Bone morphogenetic protein-2/4 signalling pathway components are expressed in the human thymus and inhibit early T-cell development

T-cell differentiation is driven by a complex network of signals mainly derived from the thymic epithelium. In this study we demonstrate in the human thymus that cortical epithelial cells produce bone morphogenetic protein 2 (BMP2) and BMP4 and that both thymocytes and thymic epithelium express all the molecular machinery required for a response to these proteins. BMP receptors, BMPRIA and BMPRII, are mainly expressed by cortical thymocytes while BMPRIB is expressed in the majority of the human thymocytes. Some thymic epithelial cells from cortical and medullary areas express BMP receptors, being also cell targets for in vivo BMP2/4 signalling. The treatment with BMP4 of chimeric human-mouse fetal thymic organ cultures seeded with CD34+ human thymic progenitors results in reduced cell recovery and inhibition of the differentiation of human thymocytes from CD4- CD8- to CD4+ CD8+ cell stages. These results support a role for BMP2/4 signalling in human T-cell differentiation.     

The unconventional role of LT alpha beta in T cell differentiation

Lymphotoxin (LT)alphabeta, a member of the tumor necrosis factor cytokine superfamily, and its receptor, the LTbeta receptor (LTbetaR), have a well defined role in secondary lymphoid organogenesis but an unexpected function in T cell differentiation. Although earlier studies indicated that conventional T cell subsets were normal in mice deficient in the LTbetaR pathway, accumulating evidence indicates that the LTalphabeta-LTbetaR pathway has a pivotal role in the ontogeny of unconventional T cells, including gammadelta T cells and invariant natural killer T cells. The LTbetaR pathway seems to operate at distinct levels during thymic development. Double positive thymocytes regulate the differentiation of early thymocyte progenitors and gammadelta T cells through a mechanism dependent on LTbetaR. In addition, LTbetaR signaling in thymic stroma was proposed to affect central tolerance to peripherally restricted antigens. These findings highlight the complex cellular crosstalk between lymphoid and stromal compartments during thymic differentiation.     

Thymic stromal organization is regulated by the specificity of T cell receptor/major histocompatibility complex interactions

The thymic architecture is normally compartmentalized into a central medulla surrounded by a peripheral cortical region. We investigated how compartmentalization of the thymic stroma is regulated using T cell receptor (TCR)-transgenic mouse models. Our studies show that the signals generated by TCR/peptide/major histocompatibility complex interactions regulate thymic stromal cell compartmentalization. In TCR-transgenic mice, normal stromal cell compartmentalization occurs when the transgenic TCR is expressed on a background that does not result in skewing toward either positive or negative selection. In models representing strong positive selection, the thymic stromal elements do not fully organize into a central medulla. Instead, small medullary foci are dispersed throughout the thymus with some regions residing directly under the capsule. The highest degree of disorganization in medullary epithelial regions is observed in TCR-transgenic mice that exhibit negative selection. Although the medullary foci lack central organization, the expression in these regions of CD80, CD86 and CD40, as well as the clustering of dendritic cells, is similar to that observed in medullae of wild-type mice. Thus, the organization of the medulla appears to occur in two stages: (1) small medullary epithelial regions that are dispersed in fetal thymi expand and associate with antigen-presenting cells, and (2) the expanded medullary foci organize into a central medullary compartment. Our data suggest a model in which this second stage of stromal cell organization is increasingly inhibited as the normal balance of TCR-mediated signals is skewed by higher-avidity interactions between thymocytes and antigen-presenting cells.     

Influence of a thymic hormone on cultured epithelial cells of the thymus

Addition of cortisone to primary cultures of mouse thymic stromal cells resulted in increased growth of medullary epithelial cells while cortical epithelial subpopulations were inhibited or destroyed. These adverse effects on cortical cells were ameliorated by the thymic hormone THF. Because thymocytes are also sensitive to both cortisone and THF we sought to eliminate them from the cultures by deoxyguanosine treatment. This treatment differentiated even more strongly between growth of cortical epithelial cells which were inhibited and medullary epithelium that was very strongly stimulated. These effects are discussed in relation to involution of the thymus with aging.     

