Development of functional thymic epithelial cells occurs independently of lymphostromal interactions

The thymus provides a specialised microenvironment for the development of T-cell precursors. This developmental programme depends upon interactions with stromal cells such as thymic epithelial cells, which provide signals for proliferation, survival and differentiation. In turn, it has been proposed that development of thymic epithelial cells themselves is regulated by signals produced by developing thymocytes. Evidence in support of this symbiotic relationship, termed thymic crosstalk, comes from studies analysing the thymus of adult mice harbouring blocks at specific stages of thymocyte development, where it is difficult to separate mechanisms regulating the initial development of thymic epithelial cells from those regulating their maintenance. To distinguish between these processes, we have analysed the initial developmental programme of thymic epithelial cells within the embryonic thymus, in either the presence or absence of normal T-cell development. We show that keratin 5+8+ precursor epithelial cells present in the early thymic rudiment differentiate into discrete cortical and medullary epithelial subsets displaying normal gene expression profiles, and acquire functional competence, independently of signals from T-cell precursors. Thus, our findings redefine current models of thymus development and argue against a role for thymocyte-epithelial cell crosstalk in the development of thymic epithelial progenitors.     

Automated technique to evaluate thymocyte adhesion to thymic epithelial cells

Thymocyte adhesion to thymic epithelial cells is a relevant issue during intrathymic T-cell differentiation, and directly intervenes in the generation and expansion of the T-cell repertoire. In view of these data, it was apparent the usefulness of an automated strategy to evaluate the degree of thymic epithelial cell-thymocyte adhesion. This prompted us to develop an ELISA procedure (using an anti-Thy1 reagent) to determine the degree of thymocyte adhesion onto cultured thymic epithelial cells. The procedure described herein is simple, non-radioactive and reproducible. Additionally, it can potentially be applied to quantitate the degree of thymocyte adhesion to any cellular or non-cellular substrate (for example, extracellular matrix). Moreover, it detected fluctuations of thymocyte adhesion secondary to glucocorticoid treatment of epithelial cells. Thus, it can be regarded as a further tool to analyze intrathymic interactions.     

胸腺髓质上皮细胞干细胞对胸腺发育的影响

来源于胸腺双能祖细胞的胸腺髓质上皮细胞干细胞(medullary thymic epithelial cell stem cells, mTECSCs),在胸腺发育过程中的胚胎早期阶段既可能发生分化,又可能在胎儿出生后维持胸腺髓质上皮细胞(medullary thymic epithelial cells, mTECs)的再生,充分发挥组织干细胞的功能,确保终身外周血中央型记忆T细胞自身耐受。mTECSCs对胸腺的发育起着重要的作用和影响,得到很多学者的关注。现就mTECSCs对胸腺功能、生理性退化及胸腺衰老和免疫稳态等发育过程中的作用和影响作一概述,并探讨其在胸腺相关疾病治疗方面的潜在价值。     

Suspension of thymic emigration promotes the maintenance of antigen-specific memory T cells and the recall responses

Thymic involution is evolutionarily conserved and occurs early in life. However, the physiological significance remains elusive of this seemingly detrimental process. The present study investigated the potential impact of altered thymic output on T cell memory using ovalbumin (OVA) expressed by Listeria monocytogenes as a model antigen. Suspension of thymic emigration by thymectomy was shown to lead to a marked increase in the frequency and total number of OVA-specific memory T cells. In contrast, oversupply of thymic emigrants through thymic grafting caused a significant decrease of such cells. When rechallenged with L. monocytogenes expressing OVA, the thymectomized mice mounted a more potent recall response as evidenced by the enlarged population of OVA-specific tetramer⁺ cells and the accelerated clearance of the bacteria. Notably, the memory-enhancing effect of thymectomy was abrogated following weekly adoptive transfer of naive T cells. Together, data from the present study indicate that reduced thymic output favors the maintenance of the memory T cell pool.     

Antigen receptor regulation of phosphoinositide-dependent kinase 1 pathways during thymocyte development

Phosphoinositide-dependent kinase 1 (PDK1) is essential for T cell development but little is know about the stimuli that regulate PDK1 signaling in vivo. The thymus contains a heterogeneous mixture of cells at different stages of development making it difficult to use biochemical techniques to examine the activity of PDK1 pathways as thymocytes develop in situ. Herein, we use a single cell assay to quantify activation of the PDK1 target kinase ribosomal S6 kinase 1 (S6K1) in different murine thymocyte subsets immediately ex vivo. This technique allows an assessment of S6K1 activation as thymocytes respond to developmental stimuli in vivo. These studies reveal that only a small percentage of thymocytes show evidence for activation of PDK1 mediated signaling in situ. The thymic subpopulations that contain active PDK1/S6K1 are those known to be responding to signaling by the pre T cell receptor and the mature alpha/beta T cell antigen receptor (TCR). Moreover, loss of antigen receptor signaling in T cell progenitors that cannot rearrange their TCR beta locus prevents in vivo activation of S6K1. The present data identifying antigen receptor signaling as a key activator of PDK1 mediated signaling afford a molecular explanation for the important role of this molecule in T cells.     

