Identification of MiR-205 As a MicroRNA That Is Highly Expressed in Medullary Thymic Epithelial Cells

Thymic epithelial cells (TECs) support T cell development in the thymus. Cortical thymic epithelial cells (cTECs) facilitate positive selection of developing thymocytes whereas medullary thymic epithelial cells (mTECs) facilitate the deletion of self-reactive thymocytes in order to prevent autoimmunity. The mTEC compartment is highly dynamic with continuous maturation and turnover, but the genetic regulation of these processes remains poorly understood. MicroRNAs (miRNAs) are important regulators of TEC genetic programs since miRNA-deficient TECs are severely defective. However, the individual miRNAs important for TEC maintenance and function and their mechanisms of action remain unknown. Here, we demonstrate that miR-205 is highly and preferentially expressed in mTECs during both thymic ontogeny and in the postnatal thymus. This distinct expression is suggestive of functional importance for TEC biology. Genetic ablation of miR-205 in TECs, however, neither revealed a role for miR-205 in TEC function during homeostatic conditions nor during recovery from thymic stress conditions. Thus, despite its distinct expression, miR-205 on its own is largely dispensable for mTEC biology.     

mTORC1 in Thymic Epithelial Cells Is Critical for Thymopoiesis,T-Cell Generation,and Temporal Control of γδT17 Development and TCRγ/δ Recombination

Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.     

Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.     

Thymic epithelial progenitor cells and thymus regeneration： an update

The thymus provides the essential microenvironment for T-cell development and maturation. Thymic epithelial cells （TECs）, which are composed of thymic cortical epithelial cells （cTECs） and thymic medullary epithelial cells （mTECs）, have been well documented to be critical for these tightly regulated processes. It has long been controversial whether the common progenitor cells of TECs could give rise to both cTECs and mTECs. Great progress has been made to characterize the common TEC progenitor cells in recent years. We herein discuss the sole origin paradigm with regard to TEC differentiation as well as these progenitor cells in thymus regeneration.     

Identification of Novel Thymic Epithelial Cell Subsets Whose Differentiation Is Regulated by RANKL and Traf6

Thymic epithelial cells (TECs) are critical for the normal development and function of the thymus. Here, we examined the developmental stages of TECs using quantitative assessment of the cortical and medullary markers Keratin 5 and Keratin 8 (K5 and K8) respectively, in normal and gain/loss of function mutant animals. Gain of function mice overexpressed RANKL in T cells, whereas loss of function animals lacked expression of Traf6 in TECs (Traf6ΔTEC). Assessment of K5 and K8 expression in conjunction with other TEC markers in wild type mice identified novel cortical and medullary TEC populations, expressing different combinations of these markers. RANKL overexpression led to expansion of all medullary TECs (mTECs) and enlargement of the thymic medulla. This in turn associated with a block in thymocyte development and loss of CD4⁺CD8⁺, CD4⁺ and CD8⁺ thymocytes. In contrast, Traf6 deletion inhibited the production of most TEC populations including cortical TECs (cTECs), defined by absence of UEA-1 binding and LY51 expression, but had no apparent effect on thymocyte development. These results reveal a large degree of heterogeneity within the TEC compartment and the existence of several populations exhibiting concomitant expression of cortical, medullary and epithelial markers and whose production is regulated by RANKL and Traf6.     

Antigen Recognition By Autoreactive Cd4+ Thymocytes Drives Homeostasis Of The Thymic Medulla

The thymic medulla is dedicated for purging the T-cell receptor (TCR) repertoire of self-reactive specificities. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process because they express numerous peripheral tissue-restricted self-antigens. Although it is well known that medulla formation depends on the development of single-positive (SP) thymocytes, the mechanisms underlying this requirement are incompletely understood. We demonstrate here that conventional SP CD4⁺ thymocytes bearing autoreactive TCRs drive a homeostatic process that fine-tunes medullary plasticity in adult mice by governing the expansion and patterning of the medulla. This process exhibits strict dependence on TCR-reactivity with self-antigens expressed by mTECs, as well as engagement of the CD28-CD80/CD86 costimulatory axis. These interactions induce the expression of lymphotoxin α in autoreactive CD4⁺ thymocytes and RANK in mTECs. Lymphotoxin in turn drives mTEC development in synergy with RANKL and CD40L. Our results show that Ag-dependent interactions between autoreactive CD4⁺ thymocytes and mTECs fine-tune homeostasis of the medulla by completing the signaling axes implicated in mTEC expansion and medullary organization.     

DKK1 Mediated Inhibition of Wnt Signaling in Postnatal Mice Leads to Loss of TEC Progenitors and Thymic Degeneration

Background

Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.

Methodology/Principal Findings

Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of ΔNP63⁺ Foxn1⁺ and Aire⁺ TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.

Conclusions/Significance

Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.     

The thymic insulin-like growth factor axis: involvement in physiology and disease.

