Floor Weerkamp

New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling

To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin− cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Dδ2-Dδ3 rearrangement occurs at the most immature CD34+CD38−CD1a− stage. TCRB rearrangement starts at the CD34+CD38+CD1a− stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTα) expression pattern show that human TCRβ-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.

Wnt3a deficiency irreversibly impairs hematopoietic stem cell self-renewal and leads to defects in progenitor cell differentiation

Canonical Wnt signaling has been implicated in various aspects of hematopoiesis. Its role is controversial due to different outcomes between various inducible Wnt-signaling loss-of-function models and also compared with gain-of-function systems. We therefore studied a mouse deficient for a Wnt gene that seemed to play a nonredundant role in hematopoiesis. Mice lacking Wnt3a die prenatally around embryonic day (E) 12.5, allowing fetal hematopoiesis to be studied using in vitro assays and transplantation into irradiated recipient mice. Here we show that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. The impaired long-term repopulation capacity of Wnt3a(-/-) HSCs could not be explained by altered cell cycle or survival of primitive progenitors. Moreover, Wnt3a deficiency affected myeloid but not B-lymphoid development at the progenitor level, and affected immature thymocyte differentiation. Our results show that Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis.

Wnt Target Genes Identified by DNA Microarrays in Immature CD34+ Thymocytes Regulate Proliferation and Cell Adhesion

The thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals induce the interaction of the cytoplasmic cofactor β-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/β-catenin/TCF pathway in the most immature (CD4−CD8−CD34+) thymocytes using Affymetrix DNA microarrays in combination with three different functional assays for in vitro induction of Wnt signaling. A relatively small number (∼30) of genes changed expression, including several proliferation-inducing transcription factors such as c-fos and c-jun, protein phosphatases, and adhesion molecules, but no genes involved in differentiation to mature T cell stages. The adhesion molecules likely confine the proliferating immature thymocytes to the appropriate anatomical sites in the thymus. For several of these target genes, we validated that they are true Wnt/β-catenin/TCF target genes using real-time quantitative PCR and reporter gene assays. The same core set of genes was repressed in Tcf-1-null mice, explaining the block in early thymocyte development in these mice. In conclusion, Wnt signals mediate proliferation and cell adhesion, but not differentiation of the immature thymic progenitor pool.

Notch and Wnt signaling in T-lymphocyte development and acute lymphoblastic leukemia

Many acute lymphoblastic leukemias can be considered as malignant counterparts of cells in the various stages of normal lymphoid development in bone marrow and thymus. T-cell development in the thymus is an ordered and tightly controlled process. Two evolutionary conserved signaling pathways, which were first discovered in Drosophila, control the earliest steps of T-cell development. These are the Notch and Wnt-signaling routes, which both are deregulated in several types of leukemias. In this review we discuss both pathways, with respect to their signaling mechanisms, functions during T-cell development and their roles in development of leukemias, especially T-cell acute lymphoblastic leukemia.

Transcriptional Control of T Lymphocyte Differentiation

Initiation of gene transcription by transcription factors (TFs) is an important regulatory step in many developmental processes. The differentiation of T cell progenitors in the thymus is tightly controlled by signaling molecules, ultimately activating nuclear TFs that regulate the expression of T lineage‐specific genes. During the last 2 years, significant progress has been made in our understanding of the signaling routes and TFs operating during the earliest stages of thymic differentiation at the CD4−CD8− double negative stage. Here we will review the TF families that play an important role in differentiation of thymocytes, particularly focusing on recent new information with respect to the Tcf, bHLH, GATA, and CBF/HES TF families.

Flow cytometric immunobead assay for the detection of BCR-ABL fusion proteins in leukemia patients

BCR–ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR–ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR–ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR–ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR–ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR–ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR–ABL immunobead assay detects all types of BCR–ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within ∼4 h, and can be run in parallel to routine immunophenotyping.

Effects of Delta1 and Jagged1 on early human hematopoiesis: correlation with expression of notch signaling-related genes in CD34+ cells.

It has been shown that Notch signaling mediated by ligands of both Jagged and Delta families expands the hematopoietic stem cell compartment while blocking or delaying terminal myeloid differentiation. Here we show that Delta1‐ and Jagged1‐expressing stromal cells have distinct effects on the clonogenic and differentiation capacities of human CD34+ CD38+ cells. Jagged1 increases the number of bipotent [colony‐forming unit‐granulocyte macrophage (CFU‐GM) and unipotent progenitors (CFU‐granulocytes and CFU‐macrophages), without quantitatively affecting terminal cell differentiation, whereas Delta1 reduces the number of CFU‐GM and differentiated monocytic cells. Expression analysis of genes coding for Notch receptors, Notch targets, and Notch signaling modulators in supernatant CD34+ cells arising upon contact with Jagged1 and Delta1 shows dynamic and differential gene expression profiles over time. At early time points, modest upregulation of Notch1, Notch3, and Hes1 was observed in Jagged1‐CD34+ cells, whereas those in contact with Delta1 strikingly upregulated Notch3 and Hes1. Later, myeloid progenitors with strong clonogenic potential emerging upon contact with Jagged1 upregulated Notch1 and Deltex and downregulated Notch signaling modulators, whereas T/NK progenitors originated by Delta1 strikingly upregulated Notch3 and Deltex and, to a lesser extent, Hes1, Lunatic Fringe, and Numb. Together, the data unravel previously unrecognized expression patterns of Notch signaling‐related genes in CD34+ CD38+ cells as they develop in Jagged1‐ or Delta1‐stromal cell environments, which appear to reflect sequential maturational stages of CD34+ cells into distinct cell lineages.

Identification of Notch target genes in uncommitted T-cell progenitors: no direct induction of a T-cell specific gene program

Deregulated Notch signaling occurs in the majority of human T-ALL. During normal lymphoid development, activation of the Notch signaling pathway poses a T-cell fate on hematopoietic progenitors. However, the transcriptional targets of the Notch pathway are largely unknown. We sought to identify Notch target genes by inducing Notch signaling in human hematopoietic progenitors using two different methods: an intracellular signal through transfection of activated Notch and a Notch-receptor dependent signal by interaction with its ligand Delta1. Gene expression profiles were generated and evaluated with respect to expression profiles of immature thymic subpopulations. We confirmed HES1, NOTCH1 and NRARP as Notch target genes, but other reported Notch targets, including the genes for Deltex1, pre-T-cell receptor α and E2A, were not found to be differentially expressed. Remarkably, no induction of T-cell receptor gene rearrangements or transcription of known T-cell specific genes was found after activation of the Notch pathway. A number of novel Notch target genes, including the transcription factor TCFL5 and the HOXA cluster, were identified and functionally tested. Apparently, Notch signaling is essential to open the T-cell pathway, but does not initiate the T-cell program itself.