Thymocyte depletion affects neurotrophin receptor expression in thymic stromal cells

Thymocytes and thymic stromal cells cross-talk in a bidirectional manner within the thymus, thus contributing to the generation of mature T-cells. The thymic stromal cells in the rat express the high- (TrkA, TrkB) and low-affinity (p75NTR) receptors for neurotrophins. In this study we analysed the regulation of TrkA, TrkB and p75NTR expression in the rat thymus by thymocytes. We induced thymocyte apoptosis by administration of corticoids in rats, and then analysed the expression and distribution of these receptors 1, 4 and 10 days later. Thymocyte death was assessed by the activation of caspase-3 in cells undergoing apoptosis. We observed massive thymocyte apoptosis 1 day after injection and, to a lesser extent, after 4 days, which was parallel with a reduction in the density of thymic epithelial cells normally expressing TrkA and p75NTR. Furthermore, TrkA expression was found in cortical thymic epithelial cells, which normally lack this receptor. The expression of TrkB was restricted to a subset of macrophage-dendritic cells, and remained unchanged with treatment. The normal pattern of neurotrophin receptor expression was almost completely restored by day 10. The results demonstrate that the expression of neurotrophin receptors by thymic epithelial cells, but not by macrophage-dendritic cells, is regulated by thymocytes.     

Thymic epithelial cells: working class heroes for T cell development and repertoire selection

The thymus represents an epithelial-mesenchymal tissue, anatomically structured into discrete cortical and medullary regions that contain phenotypically and functionally distinct stromal cells, as well as thymocytes at defined stages of maturation. The stepwise progression of thymocyte development seems to require serial migration through these distinct thymic regions, where interactions with cortical thymic epithelial cell (cTEC) and medullary thymic epithelial cell (mTEC) subsets take place. Recent work on TEC subsets provides insight into T cell development and selection, such as the importance of tumour necrosis factor (TNF) receptor superfamily members in thymus medulla development, and the specialised antigen processing/presentation capacity of the thymic cortex for positive selection. Here, we summarise current knowledge on the development and function of the thymic microenvironment, paying particular attention to the cortical and medullary epithelial compartments.     

Thymic lymphoma cells as a model for complex-formation between thymocytes and thymic medullary epithelial cells

The formation of complexes between thymocytes and thymic stromal elements is known to be involved in T cell differentiation. We have previously described one type of lympho-stromal interaction involving CD4+ CD8+ thymocytes and a medullary epithelial cell line (E-5). In this study we report the potential for complex formation of two different thymic lymphoma cell lines (Ti-6 and RDM-4). Ti-6 cells were shown to adhere to the E-5 cells, while RDM-4 cells were totally incompetent. Phenotypic characterization of these cell lines suggests that immature thymocytes are not capable of forming complexes with medullary epithelium and that a certain level of differentiation is required to do so. Comparison of their phenotypes showed that the possibility of some classical T cell surface markers being the receptor for the E-5 ligand can be dismissed.     

Contact-mediated maturational effects of thymic stromal cells on murine thymocytes in culture.

