Thymus | EPFL Graph Search

The thymus (: thymuses or thymi) is a specialized primary lymphoid organ of the immune system. Within the thymus, thymus cell lymphocytes or T cells mature. T cells are critical to the adaptive immune system, where the body adapts to specific foreign invaders. The thymus is located in the upper front part of the chest, in the anterior superior mediastinum, behind the sternum, and in front of the heart. It is made up of two lobes, each consisting of a central medulla and an outer cortex, surrounded by a capsule. The thymus is made up of immature T cells called thymocytes, as well as lining cells called epithelial cells which help the thymocytes develop. T cells that successfully develop react appropriately with MHC immune receptors of the body (called positive selection) and not against proteins of the body (called negative selection). The thymus is largest and most active during the neonatal and pre-adolescent periods. By the early teens, the thymus begins to decrease in size and a

Investigation of transcriptional partners and genes downstream of Foxn1

Zineb Bekkali

The thymus is the second fundamental organ of the immune system after the bone marrow; it is the essential for T cell maturation and repertoire selection. The function of the thymus critically depends on the thymic epithelium, which is structured in two distinct regions: The cortex and the medulla. The knowledge about molecular mechanisms involved in thymus development is limited. However, some evidences suggest that the Hox, Pax, Six, Eya, Gcm2, FGFs and Foxn1 are key players in thymus organogenesis. Foxn1 is essential for thymic epithelial cell development but its exact role is still unknown. The null mutation in the transcription factor Foxn1 generates the nude phenotype of athymia and hairlessness. The aim of our laboratory is to better understand thymus development and specially focuses on the Foxn1 function. The aim of this project is then to investigate target genes and transcription partners of Foxn1 as a transcriptional factor. As a transcription factor, Foxn1 protein should be able to bind with DNA but should also interact with other proteins such as cofactors in order to activate its target genes. At present, the transcriptional partners of Foxn1 are completely unknown in the literature and the proteinprotein interaction databases. The experimental approach proposed here is the standard method for identification of unknown transcriptional partners: Immunoprecipitation (IP) followed by a mass spectrometry analysis. The list of protein candidates can then be validated by Western blotting. We identified the right antibodies to use for this procedure and optimised protocols for the IP. We also performed a mass spectrometry analysis that was not conclusive, however, the platform is now established to complete this analysis. Only little is known about genes up- or down-stream Foxn1. Some genes such as FgfR2IIIb, DLL4 and CCL25 are believed to be up-regulated by Foxn1. Our laboratory has generated a revertible hypomorphic allele of Foxn1 and have used this to obtain a series of six allelic combinations at Foxn1. These mice, expressing different levels of Foxn1, are a very useful tool in order to investigate target genes of Foxn1. In particular, we wish to investigate whether different Foxn1 targets are activated at different Foxn1 levels. 6 To reach this objective, the proposed approach is the comparison of gene expression profiles of thymic epithelial and mesenchymal cells of the allelic series at two different time points E13.5 and E15.5 using a Solexa tool called SAGE (Serial Analysis of Gene Expression). As preliminary analysis before the global gene expression analysis, we have determined expression profiles of several genes across the allelic series by quantitative PCR. Our findings suggest that DLL4, CCL25, FgfR2IIIb, FGF10 and possible Pax1 are direct or indirect targets of Foxn1 activated at different levels of Foxn1. DLL4 and CCL25 seem to be involved in lymphocyte attraction and maturation. FgfR2IIIb appears to be important for thymic epithelium differentiation and development by increasing the sensitivity of epithelial cells to FGF10 signalling. Finally, Pax1 is possibly required downstream of Foxn1 for terminal differentiation of cortical thymic epithelial cells

2009

Molecular and Functional Characterization of Clonogenic Human Thymic Epithelial Cells

