joining (NHEJ) suppress aberrant rejoining of DNA double-strand breaks induced by recombinase-activating genes (Rags)-1/2, thus maintaining genomic stability and limiting malignant transformation during B-cell development. However, Rag deficiency does not prevent B-cell leukemogenesis in p53/NHEJ mutant mice, revealing that p53 and NHEJ also suppress Rag-independent mechanisms of B-cell leukemogenesis. Using several cytogenomic approaches, we identified a novel class of activating mutations in Fms-like tyrosine kinase 3 (Flt3), a receptor tyrosine kinase important for normal hematopoiesis in Rag/p53/NHEJ triple-mutant (TM) B-cell leukemias. These mutant Flt3 alleles were created by complex genomic rearrangements with Moloney leukemia virus (MuLV)-related endogenous retroviral (ERV) elements, generating ERV-Flt3 fusion genes encoding an N-terminally truncated mutant form of Flt3 (trFlt3) that was transcribed from ERV long terminal repeats. trFlt3 protein lacked most of the Flt3 extracellular domain and induced ligand-independent STAT5 phosphorylation and proliferation of hematopoietic progenitor cells. Furthermore, expression of trFlt3 in p53/NHEJ mutant hematopoietic progenitor cells promoted development of clinically aggressive B-cell leukemia. Thus, repetitive MuLV-related ERV sequences can participate in aberrant end-joining events that promote development of aggressive B-cell leukemia.
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http://dx.doi.org/10.1101/gad.240820.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052764PMC
June 2014

Therapeutic potential of spleen tyrosine kinase inhibition for treating high-risk precursor B cell acute lymphoblastic leukemia.

Sci Transl Med 2014 May;6(236):236ra62

Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S 1A8, Canada. Program in Genetics & Genome Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1L7, Canada.

Intensified and central nervous system (CNS)-directed chemotherapy has improved outcomes for pediatric B cell acute lymphoblastic leukemia (B-ALL) but confers treatment-related morbidities. Moreover, many patients suffer relapses, underscoring the need to develop new molecular targeted B-ALL therapies. Using a mouse model, we show that leukemic B cells require pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) signaling in vivo for survival and proliferation. In diagnostic samples from human pediatric and adult B-ALL patients, SYK and downstream targets were phosphorylated regardless of pre-BCR expression or genetic subtype. Two small-molecule SYK inhibitors, fostamatinib and BAY61-3606, attenuated the growth of 69 B-ALL samples in vitro, including high-risk (HR) subtypes. Orally administered fostamatinib reduced heavy disease burden after xenotransplantation of HR B-ALL samples into immunodeficient mice and decreased leukemia dissemination into spleen, liver, kidneys, and the CNS of recipient mice. Thus, SYK activation sustains the growth of multiple HR B-ALL subtypes, suggesting that SYK inhibitors may improve outcomes for HR and relapsed B-ALL.
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http://dx.doi.org/10.1126/scitranslmed.3008661DOI Listing
May 2014

STAT5-induced lunatic fringe during Th2 development alters delta-like 4-mediated Th2 cytokine production in respiratory syncytial virus-exacerbated airway allergic disease.

J Immunol 2014 Feb 23;192(3):996-1003. Epub 2013 Dec 23.

Department of Pathology, University of Michigan, Ann Arbor, MI 48109;

Notch activation plays an important role in T cell development and mature T cell differentiation. In this study, we investigated the role of Notch activation in a mouse model of respiratory syncytial virus (RSV)-exacerbated allergic airway disease. During RSV exacerbation, in vivo neutralization of a specific Notch ligand, Delta-like ligand (Dll)-4, significantly decreased airway hyperreactivity, mucus production, and Th2 cytokines. Lunatic Fringe (Lfng), a glycosyltransferase that enhances Notch activation by Dll4, was increased during RSV exacerbation. Lfng loss of function in Th2-skewed cells inhibited Dll4-Notch activation and subsequent IL-4 production. Further knockdown of Lfng in T cells in CD4Cre(+)Lfng(fl/fl) mice showed reduced Th2 response and disease pathology during RSV exacerbation. Finally, we identified STAT5-binding cis-acting regulatory element activation as a critical driver of Lfng transcriptional activation. These data demonstrate that STAT5-dependent amplification of Notch-modifying Lfng augments Th2 response via Dll4 and is critical for amplifying viral exacerbation during allergic airway disease.
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http://dx.doi.org/10.4049/jimmunol.1301991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946958PMC
February 2014

Notch2-dependent classical dendritic cells orchestrate intestinal immunity to attaching-and-effacing bacterial pathogens.