The effect of direct contact between thymic stromal cells and thymocytes on differentiation markers and functions of the latter was studied. The thymic stromal cells included two epithelial and one fibroblast cell lines, previously described. Murine thymocytes were incubated with confluent monolayers of these cells or their supernatants for 24 hr, using monolayers of non-thymic cells and their supernatants as controls. Then, the thymocytes were tested for changes in expression of several surface antigens [Thy-1, Lyt-1, Lyt-2, L3T4, IL-2-receptor (IL-2R)], spontaneous [3H]thymidine incorporation (STI), lectin-induced proliferative response (PR) and lymphokine (IL-2 and IL-3) production. All three thymic stromal cell lines reduced the expression of Thy-1, Lyt-1 and Lyt-2 significantly. The expression of L3T4 was also reduced in some of the experiments, while IL-2R was not expressed by the thymocytes, neither before nor after the co-culture. The thymic stromal cell lines also increased the spontaneous [3H]thymidine incorporation and lymphokine production by the thymocytes and inhibited their proliferative response to lectins. Under the same experimental conditions, the culture supernatants of the thymic stromal cells and the non-thymic cells did not have any effect on the thymocytes, either when collected and used separately or when used in a co-culture system which allowed thymocyte contact with the medium but not with the stromal cells (Transwell system). These results suggest a specific effect of thymic stromal cells, epithelial as well as fibroblasts, on thymocyte maturation. The effect is mediated by direct cell contact and not by secreted factors.     

The aged thymus shows normal recruitment of lymphohematopoietic progenitors but has defects in thymic epithelial cells

Aging is associated with reduced numbers of all thymocyte sub-populations, including early T-cell progenitors. However, it is unclear if this is due to inadequate recruitment of lymphohematopoietic progenitor cells (LPCs) to the aged thymus or to abnormal development of T cells within the thymus. We found that LPCs from young mice were recruited equally well to the thymi of young or aged mice and that thymic stromal cells (TSCs) from young and old mice expressed similar levels of P-selectin and CCL25, which are believed to mediate recruitment of LPCs to the adult thymus. However, the number of recruited thymocytes in old thymus was markedly reduced after two weeks, indicating that T-cell development or proliferation is defective in the aged thymus. We also found that LPCs from aged and young mice have similar capacities to seed a fetal thymus that was transplanted under the kidney capsule. Thymic epithelial cells (TECs) in aged mice had lower proliferative capacity and higher rate of apoptosis, compared with findings in young animals. In addition, immunofluorescence staining with antibodies to cortical and medullary TECs revealed that aged thymi had a disorganized thymic stromal architecture, combined with reduced cellularity of the medulla, and apoptosis of thymocyte sub-populations in the medullary microenvironment was increased, compared with that in young mice. We conclude that aging does not impair recruitment of LPCs to the thymus, but is characterized by abnormalities in thymic epithelial architecture, especially medullary TEC function that may provide sub-optimal support for thymic development of LPCs.     

Differential processing of self-antigens by subsets of thymic stromal cells

The stromal network of the thymus provides a unique environment that supports the development of mature CD4(+) and CD8(+) T cells expressing a very diverse repertoire of T cell receptors (TCR) with limited reactivity to self-antigens. Thymic cortical epithelial cells (cTECs) are specialized antigen-presenting cells (APCs) that promote the positive selection of developing thymocytes while medullary thymic epithelial cells (mTECs) and thymic dendritic cells (tDCs) induce central tolerance to self-antigens. Recent studies showed that cTECs express a unique set of proteases involved in the generation of self-peptides presented by major-histocompatibility encoded molecules (pMHC) and consequently may express a unique set of pMHC complexes. Conversely, the stromal cells of the medulla developed several mechanisms to mirror as closely as possible the constellation of self-peptides derived from peripheral tissues. Here, we discuss how these different features allow for the development of a highly diverse but poorly self-reactive repertoire of functional T cells.     

Overexpression of lymphotoxin in T cells induces fulminant thymic involution

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.     

The quantitative assessment of MHC II on thymic epithelium: implications in cortical thymocyte development

