Subject(s)
HIV Infections , HIV Seropositivity , HIV-1 , Hematopoietic Stem Cell Transplantation , Humans , Causality , Hematopoietic Stem Cell Transplantation/methods , Remission Induction , Transplantation Conditioning , Transplantation, Homologous , Treatment Outcome , HIV Infections/therapy , HIV Infections/virologyABSTRACT
We previously showed that immature CD166(+) osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48(-)CD166(+)CD150(+) and LSKCD48(-)CD166(+)CD150(+)CD9(+) cells, as well as human Lin(-)CD34(+)CD38(-)CD49f(+)CD166(+) cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166(-) cells. CD166(-/-) knockout (KO) LSK cells engrafted poorly in wild-type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166(-/-) hosts supported short-term, but not long-term WT HSC engraftment, confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166(-/-) mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166(-/-) cells, suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression, suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions, and suggest that CD166 expression can be modulated to enhance HSC function.
Subject(s)
Activated-Leukocyte Cell Adhesion Molecule/physiology , Biomarkers/metabolism , Bone Marrow Cells/metabolism , Hematopoietic Stem Cells/cytology , Stem Cell Niche/physiology , Animals , Antigens, CD/metabolism , Chromatin Immunoprecipitation , Flow Cytometry , Hematopoietic Stem Cells/physiology , Humans , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, SCID , Receptors, Cell Surface/metabolism , Signaling Lymphocytic Activation Molecule Family Member 1ABSTRACT
The extra-embryonic yolk sac (YS) is the first hematopoietic site in the mouse embryo and is thought to generate only primitive erythroid and myeloerythroid progenitor cells before definitive HSC emergence within the embryo on E10.5. Here, we have shown the existence of T cell-restricted progenitors in the E9.5 YS that directly engraft in recipient immunodeficient mice. T-cell progenitors were also produced in vitro from both YS and para-aortic splanchnopleura hemogenic endothelial cells, and these T-cell progenitors repopulated the thymus and differentiated into mature T-cell subsets in vivo on transplantation. Our data confirm that the YS produces T-lineage-restricted progenitors that are available to colonize the thymus and provide new insight into the YS as a definitive hematopoietic site in the mouse embryo.
Subject(s)
Hematopoietic Stem Cells/cytology , T-Lymphocytes/cytology , Yolk Sac/cytology , Yolk Sac/immunology , Animals , Animals, Newborn , Aorta/embryology , Cell Differentiation , Endothelial Cells/cytology , Endothelial Cells/metabolism , Hematopoietic Stem Cell Transplantation , Mice , Mice, Inbred C57BL , Spleen/cytology , Spleen/immunology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , T-Lymphocytes/immunology , T-Lymphocytes/transplantation , Thymus Gland/cytology , Thymus Gland/immunologyABSTRACT
Thymomas are low-grade epithelial tumors of the anterior mediastinum. The complexity of the disease and the lack of in vitro and in vivo models hamper the development of better therapeutics. In this study, we report a novel cell line, designated as IU-TAB-1, which was established from a patient with stage II thymoma (World Health Organization-type AB). The IU-TAB-1 cell line was established in vitro and characterized using histological and immunohistochemical staining, fluorescence-activated cell sorting, cytogenetic analyses and functional assays including in vitro and a NOD/SCID xenograft model. A whole-genome gene expression analysis (Illumina) was performed on the IU-TAB-1 cell line and 34 thymomas to determine the clinical relevance of the cell line. The IU-TAB-1 cell line was positive for epithelial markers (pan-cytokeratin and EpCAM/CD326) including thymic epithelial (TE) surface markers (such as CD29, CD9, CD54/ICAM-1, CD58 and CD24) and p63, and negative for B- and T-cell lineage markers. Gene expression profiling demonstrated overlapping and distinct genes between IU-TAB-1 and primary thymomas including the primary tumor (from which the cell line was derived). IU-TAB-1 cells are tumorigenic when implanted in immunodeficient mice with tumors reaching a volume of 1000 mm³ at around 130 days. The established cell line represents a biologically relevant new tool to investigate the molecular pathology of thymic malignancies and to evaluate the efficacy of novel therapeutics both in vitro and in vivo.