Melissa Maggioni

The thymus is a primary lymphoid organ where bone marrow derived T-cell progenitors come in contact with a unique microenvironment able to sustain their maturation into functional T-cells. This fundamental immunological function of the thymus is supported by thymic epithelial cells, which represent the main component of the thymic stroma. The thymic stroma is organized in a characteristic tridimensional structure that can be distinguished into two main regions, namely, the cortex and medulla. These two regions are responsible for two fundamental processes in the thymus: the positive- and negative- selection of T-cells. The first one involves the selection of T-cells able to react with antigens presented mainly on the surface of cortical cells. T-cell negative selection, meanwhile, is responsible for the elimination of self-reactive T-cells and it is mainly carried out by medullary thymic epithelial cells. The thymic epithelium derives from the primitive endoderm and cortical and medullary thymic epithelial cells have been demonstrated to originate from a common embryonic progenitor population. Currently, the presence of such population has not been described in adult animals and the mechanisms responsible for the maintenance of the post-natal thymus have yet to be clarified. Recently, Bonfanti and colleagues demonstrated that the rat thymus contains a population of clonogenic epithelial cells (rTECs), able to self-renew in the culture system developed in 1975 for epidermal stem cells [Bonfanti et al., 2010, Rheinwald and Green, 1975]. As clonogenicity is a property of skin and other stratified epithelia stem cells, the clonogenic capacity of TECs suggests them as potential thymic epithelial stem cells. Bonfanti and colleagues also demonstrated that rTECs displayed the capacity to integrate into a thymic microenvironment and to express thymic functional markers. The work described in this thesis aims to investigate the stem cell potency and the regenerative capacity of the human thymus. We demonstrated that the human thymus also contains a population of clonogenic thymic epithelial cells (hTECs) able to self-renew and to maintain a general thymic phenotype in the culture system used for skin stem cells and for rTECs. By clonal analysis, we showed that hTECs can be distinguished in three sub-populations, which displayed different molecular phenotypes and different growth and differentiation potentials in vitro. We then investigated the role of Foxn1 transcription factor in hTECs, as Foxn1 is know to be involved in thymic epithelial cells differentiation during development and also in the maintenance of the adult thymus in mice. By experimentally down-regulating Foxn1, we demonstrated that Foxn1 expression is required to sustain the growth of one of the described hTEC sub-populations. We next investigated the differentiation capacity of clonogenic hTECs. Currently, additional experiments are still required to assess hTECs ability to integrate and differentiate in a functional thymic environment. Bonfanti and colleagues also demonstrated that rTECs, if challenged in a hair follicle morphogenetic assay, can be reprogrammed into skin stem cells. Therefore, we decided to investigate the potential of hTECs in an epidermal differentiation assay. In these conditions, hTECs did not show the ability to differentiate into epidermis, probably due to the absence in this assay of the strong morphogenetic signals responsible for the reprogramming process of rTECs into skin stem cells.

EPFL2012

Exploring the potency of normal and pathological human thymic epithelial cells

Tiphaine Mélodie Chantal Arlabosse

Thymic epithelial cells (TECs) are organised in a unique 3D network that is critical for the development of efficient and self-tolerant T-cells. We report for the first time that the human thymus of all ages, even old and involuted, contains a significant number (up to 30%) of clonogenic TECs with extensive growth potential. All clonogenic TECs derive from a common EpCAM+ precursor, which within 48h in culture undergoes a molecular switch and gives rise to refringent EpCAM- colonies and stratified EpCAM+ colonies. The latter two subpopulations can be distinguished based on their morphology, their expression of genes implicated in epidermal stratification and their expression of genes implicated in epithelia-mesenchymal transition. Clonal analysis revealed that clonogenic cells are hierarchically organised, refringent cells being higher in the hierarchy than stratified cells. Although the thymus normally involutes with age, it can be the site of uncontrolled proliferation, resulting in the formation of thymic tumours including thymomas. Thymomas are classified by the WHO based on histological criteria as type A, B and AB and are frequently associated with Myasthenia Gravis, an autoimmune disease affecting the neuromuscular junction. The biology of thymomas is poorly understood, because of the rarity of the tumours and because of the lack of a reliable in vitro model. We report for the first time, that clonogenic cells can be isolated from all thymoma subtypes as well as from thymic hyperplasia and mediastinal teratoma. The latter cells displayed many similarities with normal TECs but also differences in terms of morphology, gene expression and genetic profile. Importantly, all thymic tumours studied contained refringent-like EpCAM- cells and stratified EpCAM+ cells, supporting the hypothesis that thymic tumour-initiating cells arise from clonogenic TECs. We have not succeeded yet in developing an appropriate assay to support the growth of clonogenic epithelial cells from thymic tumours in vivo. Finally, we report that the SY5 monoclonal antibody raised against human involucrin reproducibly cross-reacts with a molecule present in the Golgi apparatus of refringent cells derived from normal and pathological thymi. Immunocytochemistry and knock-down experiments revealed that the latter molecule is not involucrin. Epitope mapping by phage-display experiments and data base search identified putative candidate proteins, including nesprin-1, a player of the neuromuscular junction, which could be the key to understand the frequent association of thymomas with Myasthenia Gravis.

EPFL2017