The dynamics of MHC II expression in various thymic stromal compartments was investigated. By including MHC II in flow cytometry in addition to the cortical CDR1, medullary UEA-1 and pan-epithelial G8.8 markers, thymic stromal compartments were subdivided into at least six different populations. The total level of surface and cytoplasmic MHC II from fresh cortical thymic epithelial cells (cTECs) of normal mouse was as high as MHC II levels in medullary thymic epithelial cells (mTECs). MHC II levels as well as the percentages and cycling status of thymic epithelial cell populations expressing MHC II were not static during post-natal development, suggesting quantitative flexibility in presenting signals to the developing thymocytes. Although there was no evidence found for regulation of surface MHC II levels by TCR or by IFN-gamma, the absence of class II transactivator reduced both the level of MHC II expression and the number of MHC II+ cells. Surprisingly, MHC II molecules were found to form distinct focal aggregates on the surface of cTEC but not mTEC using high-resolution analysis by confocal microscopy. Moreover, these aggregates were formed independent of TCR or TCR-bearing cells in the thymus. These aggregates could potentially generate a functional unit containing a much higher local MHC II concentration to yield a higher avidity interaction. We discuss possible mechanisms for positive selection by weak interactions in the presence of such preformed MHC II aggregate units in cTEC.     

Development,organization and function of the thymic medulla in normal,immunodeficient or autoimmune mice

Thymopoiesis is initiated by the colonisation of the epithelial rudiment with blood-borne hemopoietic precursors. Their subsequent differentiation to the functionally mature T cell subsets is exquisitely linked to sequential interaction with a diverse array of thymic epithelial cells which form discrete microenvironments. The development and organisation of the epithelium, however, is in turn controlled by thymocyte subsets. In particular the medulla organization depends upon activating signals provided by mature thymocytes to epithelial and dendritic cells. These signals are lacking in RelB-deficient mice leading to the disorganization of the corticomedullary junction and abnormal negative selection despite normal thymocyte maturation. This thymic stromal cell architecture phenotype is found in autoimmune diseases suggesting that abnormalities in the establishment of medullary microenvironments might be linked to the development of autoimmunity.     

两种小鼠胸腺基质细胞对胸腺细胞凋亡的不同作用

采用两种体外建系的小鼠胸腺基质细胞（ＴＳＣ）系，即上皮样ＴＳＣ（ＭＴＥＣ１）和树突状ＴＳＣ（ＭＴＳＣ４），观察其对胸腺细胞凋亡的影响。小鼠胸腺细胞在体外培养过程中，可自发地出现细胞凋亡的特征，表现为ＤＮＡ呈梯度断裂片段，细胞经ＦＡＣＳ分析出现亚二倍体ＤＮＡ波峰，以及Ｆｅｕｌｇｅｎ′ｓ染色镜检所见的ＤＮＡ凝聚和断裂。胸腺细胞在与ＴＳＣ共育后，在ＭＴＥＣ１组可见其凋亡过程受到抑制和存活率的增加；在ＭＴＳＣ４组，仅在共育１２至１８小时时，见到胸腺细胞凋亡加强，而其存活率不受影响。结果提示在胸腺细胞发育过程中，其阴性选择作用的主要机制之一的ＰＣＤ过程受不同来源的胸腺基质细胞的调节。     

Phenotype of stromal cell-associated thymocytes in situ is compatible with selection of the T cell repertoire at an "immature" stage of thymic T cell differentiation

Murine thymocytes interacting with cortical macrophages, cortical epithelial cells and medullary dendritic cells in situ express T cell receptors at low to intermediate density. They co-express the lineage markers Lyt-2 and L3T4 and are not enriched for cells expressing high-density interleukin 2 and lymph node homing receptors. Thus, recognition of stromal cells in situ in distinct thymic microenvironments occurs at a common stage of T cell maturation which is phenotypically intermediate between intrathymic precursor cells and mature medullary-type thymocytes. The surface phenotype of dendritic cell-associated thymocytes indicates the presence of thymocytes with a "cortical" phenotype within the antigen-exposed ("nonsterile") phase of T cell maturation in the medulla.     