Subject(s)
Cell Line, Tumor , Thymoma/pathology , Thymus Neoplasms/pathology , Animals , Cell Proliferation , Chromosome Aberrations , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Middle AgedABSTRACT
BACKGROUND: Adoptive transfer of CD19-specific chimeric antigen receptor (CD19CAR) T cells can induce dramatic disease regression in patients with B cell malignancies. CD19CAR T cell therapy may be limited by insufficient engraftment and persistence, resulting in tumor relapse. We previously demonstrated a proof of principle that cytomegalovirus (CMV)-specific T cells can be isolated and enriched prior to CD19CAR transduction to produce CMV-CD19CAR T cells, and that these CMV-CD19CAR T cells can be expanded in vivo through CMV vaccination, resulting in better tumor control in a murine model. Here we developed a clinical platform for generating CMV-CD19CAR T cells. METHODS: Peripheral blood mononuclear cells (PBMCs) collected from CMV-seropositive healthy donors were stimulated with a good manufacturing practices-grade PepTivator overlapping CMVpp65 peptide pool and enriched for CMV-responsive interferon γ (IFNγ)+T cells using IFNγ Catchmatrix, within the CliniMACS Prodigy Cytokine Capture System (Miltenyi Biotec). Resulting CMV-specific T cells were transduced with a lentiviral vector encoding a second generation CD19R:CD28:ζ/EGFRt CAR and expanded with interleukin 2 (IL-2) and IL-15 for 15 days before characterization. RESULTS: CMV-specific T cells were enriched from 0.8%±0.5 of input PBMC to 76.3%±11.6 in nine full-scale qualification runs (absolute yield of 4.2±3.3×106 IFNγ+T cells from an input of 1×109 PBMCs). Average CD19CAR transduction efficiency of CMV-specific T cells was 27.0%±14.2 in the final products, which underwent rapid expansion, resulting in a total cell dose of 6.2±0.9 × 106 CD19CAR-tranduced T cells with CMV specificity (ie, functionally bispecific). CMV-CD19CAR T cells were polyclonal, expressed memory markers but had low expression of exhaustion markers, responded to both CD19 and CMVpp65 stimulation with rapid proliferation and exhibited antigen-specific effector functions against both CD19-expressing tumors and CMVpp65 antigen. The final products passed release criteria for clinical use. CONCLUSIONS: We demonstrated the feasibility of our large-scale platform for generating CMV-CD19CAR T cells for clinical application. We plan to initiate a clinical trial at City of Hope using CMV-CD19CAR T cells for patients with intermediate/high-grade B cell non-Hodgkin's lymphoma immediately after autologous hematopoietic cell transplantation followed by vaccination with a novel CMV vaccine based on Modified Vaccinia Ankara (Triplex) 28 days and 56 days post-T cell infusion.
Subject(s)
Adaptive Immunity/immunology , Cytomegalovirus/immunology , Leukocytes, Mononuclear/immunology , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/immunology , Animals , Cell Proliferation , Female , Humans , Male , Mice , Middle AgedABSTRACT
SARS-CoV-2 (CoV-2) viral infection results in COVID-19 disease, which has caused significant morbidity and mortality worldwide. A vaccine is crucial to curtail the spread of SARS-CoV-2, while therapeutics will be required to treat ongoing and reemerging infections of SARS-CoV-2 and COVID-19 disease. There are currently no commercially available effective anti-viral therapies for COVID-19, urging the development of novel modalities. Here, we describe a molecular therapy specifically targeted to neutralize SARS-CoV-2, which consists of extracellular vesicles (EVs) containing a novel fusion tetraspanin protein, CD63, embedded within an anti-CoV-2 nanobody. These anti-CoV-2-enriched EVs bind SARS-CoV-2 spike protein at the receptor-binding domain (RBD) site and can functionally neutralize SARS-CoV-2. This work demonstrates an innovative EV-targeting platform that can be employed to target and inhibit the early stages of SARS-CoV-2 infection.
ABSTRACT
Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue's homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs' ECs to decode mechanistic information and explore therapeutics.
Subject(s)
Bone Marrow , Endothelial Cells , Mice , Animals , Endothelial Cells/physiology , Transcriptome , Endothelium , Hematopoietic Stem Cells/metabolismABSTRACT
T cells engineered to express HIV-specific chimeric antigen receptors (CARs) represent a promising strategy to clear HIV-infected cells, but to date have not achieved clinical benefits. A likely hurdle is the limited T cell activation and persistence when HIV antigenemia is low, particularly during antiretroviral therapy (ART). To overcome this issue, we propose to use a cytomegalovirus (CMV) vaccine to stimulate CMV-specific T cells that express CARs directed against the HIV-1 envelope protein gp120. In this study, we use a GMP-compliant platform to engineer CMV-specific T cells to express a second-generation CAR derived from the N6 broadly neutralizing antibody, one of the broadest anti-gp120 neutralizing antibodies. These CMV-HIV CAR T cells exhibit dual effector functions upon in vitro stimulation through their endogenous CMV-specific T cell receptors or the introduced CARs. Using a humanized HIV mouse model, we show that CMV vaccination during ART accelerates CMV-HIV CAR T cell expansion in the peripheral blood and that higher numbers of CMV-HIV CAR T cells were associated with a better control of HIV viral load and fewer HIV antigen p24+ cells in the bone marrow upon ART interruption. Collectively, these data support the clinical development of CMV-HIV CAR T cells in combination with a CMV vaccine in HIV-infected individuals.