Immunohistochemical characterization of the thymic microenvironment

Summary The epithelial framework of the human thymus has been studied in parallel by immunohistochemical methods at the light- and electron-microscopic levels. Different monoclonal antibodies were used, reacting with components of the major histocompatibility complex, keratins, thymic hormones and other as yet antigenically undefined substances, which show specific immunoreactivities with human thymus epithelial cells.The electron-microscopic immunocytochemical observations clearly confirm microtopographical differences of epithelial cells not only between the thymic cortex and medulla, but also within the cortex itself. At least four subtypes of epithelial cells could be distinguished: 1) the cortical surface epithelium; 2) the main cortical epithelial cells and thymic nurse cells; 3) the medullary epithelial cells; and 4) the epithelial cells of Hassall's corpuscles.The various epithelial cell types of the thymus display several common features like tonofilaments, desmosomes and some surface antigens as demonstrated by anti-KiM3. In other respects, however, they differ from each other. The cortical subtype of thymic epithelial cells including the thymic nurse cells shows a distinct pattern of surface antigens reacting positively with antibodies against HLA-DR (anti-HLA-DR) and anti-21A62E. Electron-microscopic immunocytochemistry with these antibodies clearly reveals a surface labeling and a narrow contact to cortical thymocytes particularly in the peripheral cortical regions. An alternative staining pattern is realized by antibodies to some antigens associated with other subtypes of thymic epithelial cells. Medullary epithelial cells as well as the cortical surface epithelium react likewise positively with antibodies to special surface antigens (anti-Ep-1), to special epitopes of cytokeratin (anti-IV/82), and to thymic hormones (anti-FTS). The functional significance of distinct microenvironments within the thymus provided by different epithelial cells is discussed in view of the maturation of T-precursor cells.Glossary of Abbreviations Anti-X anti-X antibody - APUD-cells amine precursor uptake and decarboxylation (gastro-intestinal endocrine cells) - DAB diamino-benzidine - DMSO dimethyl sulfoxide - FTS facteur thymique sérique - HLA-A, B, C human leucocyte antigen, A, B, C-region related - HLA-DR human leucocyte antigen, D-region related - IDC interdigitating cell - MHC major histocompatibility gene complex - PBS phosphate-buffered saline - TNC thymic nurse cell This investigation was supported by grants from the Deutsche Forschungsgemeinschaft, and its Sonderforschungsbereich 111Fellow of the Alexander von Humbold-Stiftung, Institute of Pathology, University of Würzburg, Federal Republic of GermanyThe authors appreciate the contribution of human thymus tissue from Professor Alexander Bernhard, Abteilung kardiovasculäre Chirurgie der Universität Kiel; the gift of monoclonal antibodies from Dr. M.J.D. Anderson, Dr. M. Dardenne and Dr. H.J. Radzun; and the excellent technical assistence of Mrs. O.M. Bracker, Mrs. H. Hansen, Mrs. R. Köpke, Mrs. M. v. Kolszynski, Mrs. J. Quitzau, Mrs. H. Siebke, and Mrs. H. Waluk     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed. Project supported by the National Natural Science Foundation of China (Grant No. 39230320).     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

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Differential gene expression in CD3e- and RAG1-deficient thymuses: definition of a set of genes potentially involved in thymocyte maturation

 A set of 3000 mouse thymus cDNAs was analyzed by extensive measurement of expression using complex-probe hybridization of DNA arrays ("quantitative differential screening"). The complex probes were initially prepared using total thymus RNA isolated from C57BL/6 wild-type (WT), CD3e- and RAG1-deficient mice. Over 100 clones displaying over- or under-expression by at least a factor of two between WT and knockout (KO) thymuses were further analyzed by measuring hybridization signatures with probes from a wide range of KO thymuses, cell types, organs, and embryonic thymuses. A restricted set of clones was selected by virtue of their expression spectra (modulation in KO thymuses and thymocytes, lymphoid cell specificity, and differential expression during embryonic thymus development), sequenced at one extremity, and compared to sequences in databases. Clones corresponding to previously identified genes (e.g., Tcrβ, Tcf1 or CD25) showed expression patterns that were consistent with existing data. Ten distinct clones corresponding to new genes were subjected to further study: Northern blot hybridization, in situ hybridization on thymus sections, and partial or complete mRNA sequence determination. Among these genes, we report a new serine peptidase highly expressed in cortical epithelial cells that we have named thymus-specific serine peptidase (TSSP), and an acidic protein expressed in thymocytes and of unknown function that we have named thymus-expressed acidic protein (TEAP). This approach identifies new molecules likely to be involved in thymocyte differentiation and function. Received: 3 June 1999 / Revised: 3 August 1999     

