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.     

Human intrathymic T cell differentiation

The human thymus develops early on in fetal gestation with morphologic maturity reached by the beginning of the second trimester. Endodermal epithelial tissue from the third pharyngeal pouch gives rise to TE3+ cortical thymic epithelium while ectodermal epithelial tissue from the third pharyngeal cleft invaginates and splits during development to give rise to A2B5/TE4+ medullary and subcapsular cortical thymic epithelium. Fetal liver CD7+ T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression on thymocytes of other T lineage surface molecules. Human thymic epithelial cells grown in vitro bind to mature and immature thymocytes via CD2 and CD11a/CD18 (LFA-1) molecules on thymocytes and by CD58 (LFA-3) and CD54 (ICAM-1) molecules on thymic epithelial cells. Thymic epithelial cells produce numerous cytokines including IL1, IL6, G-CSF, M-CSF, and GM-CSF--molecules that likely are important in various stages of thymocyte activation and differentiation. Thymocytes can be activated via several cell surface molecules including CD2, CD3/TCR, and CD28 molecules. Finally, CD7+ CD4-CD8- CD3- thymocytes give rise to T cells of both the TCRab+ and TCR gd+ lineages.     

Thymic epithelial cells: antigen presenting cells that regulate T cell repertoire and tolerance development

During thymocyte development bone marrow-derived precursors in the thymus undergo a series of differentiation steps to produce self-tolerant, mature T lymphocytes. The thymus contains two functionally distinct anatomical compartments, consisting of a centrally located medulla surrounded by the thymic cortex. These compartments in turn are comprised of two major cellular components: (1) the T lymphoid compartment of developing thymocytes and (2) the thymic stroma consisting mainly of thymic epithelial cells (TECs). These epithelial cells are further separated into cortical and medullary TECs (cTECs and mTECs) based on their localization within the thymic cortex or medulla respectively. Reciprocal interactions between thymocytes and epithelial cells are required for the development of both cellular components into a functional thymic organ. Thymocytes provide trophic factors for the development of a complex three-dimensional epithelial cell network, while epithelial cells regulate T cell development through expression and presentation of self-antigens on major histocompatibility molecules. Our work focuses on how thymic epithelial cells regulate T cell development and function and on elucidating the mechanisms of thymic epithelial cell differentiation. Here we review current knowledge and provide our own insight into the development, differentiation and antigen presenting properties of TECs. We focus specifically on how mTECs regulate T cell repertoire selection and central tolerance.     

Human epithelial thymic tumours: heterogeneity in immunostaining of epithelial cell markers and thymic hormones.

Different hormones (thymulin, thymosin alpha 1, vasopressin), antigenic markers of cortical and subcapsular/medullary thymic areas and tumour associated antigens were studied on paraffin or frozen section and cultures of human epithelial thymic tumours ('thymomas'). Thymulin, thymosin alpha 1 and for the first time vasopressin are found in most tumours. The epithelial cells of five 'thymomas' had markers of both cortical (TE3) and subcapsular/medullary thymic regions (A2B5 and/or TE4 and/or anti-p19). Leu-7, a marker of subcapsular epithelial cells was positive only in two tumours. The histological classification into cortical and medullary tumours does not correspond to our immunofluorescence results. The presence of these markers does not support the theory of different embryologic origin of the cortical and subcapsular/medullary epithelial cells. Transferrin receptors were detected on only some epithelial cells of thymic 'carcinomas'. Adenocarcinoma related antigen and carcino embryonic antigen only stained a few epithelial cells of all the tumours. There is no expected correlation between the presence of epidermal growth factor receptors on cell membranes and the number of proliferative cells stained by the anti-Ki67 antibodies. Immunostainings were heterogeneous according to the epithelial thymic tumours, independent of histological classification and not yet useful for prognosis.     