ABSTRACT
The COVID-19 pandemic has triggered the first widespread vaccination campaign against a coronavirus. Most vaccinated subjects are naïve to SARS-CoV-2, however almost all have previously encountered other coronaviruses (CoVs) and the role of this immunity in shaping the vaccine response remains uncharacterized. Here we use longitudinal samples and highly-multiplexed serology to identify mRNA-1273 vaccine-induced antibody responses against a range of CoV Spike epitopes and in both phylogenetically conserved and non-conserved regions. Whereas reactivity to SARS-CoV-2 epitopes showed a delayed but progressive increase following vaccination, we observed distinct kinetics for the endemic CoV homologs at two conserved sites in Spike S2: these became detectable sooner, and decayed at later timepoints. Using homolog-specific depletion and alanine-substitution experiments, we show that these distinctly-evolving specificities result from cross-reactive antibodies as they mature against rare, polymorphic residues within these epitopes. Our results reveal mechanisms for the formation of antibodies with broad reactivity against CoVs.
ABSTRACT
The COVID-19 pandemic has triggered the first widespread vaccination campaign against a coronavirus. Many vaccinated subjects are previously naive to SARS-CoV-2; however, almost all have previously encountered other coronaviruses (CoVs), and the role of this immunity in shaping the vaccine response remains uncharacterized. Here, we use longitudinal samples and highly multiplexed serology to identify mRNA-1273 vaccine-induced antibody responses against a range of CoV Spike epitopes, in both phylogenetically conserved and non-conserved regions. Whereas reactivity to SARS-CoV-2 epitopes shows a delayed but progressive increase following vaccination, we observe distinct kinetics for the endemic CoV homologs at conserved sites in Spike S2: these become detectable sooner and decay at later time points. Using homolog-specific antibody depletion and alanine-substitution experiments, we show that these distinct trajectories reflect an evolving cross-reactive response that can distinguish rare, polymorphic residues within these epitopes. Our results reveal mechanisms for the formation of antibodies with broad reactivity against CoVs.
Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , 2019-nCoV Vaccine mRNA-1273 , Antibodies, Viral , Antibody Formation , COVID-19/prevention & control , COVID-19 Vaccines , Epitopes , Humans , Pandemics , SARS-CoV-2 , VaccinationABSTRACT
BACKGROUND: Local modulation of vascular mammalian target of rapamycin (mTOR) signaling reduces smooth muscle cell (SMC) proliferation after endovascular interventions but may be associated with endothelial cell (EC) toxicity. The trilaminate vascular architecture juxtaposes ECs and SMCs to enable complex paracrine coregulation but shields SMCs from flow. We hypothesized that flow differentially affects mTOR signaling in ECs and SMCs and that SMCs regulate mTOR in ECs. METHODS AND RESULTS: SMCs and/or ECs were exposed to coronary artery flow in a perfusion bioreactor. We demonstrated by flow cytometry, immunofluorescence, and immunoblotting that EC expression of phospho-S6 ribosomal protein (p-S6RP), a downstream target of mTOR, was doubled by flow. Conversely, S6RP in SMCs was growth factor but not flow responsive, and SMCs eliminated the flow sensitivity of ECs. Temsirolimus, a sirolimus analog, eliminated the effect of growth factor on SMCs and of flow on ECs, reducing p-S6RP below basal levels and inhibiting endothelial recovery. EC p-S6RP expression in stented porcine arteries confirmed our in vitro findings: Phosphorylation was greatest in ECs farthest from intact SMCs in metal stented arteries and altogether absent after sirolimus stent elution. CONCLUSIONS: The mTOR pathway is activated in ECs in response to luminal flow. SMCs inhibit this flow-induced stimulation of endothelial mTOR pathway. Thus, we now define a novel external stimulus regulating phosphorylation of S6RP and another level of EC-SMC crosstalk. These interactions may explain the impact of local antiproliferative delivery that targets SMC proliferation and suggest that future stents integrate design influences on flow and drug effects on their molecular targets.