Regulation of chromatin structure during thymic T cell development

Development is the process whereby a multipotent cell gives rise, through series of divisions, to progeny with successively restricted potentials. During T cell development, the process begins with a multipotent hematopoietic stem cell (HSC) in the bone marrow, moves to the thymus where early T cells or thymocytes pass through signal‐initiated developmental checkpoints, and ends in the periphery where mature T cells reside. At each step along this developmental pathway, T lymphocyte progenitors must be able to turn genes on and off, creating a specialized program of gene expression, to allow further development. How is gene expression coordinated? This review will summarize what has been learned about the function of chromatin structure in generating a “blueprint” of gene expression during T cell development. This will include discussion of mechanisms of chromatin remodeling, histone modification, and heritable gene silencing. In many cases, these processes are carried out by multi‐protein complexes whose components are largely ubiquitously expressed. The spatial and temporal specificity of these complexes is contributed by sequence specific DNA binding factors, some of which are cell type restricted in their expression. This review will summarize research underway to identify these key genetic “targeters.” Taken together, the research reviewed here provides a glimpse into the importance of regulation of chromatin structure in T cell development and the “players” involved. © 2005 Wiley‐Liss, Inc.     

An immuno-electron-microscopic study of human thymic B cells

Thymic B cells are a constituent of normal human thymic medulla. They are supposed to play a role in T cell maturation. Thymic B cells have been characterized morphologically and immunohistochemically at the light-microscopic level. Their ultrastructural appearance in vivo has not been demonstrated. Six normal infantile thymi were immunolabelled with the pan-B cell marker CD20 using a pre-embedding technique and viewed at the electron-microscopic level. Cells expressing CD20 had long cytoplasmic processes. They were all ”asteroid” in shape and in close contact with thymocytes. Also, their long cytoplasmic processes intermingled with cytoplasmic processes of cells that were presumed to be interdigitating reticulum cells (IDC) based on morphological criteria. Thymic B cells may act in concert with IDC during T cell maturation. Received: 20 October 1995 / Accepted: 10 January 1996     

Isolation,Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

The identification of thymic nurse cells in vivo and the role of cytoskeletal proteins in thymocyte internalization

Much debate has been generated about the existence of thymic nurse cells within the thymus. Until now, the authenticity of an epithelial cell capable of internalizing developing thymocytes within the thymic cortex has been in question. Here, we use the thymic nurse cell-specific monoclonal antibody, ph91, to define the in vivo location of thymic nurse cells. For the first time, thymic nurse cells enclosing several thymocytes were detected in the subcapsular region of the thymic cortex in a “honeycomb-like” configuration. In vitro studies show the internalization process using digitalized time-lapse microscopy. Internalized thymocytes have also been reported to interact with macrophages within the TNC complex. The cytoplasmic interaction between thymocytes and macrophages was detected using time-lapse microscopy. Using fluorescence microscopy, we show polymerization of actin within macrophages at the contact point with thymocytes, which is indicative of an immunological synapse. Microfilaments and microtubules within TNCs were shown to be associated with thymocyte binding and internalization, but neither interacted with macrophages. Also, we provide data to show that thymocytes are actively involved in the internalization process. These experiments show for the first time the existence of thymic nurse cells within the thymic microenvironment. They provide a visual documentation of thymocyte uptake by thymic nurse cells, and define an interaction between thymocytes and macrophages within the TNC complex.     