Nat Immunol 2013 Sep 4;14(9):937-48. Epub 2013 Aug 4.

Department of Pathology and Immunology, School of Medicine, Washington University in St. Louis, Missouri, USA.

Defense against attaching-and-effacing bacteria requires the sequential generation of interleukin 23 (IL-23) and IL-22 to induce protective mucosal responses. Although CD4(+) and NKp46(+) innate lymphoid cells (ILCs) are the critical source of IL-22 during infection, the precise source of IL-23 is unclear. We used genetic techniques to deplete mice of specific subsets of classical dendritic cells (cDCs) and analyzed immunity to the attaching-and-effacing pathogen Citrobacter rodentium. We found that the signaling receptor Notch2 controlled the terminal stage of cDC differentiation. Notch2-dependent intestinal CD11b(+) cDCs were an obligate source of IL-23 required for survival after infection with C. rodentium, but CD103(+) cDCs dependent on the transcription factor Batf3 were not. Our results demonstrate a nonredundant function for CD11b(+) cDCs in the response to pathogens in vivo.
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http://dx.doi.org/10.1038/ni.2679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3788683PMC
September 2013

Lunatic fringe deficiency cooperates with the Met/Caveolin gene amplicon to induce basal-like breast cancer.

Cancer Cell 2012 May;21(5):626-41

Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, ON, M5G 1L7, Canada.

Basal-like breast cancers (BLBC) express a luminal progenitor gene signature. Notch receptor signaling promotes luminal cell fate specification in the mammary gland, while suppressing stem cell self-renewal. Here we show that deletion of Lfng, a sugar transferase that prevents Notch activation by Jagged ligands, enhances stem/progenitor cell proliferation. Mammary-specific deletion of Lfng induces basal-like and claudin-low tumors with accumulation of Notch intracellular domain fragments, increased expression of proliferation-associated Notch targets, amplification of the Met/Caveolin locus, and elevated Met and Igf-1R signaling. Human BL breast tumors, commonly associated with JAGGED expression, elevated MET signaling, and CAVEOLIN accumulation, express low levels of LFNG. Thus, reduced LFNG expression facilitates JAG/NOTCH luminal progenitor signaling and cooperates with MET/CAVEOLIN basal-type signaling to promote BLBC.
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http://dx.doi.org/10.1016/j.ccr.2012.03.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3603366PMC
May 2012

Zebrafish screen identifies novel compound with selective toxicity against leukemia.

Blood 2012 Jun 9;119(24):5621-31. Epub 2012 Apr 9.

Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT 84112, USA.

To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces long-term remission in adult zebrafish with cMYC-induced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells.
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http://dx.doi.org/10.1182/blood-2011-12-398818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3382926PMC
June 2012

Proteomic analyses reveal high expression of decorin and endoplasmin (HSP90B1) are associated with breast cancer metastasis and decreased survival.

PLoS One 2012 20;7(2):e30992. Epub 2012 Feb 20.

Campbell Family Institute for Breast Cancer Research, University Health Network, Toronto, Ontario, Canada.

Background: Breast cancer is the most common malignancy among women worldwide in terms of incidence and mortality. About 10% of North American women will be diagnosed with breast cancer during their lifetime and 20% of those will die of the disease. Breast cancer is a heterogeneous disease and biomarkers able to correctly classify patients into prognostic groups are needed to better tailor treatment options and improve outcomes. One powerful method used for biomarker discovery is sample screening with mass spectrometry, as it allows direct comparison of protein expression between normal and pathological states. The purpose of this study was to use a systematic and objective method to identify biomarkers with possible prognostic value in breast cancer patients, particularly in identifying cases most likely to have lymph node metastasis and to validate their prognostic ability using breast cancer tissue microarrays.