Subject(s)
Arteries/physiology , Cell Communication/physiology , Endothelial Cells/metabolism , Endothelium, Vascular/injuries , Intracellular Signaling Peptides and Proteins/metabolism , Myocytes, Smooth Muscle/metabolism , Protein Serine-Threonine Kinases/metabolism , Ribosomal Protein S6/metabolism , Animals , Aorta/physiology , Arteries/physiopathology , Cells, Cultured , Coronary Vessels/physiology , Endothelium, Vascular/pathology , Endothelium, Vascular/physiopathology , Humans , In Vitro Techniques , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Regional Blood Flow/drug effects , Regional Blood Flow/physiology , Signal Transduction , Sirolimus/analogs & derivatives , Sirolimus/pharmacology , Stents/adverse effects , Swine , Swine, Miniature , TOR Serine-Threonine Kinases , Transcription Factors/metabolismABSTRACT
Cyclin-dependent kinase inhibitors (CKIs) and Notch receptor activation have been shown to influence adult stem cells and progenitors by altering stem cell self-renewal and proliferation. Yet, no interaction between these molecular pathways has been defined. Here we show that ligand-independent and ligand-dependent activation of Notch1 induces transcription of the S phase kinase-associated protein 2 (SKP2), the F-box subunit of the ubiquitin-ligase complex SCF(SKP2) that targets proteins for degradation. Up-regulation of SKP2 by Notch signaling enhances proteasome-mediated degradation of the CKIs, p27 Kip1 and p21 Cip1, and causes premature entry into S phase. Silencing of SKP2 by RNA interference in G1 stabilizes p27 Kip1 and p21 Cip1 and abolishes Notch effect on G1-S progression. Thus, SKP2 serves to link Notch1 activation with the cell cycle machinery. This novel pathway involving Notch/SKP2/CKIs connects a cell surface receptor with proximate mediators of cell cycle activity, and suggests a mechanism by which a known physiologic mediator of cell fate determination interfaces with cell cycle control.
Subject(s)
Cell Cycle Proteins/metabolism , G1 Phase/physiology , Receptors, Cell Surface/metabolism , S Phase/physiology , S-Phase Kinase-Associated Proteins/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , 3T3 Cells , Animals , Base Sequence , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , DNA/genetics , Humans , Mice , Mice, Knockout , Mice, Transgenic , Models, Biological , RNA Interference , Receptor, Notch1 , Transcription, Genetic , Ubiquitin/metabolismABSTRACT
Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.
Subject(s)
PTEN Phosphohydrolase/physiology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Cell Line, Tumor , Cell Survival/genetics , Humans , Models, Biological , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Proto-Oncogene Proteins c-akt/geneticsABSTRACT
BACKGROUND: The interactions of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival. In the present study, we proposed to identify and investigate the role of molecules critically involved in leukemia--microenvironment crosstalk. PROCEDURE: Gene expression profiling analyses of BM mesenchymal stem cells (BMMSC) were performed following stimulation by ALL cells. CCL2 and IL-8 plasma levels were evaluated from ALL patients and controls. Expression of the CCL2 and IL-8 receptors in ALL was determined by RT-PCR. The biological effects of CCL2, IL-8 or its neutralizing antibodies in primary precursor-B ALL and BMMSC cells were evaluated using in vitro assays. RESULTS: Leukemia stimulation of BMMSC upregulated the expression of several inflammatory chemokines, including CCL2 and IL-8. The BM plasma levels of CCL2 and IL-8 in children at diagnosis were significantly higher than in healthy controls (P < 0.001). Functional studies revealed that CCL2 and IL-8 enhanced the capacity of BMMSC to support adhesion of ALL cells. CCL2 and IL-8 were also found to enhance BMMSC survival and to increase their proliferation. ALL cells were not directly affected by CCL2 or IL-8. CONCLUSIONS: The leukemic BM microenvironment had increased levels of CCL2 and IL-8. These chemokines are known to have suppressive effects in normal hematopoiesis. Our data indicate that CCL2 and IL-8 have a positive impact on BMMSC survival, proliferation, and adhesiveness to ALL cells. Leukemia-associated CCL2 and IL-8 upregulation may represent one possible mechanism of microenvironment perversion in favor of ALL cells.
