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 beta-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/beta-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 (approximately 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/beta-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

iPSC-Based Modeling of RAG2 Severe Combined Immunodeficiency Reveals Multiple T Cell Developmental Arrests

RAG2 severe combined immune deficiency (RAG2-SCID) is a lethal disorder caused by the absence of functional T and B cells due to a differentiation block. Here, we generated induced pluripotent stem cells (iPSCs) from a RAG2-SCID patient to study the nature of the T cell developmental blockade. We observed a strongly reduced capacity to differentiate at every investigated stage of T cell development, from early CD7−CD5− to CD4+CD8+. The impaired differentiation was accompanied by an increase in CD7−CD56+CD33+ natural killer (NK) cell-like cells. T cell receptor D rearrangements were completely absent in RAG2SCID cells, whereas the rare T cell receptor B rearrangements were likely the result of illegitimate rearrangements. Repair of RAG2 restored the capacity to induce T cell receptor rearrangements, normalized T cell development, and corrected the NK cell-like phenotype. In conclusion, we succeeded in generating an iPSC-based RAG2-SCID model, which enabled the identification of previously unrecognized disorder-related T cell developmental roadblocks.In this article, Mikkers

Olfactomedin 4 serves as a marker for disease severity in pediatric respiratory syncytial virus (RSV) infection

Background: Respiratory viral infections follow an unpredictable clinical course in young children ranging from a common cold to respiratory failure. The transition from mild to severe disease occurs rapidly and is difficult to predict. The pathophysiology underlying disease severity has remained elusive. There is an urgent need to better understand the immune response in this disease to come up with biomarkers that may aid clinical decision making. Methods: In a prospective study, flow cytometric and genome-wide gene expression analyses were performed on blood samples of 26 children with a diagnosis of severe, moderate or mild Respiratory Syncytial Virus (RSV) infection. Differentially expressed genes were validated using Q-PCR in a second

Canonical Wnt signaling negatively modulates regulatory T cell function

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that Tcell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both invitro and invivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector Tcells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity

The canonical Wnt signaling pathway plays an important role in lymphopoiesis and hematopoiesis

The evolutionarily conserved canonical Wnt-β-catenin-T cell factor (TCF)/lymphocyte enhancer binding factor (LEF) signaling pathway regulates key checkpoints in the development of various tissues. Therefore, it is not surprising that a large body of gain-of-function and loss-of-function studies implicate Wnt-β-catenin signaling in lymphopoiesis and hematopoiesis. In contrast, recent papers have reported that Mx-Cre-mediated conditional deletion of β-catenin and/ or its homolog γ-catenin (plakoglobin) did not impair hematopoiesis or lymphopoiesis. However, these studies also report that TCF reporter activity remains active in β-catenin- and γ-catenin-deficient hematopoietic stem cells and all cells derived from these precursors, indicating that the canonical Wnt signaling pathway was not abrogated. Therefore, these studies in fact show that the canonical Wnt signaling pathway is important in hematopoiesis and lymphopoiesis, even though the molecular basis for the induction of the reporter activity is currently unknown. In this perspective, we provide a broad background to the field with a discussion of the available data and create a framework within which the available and future studies may be evaluated

Wnt signaling strength regulates normal hematopoiesis and its deregulation is involved in leukemia development

A strict balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is required in orde

Dialysis-related systemic microinflammation is associated with specific genomic patterns

Background. Although several reports have focused on the clinical importance of the systemic microinflammatory state in the uraemic population, the relationship between the activation of a specific transcriptome and the development of this condition is still not completely defined. Methods. Thirty haemodialysis (HD), 30 peritoneal dialysis (PD) and 30 chronic kidney disease (CKD) patients were enrolled in our study. For all patients, serum C-reactive protein (CRP) and ferritin levels were determined. In addition, the expression level of 234 inflammatory responses and oxidative stress pathway genes was measured, using oligonucleotide microarray chips (HG-U133A, Affymetrix), in peripheral blood mononuclear cells of 24 randomly selected patients (8 HD, 8 PD and 8 CKD). Results. HD patients demonstrated higher CRP and ferritin levels compared to PD and CKD patients (P < 0.001). Statistical analysis identified 10 genes able to discriminate CKD from HD and PD patients (FDR = 5%, P < 0.001) and significantly correlated to CRP levels. All together, these genes were able to predict inflammation with an accuracy of 87% (P < 0.001). Among the selected genes there were those encoding for key regulators of inflammation and oxidative stress (e.g. RELA, GSS). Interestingly, only three inflammatory genes (MIF, IL8RB and CXCL12) were still significantly associated with inflammation when included in a multivariate analysis. RT-PCR for RELA, MIF, CXCL12 and western blots for IL8RB and GSS, using 66 patients, validated the microarray results. Conclusions. This study may help to better understand the physiopathology of t

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

The peripheral blood compartment in patient with Graves’ disease: activated T lymphocytes and increased transitional and pre-naive mature B lymphocytes

Graves' disease (GD) is an autoimmune disease that involves aberrant B and T lymphocyte responses. Detailed knowledge about lymphocyte subpopulation composition will therefore enhance our understanding of the pathogenesis of GD and might support the development of new immunomodulatory treatment approaches. The aim of this study was to gain detailed insight into the composition of the peripheral blood lymphocyte compartment in GD before and during anti-thyroid drug therapy. Major B and T lymphocyte subpopulations were investigated by flow cytometry in peripheral blood from newly diagnosed GD patients (n=5), GD patients treated wit

Deregulated WNT signaling in childhood T-cell acute lymphoblastic leukemia

WNT signaling has been implicated in the regulation of hematopoietic stem cells and plays an important role during T-cell development in thymus. Here we investigated WNT pathway activation in childhood T-cell acute lymphoblastic leukemia (T-ALL) patients. To evaluate the potential role of WNT signaling in T-cell leukomogenesis, we performed expression analysis of key components of WNT pathway. More than 85% of the childhood T-ALL patients showed upregulated β-catenin expression at the protein level compared with normal human thymocytes. The impact of this upregulation was reflected in high expression of known target genes (AXIN2, c-MYC, TCF1 and LEF). Especially AXIN2, the universal target gene of WNT pathway, was upregulated at both mRNA and