基因打靶突变小鼠的早期T细胞发育

早期Ｔ细胞的发育是一个受到多种分子精确调控的过程,基因打靶技术的建立和发展 内研究上述分子的作用提供了有效的手段。对ＴＣＲ、ＣＤ３基因打靶小鼠的研究表明,ＣＤ４４－ＣＤ２５阶段是早期Ｔ细胞发育的重要调控点,在此发育阶段,由ＴＣＲβ、ＴＣＲα和ＣＤ３成分组成的ｐｒｅ－ＴＣＲ复俣体的表达或其与未知配体的结合通过ｐ５６ｌｃｋ传递信号,介导ＣＤ４４－ＣＤ２５细胞的进一上发育,该复全体任何成分的缺失都将使Ｔ     

Age-related hyperplasia of the thymus and T-cell system in the Buffalo rat

This report describes the development of hyperplasia of both the thymus and the peripheral T-cell system with advancing age in the Buffalo rat. Buffalo/Mna rats do not show age-related thymic involution, but rather develop thymic hyperplasia with advancing age. This thymic growth is expansile and there is no infiltration of the surrounding tissues. Because the enlarging thymus occupies the thoracic cavity, most of the rats die of respiratory failure by the age of 24 months. Thymic enlargement is due to primary hyperplasia of cortical epithelial cells and the large number of proliferating lymphocytes. The hyperplastic epithelial cells are bizarre in shape and strongly positive when stained with Th-3 monoclonal antibody (MoAb), anti-thymosin antibody and anti-EGF antibody, but negative with Th-4 MoAb. The patterns of distribution of CD-5+, CD-4+ and CD-8+ lymphocytes within the hyperplastic thymus are similar to those seen in young rats of other species. The high level of T-cell emigration from the thymus to the periphery appears to persist throughout life, since the percentage of normal splenic T-cells also increase with advancing age and exceed 70% of the total by 24 months of age. This thymic enlargement with abnormal hyperplasia of cortical epithelial cells can be prevented by hypophysectomy.     

Histochemical and molecular overview of the thymus as site for T-cells development

The thymus represents the primary site for T cell lymphopoiesis, providing a coordinated set for critical factors to induce and support lineage commitment, differentiation and survival of thymus-seeding cells. One irrefutable fact is that the presence of non-lymphoid cells through the thymic parenchyma serves to provide coordinated migration and differentiation of T lymphocytes. Moreover, the link between foetal development and normal anatomy has been stressed in this review. Regarding thymic embryology, its epithelium is derived from the embryonic endodermal layer, with possible contributions from the ectoderm. A series of differentiating steps is essential, each of which must be completed in order to provide the optimum environment for thymic development and function. The second part of this article is focused on thymic T-cell development and differentiation, which is a stepwise process, mediated by a variety of stromal cells in different regions of the organ. It depends strongly on the thymic microenvironment, a cellular network formed by epithelial cells, macrophages, dendritic cells and fibroblasts, that provide the combination of cellular interactions, cytokines and chemokines to induce thymocyte precursors for the generation of functional T cells. The mediators of this process are not well defined but it has been demonstrated that some interactions are under neuroendocrine control. Moreover, some studies pointed out that reciprocal signals from developing T cells also are essential for establishment and maintenance of the thymic microenvironment. Finally, we have also highlighted the heterogeneity of the lymphoid, non-lymphoid components and the multi-phasic steps of thymic differentiation. In conclusion, this review contributes to an understanding of the complex mechanisms in which the foetal and postnatal thymus is involved. This could be a prerequisite for developing new therapies specifically aimed to overcome immunological defects, linked or not-linked to aging.     

Phenotype and topography of human thymic B cells

Using single and double labeling immunohistochemical techniques and a large panel of monoclonal antibodies against B-cell differentiation antigens, including those newly defined at the Fourth International Leucocyte Typing Workshop, we have examined the immunophenotype and tissue distribution of human thymic B-cells. The existence of a distinct B-cell population as a constant constituent of the thymic microenvironment has been noted only recently. We found a singificant population of B-lymphocytes in the thymic medulla expressing the B-cell restricted antigens CD19, CD20, CD22, CD37, CD72, CD76 and IgM and IgD. As with other extrafollicular B-lymphocytes, they differ significantly from both follicle mantle and germinal center cells in morphology and immunophenotype, which points to alternative modes of B-cell differentiation. Thymic B-cells themselves show considerable heterogeneity and a subpopulation with dendritic features and the expression of CD23 has been referred to as “asteroid” cells. Their close association with T-cells and medullary epithelial cells points to a functional role for B-cells in the thymus. A second population of B-lymphocytes together with frequent lymph follicles is found within the extrathymic perviascular space. Though separated from the medulla by a layer of epithelial cells, a clear distinction between the B-cells of these two compartments is not always possible. The intramedullary B-cell compartment shows a parallel numeric increase with the occurrence of germinal centers in the perivascular space, mostly due to an accumulation of B-cells in the medulla adjacent to these lymph follicles. Thus a close relationship between the intra-and extramedullary B-cell population of the thymus seems likely. Presented in part in Leucocyte Typing IV (1989) Knapp W et al. (eds) Oxford University Press, Oxford, pp 221–222