Subject(s)
Bone Marrow Cells/metabolism , Chemokine CCL2/metabolism , Interleukin-8/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Cell Adhesion , Cell Survival , Chemokine CCL2/genetics , Chemokine CCL2/physiology , Child , Child, Preschool , Female , Humans , Infant , Interleukin-8/genetics , Interleukin-8/physiology , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/physiopathology , Prognosis , Signal TransductionABSTRACT
PURPOSE OF REVIEW: In the postnatal life, hematopoietic stem cell (HSC) niches are specialized microenvironments in the bone marrow that are essential for the maintenance and function of HSCs. The purpose of this review is to discuss the concept of HSC niche in light of recent studies that broaden its complexity and better define its molecular regulation. Also, we will discuss recent studies addressing the impact of leukemia development on HSC regulation and normal hematopoiesis, while discussing the potential regulation of leukemia-initiating cells by bone marrow niches. RECENT FINDINGS: Recent studies have identified new cellular and molecular components of the HSC niche and highlighted reciprocal interactions between the hematopoietic cells and their niches. These studies indicate that the HSC niche is not constituted by a single cell type but rather should be considered as a multicellular functional unit. Finally, advances have been made that provide promising insights into the the instructive role of the bone marrow microenvironment in hematological malignancies. SUMMARY: Increasing insights into the cell-cell cross talk between the hematopoietic system and its microenvironment in the bone marrow, and in particular in the interplay of HSCs with their niche(s), should provide new tools for combinatorial therapies in bone marrow failure and bone marrow cancers.
Subject(s)
Bone Marrow Cells/physiology , Bone Marrow Neoplasms/physiopathology , Hematopoiesis/physiology , Stem Cell Niche/physiology , Animals , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Bone Marrow Neoplasms/genetics , Bone Marrow Neoplasms/pathology , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Lineage/genetics , Cell Lineage/physiology , Gene Expression Regulation, Developmental , Hematopoiesis/genetics , Humans , Stem Cell Niche/cytology , Stem Cell Niche/metabolismABSTRACT
Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients. About 10-15% of pediatric ALL belong to T-cell ALL (T-ALL), which is characterized by aggressive expansion of immature T-lymphoblasts and is categorized as high-risk leukemia. Leukemia initiating cells represent a reservoir that is responsible for the initiation and propagation of leukemia. Its perinatal origin has been suggested in some childhood acute B-lymphoblastic and myeloblastic leukemias. Therefore, we hypothesized that child T-ALL initiating cells also exist during the perinatal period. In this study, T-ALL potential of the hematopoietic precursors was found in the para-aortic splanchnopleura (P-Sp) region, but not in the extraembryonic yolk sac (YS) of the mouse embryo at embryonic day 9.5. We overexpressed the Notch intracellular domain (NICD) in the P-Sp and YS cells and transplanted them into lethally irradiated mice. NICD-overexpressing P-Sp cells rapidly developed T-ALL while YS cells failed to display leukemia propagation despite successful NICD induction. These results suggest a possible role of fetal-derived T-cell precursors as leukemia-initiating cells.
ABSTRACT
Interleukin (IL)-7 is essential for normal T cell development. Previously, we have shown that IL-7 increases viability and proliferation of T cell acute lymphoblastic leukemia (T-ALL) cells by up-regulating Bcl-2 and down-regulating the cyclin-dependent kinase inhibitor p27kip1. Here, we examined the signaling pathways via which IL-7 mediates these effects. We investigated mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)-Akt (protein kinase B) pathways, which have active roles in T cell expansion and have been implicated in tumorigenesis. IL-7 induced activation of the MEK-Erk pathway in T-ALL cells; however, inhibition of the MEK-Erk pathway by the use of the cell-permeable inhibitor PD98059, did not affect IL-7-mediated viability or cell cycle progression of leukemic cells. IL-7 induced PI3K-dependent phosphorylation of Akt and its downstream targets GSK-3, FOXO1, and FOXO3a. PI3K activation was mandatory for IL-7-mediated Bcl-2 up-regulation, p27kip1 down-regulation, Rb hyperphosphorylation, and consequent viability and cell cycle progression of T-ALL cells. PI3K signaling was also required for cell size increase, up-regulation of CD71, expression of the glucose transporter Glut1, uptake of glucose, and maintenance of mitochondrial integrity. Our results implicate PI3K as a major effector of IL-7-induced viability, metabolic activation, growth and proliferation of T-ALL cells, and suggest that PI3K and its downstream effectors may represent molecular targets for therapeutic intervention in T-ALL.