Methods And Findings: Differential proteomic analyses were employed to identify candidate biomarkers in primary breast cancer patients. These analyses identified decorin (DCN) and endoplasmin (HSP90B1) which play important roles regulating the tumour microenvironment and in pathways related to tumorigenesis. This study indicates that high expression of Decorin is associated with lymph node metastasis (p<0.001), higher number of positive lymph nodes (p<0.0001) and worse overall survival (p = 0.01). High expression of HSP90B1 is associated with distant metastasis (p<0.0001) and decreased overall survival (p<0.0001) these patients also appear to benefit significantly from hormonal treatment.

Conclusions: Using quantitative proteomic profiling of primary breast cancers, two new promising prognostic and predictive markers were found to identify patients with worse survival. In addition HSP90B1 appears to identify a group of patients with distant metastasis with otherwise good prognostic features.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030992PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3282708PMC
June 2012

Jagged2 acts as a Delta-like Notch ligand during early hematopoietic cell fate decisions.

Blood 2011 Apr 3;117(17):4449-59. Epub 2011 Mar 3.

Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Ghent University Hospital, Ghent, Belgium.

Notch signaling critically mediates various hematopoietic lineage decisions and is induced in mammals by Notch ligands that are classified into 2 families, Delta-like (Delta-like-1, -3 and -4) and Jagged (Jagged1 and Jagged2), based on structural homology with both Drosophila ligands Delta and Serrate, respectively. Because the functional differences between mammalian Notch ligands were still unclear, we have investigated their influence on early human hematopoiesis and show that Jagged2 affects hematopoietic lineage decisions very similarly as Delta-like-1 and -4, but very different from Jagged1. OP9 coculture experiments revealed that Jagged2, like Delta-like ligands, induces T-lineage differentiation and inhibits B-cell and myeloid development. However, dose-dependent Notch activation studies, gene expression analysis, and promoter activation assays indicated that Jagged2 is a weaker Notch1-activator compared with the Delta-like ligands, revealing a Notch1 specific signal strength hierarchy for mammalian Notch ligands. Strikingly, Lunatic-Fringe- mediated glycosylation of Notch1 potentiated Notch signaling through Delta-like ligands and also Jagged2, in contrast to Jagged1. Thus, our results reveal a unique role for Jagged1 in preventing the induction of T-lineage differentiation in hematopoietic stem cells and show an unexpected functional similarity between Jagged2 and the Delta-like ligands.
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http://dx.doi.org/10.1182/blood-2010-06-290049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3673751PMC
April 2011

Lunatic Fringe prolongs Delta/Notch-induced self-renewal of committed αβ T-cell progenitors.

Blood 2011 Jan 19;117(4):1184-95. Epub 2010 Nov 19.

Program in Developmental and Stem Cell Biology, Hospital for Sick Children Research Institute, 101 College Street, Toronto, Ontario, Canada.

Lunatic Fringe (Lfng) enhances Notch1 activation by Delta-like 4 (DL4) to promote Notch1-dependent T-lineage commitment of thymus-seeding progenitors. Subsequently, Notch1 and T-cell receptor-β (TCRβ)-containing pre-TCR complexes signal CD4/CD8 double-negative 3 (DN3) committed T-cell progenitors to survive, proliferate, and differentiate into CD4/CD8 double-positive (DP) αβ T-cell precursors. Few DP thymocytes develop without Notch1 or pre-TCR signals, whereas ectopic Notch1 activation causes T-cell leukemia. However, mechanisms of a Notch-pre-TCR collaboration during this "β-selection" process are poorly understood. We genetically manipulated Lfng to attenuate or enhance Notch1 activation in DN3 thymocytes without inducing leukemogenesis. We show that Lfng temporally sustains DL-induced Notch1 signaling to prolong proliferative self-renewal of pre-DP thymocytes. Pre-TCR signaling greatly augmented Notch trophic functions to promote robust proliferation of pre-DP progenitors. In contrast, in the absence of DL/Notch signaling, pre-TCR-expressing progenitors rapidly atrophied and differentiated into DP thymocytes. Thus, Lfng prolongs Notch1 signaling to promote self-renewal more than differentiation during the early stages of β-selection. Our data provide novel insights into the Notch-pre-TCR collaboration, and suggest that decreasing Lfng expression during the DN3-DP transition minimizes the potent leukemogenic potential of Notch1 signaling.
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http://dx.doi.org/10.1182/blood-2010-07-296616DOI Listing
January 2011