A repertoire of neuroendocrine-related genes is transcribed in the non-lymphoid compartment of the thymus, transposing the dual physiological role of this organ at the molecular level in T-cell development towards the establishment of central T-cell self-tolerance. The "neuroendocrine self" has been defined as a series of antigen sequences processed from precursors predominantly expressed in the thymus and first encountered by differentiating T-lymphocytes in their early life. All the members of the insulin gene family are expressed in the thymus according to a precise hierarchy and cellular topography, whereby IGF-II (epithelium of the subcapsular cortex and medulla) exceeds IGF-I (macrophages), which in turn far exceeds INS (rare subsets of medullary epithelial cells). This hierarchy in the degree of their respective thymic expression explains why IGF-II is more tolerated than IGF-I, and much more so than insulin. Evidence has been found for significant regulatory/tolerogenic properties in the IGF-II B:11 - 25 sequence after analysis of the cytokine secretion profile in peripheral blood mononuclear cells isolated from ten DQ8+ type 1 diabetic adolescents. In the thymus, IGF ligands and receptors also intervene in the control of T-cell proliferation and differentiation. Here, we also discuss how a disturbance in the intrathymic IGF-mediated signaling could contribute to the pathogenesis of T-cell leukemia.     

A novel role for autophagy in T cell education

During T cell development in the thymus, scanning of peptide/major histocompatibility (MHC) molecule complexes on the surface of thymic epithelial cells ensures that only useful (self-MHC restricted) and harmless (self-tolerant) thymocytes survive. In recent years, a number of distinct cell-biological features of thymic epithelial cells have been unraveled that may have evolved to render these cells particularly suited for T cell selection, e.g., cortical epithelial cells use unique proteolytic enzymes for the generation of MHC/peptide complexes, whereas medullary epithelial cells "promiscuously" express otherwise tissue-restricted self-antigens. We recently showed that macroautophagy in thymic epithelial cells contributes to CD4 T cell selection and is essential for the generation of a self-tolerant T cell repertoire. We propose that the unusually high constitutive levels of autophagy in thymic epithelial cells deliver endogenous proteins to MHC class II molecules for both positive and negative selection of developing thymocytes.     

Stages of the rat thymic medulla development in foetal period

Development of thymic medulla was examined on consecutive gestational days (GD) in Wistar rats. Medullary thymic epithelial cells (TEC) were identified by immunocytochemical localisation of neuron-specific enolase (NSE). Organisation of thymic medullary architecture was determined by interaction of thymocytes with NSE-positive TEC, that led to formation of lymphoepithelial complexes (GD 19), in which the cells exhibited proliferative activity or traits of apoptosis. The studies indicated that differentiation events and organisation of thymic medulla require stage-specific interactions between TEC and thymocytes.     

Differential expression and regulation of cyclooxygenase isozymes in thymic stromal cells

Prostaglandins (PGs) are lipid-derived mediators of rapid and localized cellular responses. Given the role of PG in supporting thymic T cell development, we investigated the expression of the PG synthases, also known as cyclooxygenases (COX)-1 and -2, in the biosynthesis of PGs in thymic stromal cell lines. The predominant isozyme expressed in cortical thymic epithelial cells was COX-1, while COX-2 predominated in the medulla. IFN-gamma up-regulated expression and activity of COX-2 in medullary cells, in which COX-2 was expressed constitutively. In contrast, IFN-gamma down-regulated COX-1 activity, but not expression, in cortical cells. Stromal cells support T cell development in the thymus, although the mediators of this effect are unknown. Selective inhibition of COX-2, but not COX-1, blocked the adhesion of CD4+CD8+ and CD4+CD8- thymocytes to medullary cell lines. No effect of the inhibitors was observed on the interactions of thymocytes with cortical epithelial lines. These data further support the differential regulation of COX-1 and COX-2 expression and function in thymic stromal cells. PGs produced by COX-2 in the medullary thymic stroma may regulate the development of thymocytes by modulating their interaction with stromal cells.     

Fine structure of the thymus in the adult cling fish Sicyases sanguineus (Pisces, Gobiesocidae)

The structure of the thumus in adult specimens of a marine teleost, the cling fish Sicyases sanguineus, has been studied by light and transmission electron microscopy. Most cling fishes have an outer thymus located beneath the opercular epithelium. A few of them, however, have a large inner thymus besides a poorly developed outer thymus. In the well-developed outer thymus of cling fish there are three different zones: outer cortex, inner cortex, and medulla. The inner cortex is similar to the cortical region of the thumus in other vertebrates, whereas the outer cortex is a specialized lympho-epithelial zone containing cystic cells (also present in medullary region) and true Hassall's corpuscles. In accordance with the development of the thymic parenchyma, the medullary or basal region may appear either like a true thymic medulla or like a subcapsular region. In the inner thymus, a subcapsular or peripheral "medullary" region and a central area (inverted cortex) show structural features like those of the medullary (basal) and deep cortical regions of the outer thymus, respectively. In addition to the above regions, sometimes there is a lymphomyeloid perithymic infiltration that often extends along connective tissue septa into the perivascular spaces of the gland. Reticuloepithelial, mesenchymal, and unidentified types of stromal cells within the thymus are described. Some erythrocytes, granulocytes, and monocytoid cells are found, but no plasma cells nor erythropoietic foci are evident. The probable significance of these findings is discussed.     