Subject(s)
Glucose/metabolism , Interleukin-7/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , T-Lymphocytes/metabolism , Antigens, CD/biosynthesis , Antigens, Differentiation, B-Lymphocyte/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , Cell Cycle , Cell Cycle Proteins/metabolism , Cell Division , Cell Survival , Cyclin-Dependent Kinase Inhibitor p27 , Down-Regulation , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Glucose Transporter Type 1 , Humans , Immunoblotting , Immunophenotyping , Lectins, C-Type , Membrane Potentials , Mitochondria/pathology , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/metabolism , Monosaccharide Transport Proteins/metabolism , Phosphorylation , Precipitin Tests , Receptors, Transferrin , Signal Transduction , Time Factors , Tumor Suppressor Proteins/metabolism , Up-RegulationABSTRACT
Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).
Subject(s)
Apoptosis , Cell Proliferation , Gene Expression Regulation, Neoplastic , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Protein Kinase Inhibitors/pharmacology , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , S-Phase Kinase-Associated Proteins/physiology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: Homeostasis of the hematopoietic compartment is challenged and maintained during conditions of stress by mechanisms that are poorly defined. To understand how the bone marrow (BM) microenvironment influences hematopoiesis, we explored the role of Notch signaling and BM endothelial cells in providing microenvironmental cues to hematopoietic cells in the presence of inflammatory stimuli. MATERIALS AND METHODS: The human BM endothelial cell line (BMEC) and primary human BM endothelial cells were analyzed for expression of Notch ligands and the ability to expand hematopoietic progenitors in an in vitro coculture system. In vivo experiments were carried out to identify modulation of Notch signaling in BM endothelial and hematopoietic cells in mice challenged with tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS), or in Tie2-tmTNF-alpha transgenic mice characterized by constitutive TNF-alpha activation. RESULTS: BM endothelial cells were found to express Jagged ligands and to greatly support progenitor's colony-forming ability. This effect was markedly decreased by Notch antagonists and augmented by increasing levels of Jagged2. Physiologic upregulation of Jagged2 expression on BMEC was observed upon TNF-alpha activation. Injection of TNF-alpha or LPS upregulated three- to fourfold Jagged2 expression on murine BM endothelial cells in vivo and resulted in increased Notch activation on murine hematopoietic stem/progenitor cells. Similarly, constitutive activation of endothelial cells in Tie2-tmTNF-alpha mice was characterized by increased expression of Jagged2 and by augmented Notch activation on hematopoietic stem/progenitor cells. CONCLUSIONS: Our results provide the first evidence that BM endothelial cells promote expansion of hematopoietic progenitor cells by a Notch-dependent mechanism and that TNF-alpha and LPS can modulate the levels of Notch ligand expression and Notch activation in the BM microenvironment in vivo.
Subject(s)
Bone Marrow/immunology , Endothelial Cells/immunology , Inflammation/immunology , Receptors, Notch/metabolism , Signal Transduction/immunology , Tumor Necrosis Factor-alpha/physiology , Animals , Bone Marrow/blood supply , Bone Marrow/drug effects , Cells, Cultured , Endothelial Cells/drug effects , Hematopoietic Stem Cells/immunology , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Jagged-2 Protein , Ligands , Lipopolysaccharides/pharmacology , Membrane Proteins/drug effects , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred Strains , Mice, Transgenic , Receptors, Notch/drug effects , Receptors, Notch/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation/drug effects , Up-Regulation/immunologyABSTRACT
Cognition has been found to constrain several aspects of human behaviour, such as the number of friends and the number of favourite places a person keeps stable over time. This limitation has been empirically defined in the physical and social spaces. But do people exhibit similar constraints in the digital space? We address this question through the analysis of pseudonymised mobility and mobile application (app) usage data of 400,000 individuals in a European country for six months. Despite the enormous heterogeneity of apps usage, we find that individuals exhibit a conserved capacity that limits the number of applications they regularly use. Moreover, we find that this capacity steadily decreases with age, as does the capacity in the physical space but with more complex dynamics. Even though people might have the same capacity, applications get added and removed over time. In this respect, we identify two profiles of individuals: app keepers and explorers, which differ in their stable (keepers) vs exploratory (explorers) behaviour regarding their use of mobile applications. Finally, we show that the capacity of applications predicts mobility capacity and vice-versa. By contrast, the behaviour of keepers and explorers may considerably vary across the two domains. Our empirical findings provide an intriguing picture linking human behaviour in the physical and digital worlds which bridges research studies from Computer Science, Social Physics and Computational Social Sciences.