Cytokine crosstalk for thymic medulla formation

The medullary microenvironment of the thymus plays a crucial role in the establishment of self-tolerance through the deletion of self-reactive thymocytes and the generation of regulatory T cells. Crosstalk or bidirectional signal exchanges between developing thymocytes and medullary thymic epithelial cells (mTECs) contribute to the formation of the thymic medulla. Recent studies have identified the molecules that mediate thymic crosstalk. Tumor necrosis factor superfamily cytokines, including RANKL, CD40L, and lymphotoxin, produced by positively selected thymocytes and lymphoid tissue inducer cells promote the proliferation and differentiation of mTECs. In return, CCR7 ligand chemokines produced by mTECs facilitate the migration of positively selected thymocytes to the medulla. The cytokine crosstalk between developing thymocytes and mTECs nurtures the formation of the thymic medulla and thereby regulates the establishment of self-tolerance.     

The contribution of thymic stromal abnormalities to autoimmune disease

In essence, normal thymus function involves the production of a broad repertoire of αβT cells capable of responding to foreign antigens with low risk of autoreactivity. Thymic epithelial cells are an essential component of the thymic stromal microenvironment, promoting the growth and export of self-tolerant thymocytes. Autoimmune disease, resulting from a loss of self-tolerance, is clinically and genetically complex, and accordingly has many potential etiological origins. However, it is commonly linked to defects in the thymic epithelial microenvironment. The study of autoimmune-linked thymic stromal dysfunction has indisputably advanced our understanding of T cell tolerance; notably, a field-wide paradigm shift occurred when autoimmune regulator (Aire) was found to drive expression of a multitude of peripheral tissue-restricted antigens in medullary thymic epithelial cells. Many other associations with polygenically controlled autoimmune diseases have been reported but are more difficult to definitively dissect. Paradoxically, immunodeficiency and age-related immunosenescence are also linked with increased autoimmunity. Here we discuss the theoretical basis and the evidence gathered thus far to support these associations.     

大鼠抗小鼠胸腺基质细胞单克隆抗体的鉴定

根据小鼠胸腺冰冻切片免疫组织化学染色，将已获得的３０种大鼠抗小鼠胸腺基质细胞单克隆抗体分为８组：１．髓质胸腺基质细胞（ＴＳＣ）阳性，皮质ＴＳＣ阴性；２．髓质部分ＴＳＣ阳性，皮质ＴＳＣ阴性；３．皮、髓交界部分ＴＳＣ阳性；４．皮、髓质部分ＴＳＣ阳性；５．髓质ＴＳＣ阳性，皮质部分ＴＳＣ阳性；６．皮、髓质大部分ＴＳＣ阳性；７．被膜和血管内皮细胞呈阳性；８．髓质ＴＳＣ和皮质胸腺细胞为阳性。用这８组单抗对本室     

Expression of the HPV16E7 oncoprotein by thymic epithelium is accompanied by disrupted T cell maturation and a failure of the thymus to involute with age

Transgenic mice expressing the E7 protein of HPV16 from the keratin 14 promoter demonstrate increasing thymic hypertrophy with age. This hypertrophy is associated with increased absolute numbers of all thymocyte types, and with increased cortical and medullary cellularity. In the thymic medulla, increased compartmentalization of the major thymic stromal cell types and expansion of thymic epithelial cell population is observed. Neither an increased rate of immature thymocyte division nor a decreased rate of immature thymocyte death was able to account for the observed hypertrophy. Thymocytes with reduced levels of expression of CD4 and/or CD8 were more abundant in transgenic (tg) mice and became increasingly more so with age. These thymic SP and DP populations with reduced levels of CD4 and/or CD8 markers had a lower rate of apoptosis in the tg than in the non-tg mice. The rate of export of mature thymocytes to peripheral lymphoid organs was less in tg animals relative to the pool of available mature cells, particularly for the increasingly abundant CD4lo population. We therefore suggest that mature thymocytes that would normally die in the thymus gradually accumulated in E7 transgenic animals, perhaps as a consequence of exposure to a hypertrophied E7-expressing thymic epithelium or to factors secreted by this expanded thymic stromal cell population. The K14E7 transgenic mouse thus provides a unique model to study effects of the thymic epithelial cell compartment on thymus development and involution.