Ontogeny and organization of the stationary non-lymphoid cells in the human thymus

Summary Ontogenetic differentiation of the human thymus was investigated in 50 embryos by means of light and electron microscopic methods in an attempt to clarify the morphogenesis of the complicated microecology of thymic tissue. At the 8th gestational week (g.w.), the primordium of the thymus contains almost exclusively undifferentiated epithelial cells. At the 10th g.w., the epithelial cells in the central part are spindle-shaped. During the subsequent weeks the cortical region of the thymus becomes separated into lobes by mesenchymal septa containing hemopoietic precursor cells and large electronlucent cells with irregularly shaped nuclei. The latter cells are also found in the deeper presumptive medullary regions of the thymus; they differentiate into interdigitating reticulum cells (IDC). The permeation of the medulla of the thymus by non-epithelial IDC occurs concurrently with the formation of cortical and medullary epithelial cells. Between the 12th and 14th g.w. the cortical and medullary differentiation is completed. At this time-stage cortical small lymphocytes differ in morphological shape from medullary lymphocytes, the latter acquiring the appearance of immunocompetent T cells and establishing intimate contact with the IDC.These findings indicate that the thymic cortex and medulla contain different epithelial cells. In addition, the thymic medulla displays cells characterized by the morphology of typical interdigitating reticulum cells of peripheral lymphoid tissue. The structural pattern of the thymus is correlated to morphologically differing lymphoid cell populations in the cortical and medullary regions.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft and by the Sonderforschungsbereich 111The authors dedicate this paper to Professor Helmut Leonhardt on the occasion of his 60th birthday. The authors also appreciate the excellent technical assistance of Mrs. I. Knauer, Mrs. H. Waluk and Mrs. H. Siebke     

The thymus microenvironment in regulating thymocyte differentiation

The thymus plays a crucial role in the development of T lymphocytes providing an inductive microenvironment in which committed progenitors undergo proliferation, T-cell receptor gene rearrangements and thymocyte differentiation into mature T-cells. The thymus microenvironment forms a complex network of interaction that comprises non lymphoid cells (e.g., thymic epithelial cells, TEC), cytokines, chemokines, extracellular matrix elements (ECM), matrix metalloproteinases and other soluble proteins. The thymic epithelial meshwork is the major component of thymic microenvironment, both morphologically and phenotypically limiting heterogeneous regions in thymic lobules and fulfilling an important role during specific stages of T-cell maturation. The process starts when bone marrow–derived lymphocyte precursors arrive at the outer cortical region of the thymic gland and begin to mature into functional T lymphocytes that will finally exit the thymus and populate the peripheral lymphoid organs. During their journey inside the thymus, thymocytes must interact with stromal cells (and their soluble products) and extracellular matrix proteins to receive appropriate signals for survival, proliferation and differentiation. The crucial components of the thymus microenvironment and their complex interactions during the T-cell maturation process with the objective of contributing to a better understanding of the function of the thymus as well as assist in the search for new therapeutic approaches to improve the immune response in various pathological conditions are summarized here.     

Ontogeny and regulation of IL-7-expressing thymic epithelial cells

Epithelial cells in the thymus produce IL-7, an essential cytokine that promotes the survival, differentiation, and proliferation of thymocytes. We identified IL-7-expressing thymic epithelial cells (TECs) throughout ontogeny and in the adult mouse thymus by in situ hybridization analysis. IL-7 expression is initiated in the thymic fated domain of the early primordium by embryonic day 11.5 and is expressed in a Foxn1-independent pathway. Marked changes occur in the localization and regulation of IL-7-expressing TECs during development. IL-7-expressing TECs are present throughout the early thymic rudiment. In contrast, a major population of IL-7-expressing TECs is localized to the medulla in the adult thymus. Using mouse strains in which thymocyte development is arrested at various stages, we show that fetal and postnatal thymi differ in the frequency and localization of IL-7-expressing TECs. Whereas IL-7 expression is initiated independently of hemopoietic-derived signals during thymic organogenesis, thymocyte-derived signals play an essential role in regulating IL-7 expression in the adult TEC compartment. Moreover, different thymocyte subsets regulate the expression of IL-7 and keratin 5 in adult cortical epithelium, suggesting that despite phenotypic similarities, the cortical TEC compartments of wild-type and RAG-1(-/-) mice are developmentally and functionally distinct.