An early decrease in Notch activation is required for human TCR- lineage differentiation at the expense of TCR- T cells

Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Ghent University Hospital, Ghent, Belgium.
Blood (Impact Factor: 10.45). 03/2009; 113(13):2988-98. DOI: 10.1182/blood-2008-06-164871
Source: PubMed


Although well characterized in the mouse, the role of Notch signaling in the human T-cell receptor alphabeta (TCR-alphabeta) versus TCR-gammadelta lineage decision is still unclear. Although it is clear in the mouse that TCR-gammadelta development is less Notch dependent compared with TCR-alphabeta differentiation, retroviral overexpression studies in human have suggested an opposing role for Notch during human T-cell development. Using the OP9-coculture system, we demonstrate that changes in Notch activation are differentially required during human T-cell development. High Notch activation promotes the generation of T-lineage precursors and gammadelta T cells but inhibits differentiation toward the alphabeta lineage. Reducing the amount of Notch activation rescues alphabeta-lineage differentiation, also at the single-cell level. Gene expression analysis suggests that this is mediated by differential sensitivities of Notch target genes in response to changes in Notch activation. High Notch activity increases DTX1, NRARP, and RUNX3 expression, genes that are down-regulated during alphabeta-lineage differentiation. Furthermore, increased interleukin-7 levels cannot compensate for the Notch dependent TCR-gammadelta development. Our results reveal stage-dependent molecular changes in Notch signaling that are critical for normal human T-cell development and reveal fundamental molecular differences between mouse and human.

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    • "It is unclear whether the Notch signaling is associated with changes in the immune status observed in children with HFMD. Previous studies have shown that the Notch signaling pathway possesses a crucial role in differentiations of a variety of immune cells [13,14]. Mukuherjee et al. [15] found that the Notch ligand Dll4 promoted T-cell differentiation through increased expression of IL-17 and RORγ T. Schaller et al. [10] also found that Dll4 expression on bone marrow-derived dendritic cells (DCs) increased significantly after infection of mice with respiratory syncytial virus, accompanied by increased secretion of Th2 cytokines and reduced production of INF-γ. "
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    ABSTRACT: Background Hand, foot and mouth disease (HFMD), a virus-induced infectious disease that usually affects infants and children, has an increased incidence in China in recent years. This study attempted to investigate the role of the Notch signaling pathway in the pathogenesis of HFMD. Methods Eighty-two children diagnosed with HFMD were enrolled into this study. The HFMD group was further divided into the uncomplicated HFMD and HFMD with encephalitis groups. The control group included 40 children who underwent elective surgery for treatment of inguinal hernias. Results Children with HFMD displayed significantly reduced CD3+, CD3+CD4+ and CD3+CD8+ cell subsets, but substantially enhanced CD3−CD19+ cell subset (p < 0.05 versus control subjects). The expression levels of Notch ligands Dll1 and Dll4 in the peripheral blood of the HFMD group were significantly higher than those in the control group (p < 0.05). There were statistically significant differences in CD3+, CD3+CD4+ and CD3−CD19+ cell subsets, but not in Notch ligand expression, between the uncomplicated HFMD and HFMD with encephalitis groups. Dll4 expression in HFMD subjects correlated negatively with the CD3+ and CD3+CD8+ cell subsets (p < 0.05), but positively with the CD3−CD19+ cell subset (p < 0.05). Furthermore, Dll4 expression in HFMD with encephalitis subjects correlated positively with total white blood cell (WBC) counts and total protein contents in cerebrospinal fluid (CSF) (p < 0.05). Conclusions The Notch ligand Dll4 exhibits a strong correlation with the CD3+, CD3+CD8+ and CD3−CD19+ cell subsets in children with HFMD, indicating that the Notch signaling may be involved in the development of HFMD by affecting the number and status of peripheral lymphocytes.
    BMC Infectious Diseases 06/2014; 14(1):337. DOI:10.1186/1471-2334-14-337 · 2.61 Impact Factor
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    • "Our data reveal that the strength of the Notch signal is an important parameter in this decision, with lower Notch signals mediating T cell differentiation, while strong Notch signals induce ILC2 differentiation. Similar dosage dependent outcomes of Notch signaling have been reported before (64), for instance in the lineage decision between αβ and γδ T cells (52, 65). While we did not observe γδ T cells in our cultures, possibly due to differences in culture conditions and progenitor sort strategies, the block in αβ T cell development observed here with high levels of Notch activation is in line with these studies (65). "
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    ABSTRACT: Innate lymphoid cells (ILCs) are emerging key players of the immune system with close lineage relationship to T cells. ILC2 play an important role in protective immunity against multicellular parasites, but are also involved in the pathogenesis of type 2 immune diseases. Here, we have studied the developmental requirements for human ILC2. We report that ILC2 are present in the thymus of young human donors, possibly reflecting local differentiation. Furthermore, we show that uncommitted lineage(-)CD34(+)CD1a(-)human thymic progenitors have the capacity to develop into ILC2 in vitro under the influence of Notch signaling, either by stimulation with the Notch ligand Delta like 1 (Dll1) or by expression of the active intracellular domain of NOTCH1 (NICD1). The capacity of NICD1 to mobilize the ILC2 differentiation program was sufficiently potent to override commitment to the T cell lineage in CD34(+)CD1a(+) progenitors and force them into the ILC2 lineage. As Notch is an important factor also for T cell development, these results raise the question how one and the same signaling pathway can elicit such distinct developmental outcomes from the same precursors. We provide evidence that Notch signal strength is a critical determinant in this decision: by tuning signal amplitude, Notch can be converted from a T cell inducer (low signal strength) to an ILC2 inducer (high signal strength). Thus, this study enhances our understanding of human ILC2 development and identifies a mechanism determining specificity of Notch signal output during T cell and ILC2 differentiation.
    Frontiers in Immunology 10/2013; 4:334. DOI:10.3389/fimmu.2013.00334
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    • "Future experiments in which MS5/DL1ind100 cells will be sorted according to low or high levels of DL1 expression will be extremely interesting to fully explore the relation between NOTCH ligand expression levels and hematopoietic cell differentiation. Inversely, washing out doxycyclin during T cell culture led to fast DL1 down-regulation interrupting NOTCH signalling with important effects such as accelerated DP generation and decreased proliferation of such cells were observed in accordance with other studies [12]. Studying the effect of lower NOTCH ligand expression levels on T cell development upon addition/removal of graded amounts of doxycyclin is also an interesting question to address in such TET/on/off system. "
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    ABSTRACT: Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34(+)CD38(-/low)(CD45RA(-)CD90(+)) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.
    PLoS ONE 11/2012; 7(11):e50495. DOI:10.1371/journal.pone.0050495 · 3.23 Impact Factor
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