Molecular Regulation of Early T-Cell Development in the Thymus

The human body is under constant siege of pathogens - bacteria, viruses, fungi and parasites. We can only survive because these attackers are continuously fought off by our immune system. Important tasks within the immune system of vertebrates are performed by T lymphocytes, the executors of speci.c, cellular immunity. The speci.city of T lymphocytes lies in their T-cell receptor (TCR), through which they sense the presence of antigens in their environment. Each T cell expresses a TCR with a unique antigen-recognition site, so all T cells together can respond to an enormous variety of antigens. The highly diverse T-cell repertoire is generated by random recombination of discrete TCR gene segments. Via the TCR, T cells recognize peptide antigens that are displayed by antigen presenting cells (APCs), in the context of major histocompatibility complex (MHC) class I or class II molecules. Mature T cells carry out their function in cellular immunity as either CD8+ cytotoxic T cells or as CD4+ helper T cells. The humoral part of the speci.c immune system is supplied by B lymphocytes, which can secrete their antigen receptors in the form of antibodies. T cells develop from multipotent precursors via a highly ordered, but complex differentiation pathway. A number of critical events occur during this T-cell development process. Cells proliferate, adopt a T-cell fate, and produce a TCR molecule via a strictly ordered process of gene rearrangements. Stringent selection processes make sure that the produced TCR molecule is self-MHC restricted but not reactive to self-antigens. Finally, the selected T cells are allowed to mature into functional effector T cells. To become highly differentiated and thoroughly ‘educated’ cells, T cells need a specialized microenvironment for their development. The organ which has evolved to ful.ll this task and which also gave T cells their name, is the Thymus. The hematopoietic cells that undergo T-cell development in the thymus are called thymocytes. The importance of the thymus as essential microenvironment for T-cell development is illustrated by children af.icted by the DiGeorge syndrome, who sometimes completely lack a thymus. These children have severely reduced T cell numbers and suffer from recurrent life-threatening infections. DiGeorge syndrome also illustrates the fact that T cells are the only hematopoietic cell type that absolutely require the thymus for their development. Although human T-cell development is the main theme of this thesis, most of the published knowledge about T-cell differentiation has been acquired by studies in the mouse

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

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

Clinical characteristics of women captured by extending the definition of severe postpartum haemorrhage with 'refractoriness to treatment': a cohort study

Background: The absence of a uniform and clinically relevant definition of severe postpartum haemorrhage hampers comparative studies and optimization of clinical management. The concept of persistent postpartum haemorrhage, based on refractoriness to initial first-line treatment, was proposed as an alternative to common definitions that are either based on estimations of blood loss or transfused units of packed red blood cells (RBC). We compared characteristics and outcomes of women with severe postpartum haemorrhage captured by these three types of definitions. Methods: In this large retrospective cohort study in 61 hospitals in the Netherlands we included 1391 consecutive women with postpartum haemorrhage who received either ≥4 units of RBC or a multicomponent transfusion. Clinical characteristics and outcomes of women with severe postpartum haemorrhage defined as persistent postpartum haemorrhage were compared to definitions based on estimated blood loss or transfused units of RBC within 24 h following birth. Adverse maternal outcome was a composite of maternal mortality, hysterectomy, arterial embolisation and intensive care unit admission. Results: One thousand two hundred sixty out of 1391 women (90.6%) with postpartum haemorrhage fulfilled the definition of persistent postpartum haemorrhage. The majority, 820/1260 (65.1%), fulfilled this definition within 1 h following birth, compared to 819/1391 (58.7%) applying the definition of ≥1 L blood loss and 37/845 (4.4%) applying the definition of ≥4 units of RBC. The definition persistent postpartum haemorrhage captured 430/471 adverse maternal outcomes (91.3%), compared to 471/471 (100%) for ≥1 L blood loss and 383/471 (81.3%) for ≥4 units of RBC. Persistent postpartum haemorrhage did not capture all adverse outcomes because of missing data on timing of initial, first-line treatment. Conclusion: The definition persistent postpartum haemo