Lunatic fringe enhances competition for delta-like Notch ligands but does not overcome defective pre-TCR signaling during thymocyte beta-selection in vivo.

J Immunol 2010 Oct 15;185(8):4609-17. Epub 2010 Sep 15.

Program in Stem Cell and Developmental Biology, Hospital for Sick Children Research Institute, University of Toronto, Toronto, Ontario, Canada.

Notch1 activation by Delta-like (DL) Notch ligands is essential to induce T cell commitment and to suppress B cell development from thymus-seeding progenitors. Thymus-seeding progenitor competition for DL4 is critically regulated by Lunatic Fringe (Lfng), which glycosylates epidermal growth factor repeats in the Notch1 extracellular domain to enhance binding avidity for DL ligands. Notch1 activation is also essential for the process of β-selection, which drives TCRβ(+) CD4/CD8 double-negative 3 (DN3) precursors to proliferate and generate a large pool of CD4/CD8 double-positive thymocytes. We have used several genetic approaches to determine the importance of Lfng-Notch1 interactions in regulating competition of preselection and postselection DN3 thymocytes for DL ligands in vivo. Surprisingly, although Lfng overexpression enhanced DL4 binding by preselection DN3a thymocytes, it did not confer them with a competitive advantage in mixed chimeras. In contrast, Lfng overexpression enhanced competition of post-β-selection DN3b precursors for DL ligands. Lfng modification of O-fucose in the Notch1 ligand-binding domain contributed to but was not solely responsible for the developmental effects of Lfng overexpression. Although previous studies have suggested that pre-TCR-deficient DN3 thymocytes compete poorly for DL ligands, Lfng overexpression did not fully restore double-positive thymocyte pools from DN3b cells with pre-TCR signaling defects. Thus, pre-TCR and Notch signaling have largely nonoverlapping functions in β-selection. Collectively, our data reveal that Lfng enhances DN3b precursor competition for intrathymic DL ligands to maximize Notch-induced clonal expansion during the earliest stage of β-selection.
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http://dx.doi.org/10.4049/jimmunol.1002008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3579566PMC
October 2010

Functions of notch signaling in the immune system: consensus and controversies.

Annu Rev Immunol 2010 ;28:343-65

Hospital for Sick Children Research Institute, Department of Immunology, University of Toronto, Ontario, Canada.

Mammalian genomes encode up to four Notch receptors (Notch1-4) and five Notch ligands of the DSL (Delta/Serrate/Lag-2) family, and Notch signaling controls a wide spectrum of developmental processes. Intrathymic Notch1 signaling is essential for several distinct aspects of early T cell development. Notch signaling has also been implicated as a key regulator of peripheral T cell activation and effector cell differentiation, but its functions in these processes remain poorly understood. Notch signaling is dispensable for B cell development in the bone marrow, but it is required to generate the innate-like marginal zone B cell subset in the spleen and may also regulate plasma cell functions. Modification of Notch receptors by fringe glycosyltransferases influences many Notch-dependent aspects of hematopoiesis by altering Notch responsiveness to Delta-like versus Jagged DSL ligands. Here we review recent advances in general aspects of Notch signaling, as well as studies probing Notch functions in these immunological processes.
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http://dx.doi.org/10.1146/annurev.immunol.021908.132719DOI Listing
May 2010