The intrathymic microenvironment: expression of lectin receptors and lectin-like molecules of differentiation antigens and MHC gene products

The expression of MHC products, differentiation antigens and lectin receptors has been investigated in the various cell types populating different compartments of the thymus. The ultrastructural classification of suspended thymic epithelial cells was facilitated by using a technique that preserves cortical nursing cells or medullary epithelial cell clusters. A subset of peanut lectin positive lymphocytes could be distinguished by their ability to bind soybean lectin also. This subset corresponds to the large proliferating lymphocytes that populate the area between the thymic capsule and the cortex. Ia and H-2 D/K antigens could be detected on nearly all epithelial and lymphoid cells. Expression of H-2 antigens, however, is more pronounced on medullary epithelial cells. T-cell differentiation antigens such as Thy-1 and Lyt-1 could be demonstrated not only on lymphocytes, but, interestingly enough, on cortical epithelial cells as well. These latter cells, in addition, exhibit a cell membrane-bound lectin with a specificity for D-galactose which might well be the structure responsible for binding the galactosyl residues of the peanut lectin receptor of thymic lymphocytes. Binding sites for a large set of lectins could be demonstrated on both, thymic lymphocytes and epithelium. The intrathymic differentiation pathway of T-lineage cells is discussed with regard to those lymphocytic and epithelial cell surface structures considered to enable cellular interaction.     

Cytoskeletal proteins in thymic epithelial cells of the Australian lungfish Neoceratodus forsteri

The vertebrate thymus consists of distinctive subpopulations of epithelial cells that contain a diverse repertoire of cytoskeletal proteins. In this study of the thymus in the Australian lungfish, Neoceratodus forsteri , immunohistochemistry was used to distinguish the cytoskeletal proteins present in each class of thymic epithelial cell. A panel of antibodies (Abs), each specific for a different cytoskeletal polypeptide (keratins, vimentin, desmin, actin and tubulins), was used on paraffin and ultrathin resin sections of thymus. Ab AE I (reactive against human type I cytokeratins (CK) 14, 16 and 19) selectively stained the cytoplasm of capsular, trabecular and the outermost epithelial cells of Hassall's corpuscles. Anti-CK 10 Abs strongly labelled the capsular epithelial cells and less than 20% of cortical and medullary epithelial cells. The anti-50-kDa desmin Ab did not react with any thymic cells, whereas the anti-53-kDa desmin Ab labelled some capsular, cortical and medullary thymic epithelial cells. The anti-vimentin Ab stained most of the capsular and ~60% of the cortical epithelium. Thymic nurse cells and Hassall's corpuscles were found to be devoid of actin, which was strongly detected in medullary and perivascular epithelium. Both α and β tubulins were detected in all thymic cells. This study extends the concept of thymic epithelial heterogeneity. The complexity of thymic epithelium in N. forsteri may indicate a relationship between thymic epithelial subpopulations and the thymic microenvironment. These data identify anti-keratin Abs as a valuable tool for studying differentiation and ontogeny of the thymic epithelium in N. forsteri .     

Immunohistochemical and in situ hybridization detection of growth-hormone-producing cells in human thymoma.

We have studied 25 thymomas by both immunohistochemistry and in situ hybridization for the presence of growth hormone (GH)-producing cells. Our results indicate that 1) GH-immunoreactive cells were present in 13 of 17 thymomas of cortical and predominantly cortical type but not in medullary (spindled) thymomas (n = 3) or low- to high-grade thymic carcinomas (n = 5), 2) GH-positive cells were mainly located at the periphery of the neoplastic lobules, at the periphery of the perivascular spaces and in the areas of medullary differentiation, 3) cells containing GH mRNA appeared at locations similar to those of GH-immunoreactive cells, and 4) GH-immunoreactive material was present only in the epithelial cell component as revealed by immunoelectron microscopy. In conclusion, this paper demonstrates the occurrence of GH-producing cells in noncarcinoid thymic tumors. The relevance of GH in thymoma cell biology requires additional investigations.     

Expression of cortical and medullary thymic epithelial antigens in thymomas. An immunohistological study of 14 cases including a characterization of the lymphocytic compartment

Four monoclonal antibodies against antigens expressed differentially by the normal thymus epithelium, which define the cortical, medullary and subcapsular compartments, were used for immunohistological characterization of the epithelial cells in 14 thymomas. Furthermore, thymoma-associated lymphocytes were studied with monoclonal antibodies directed against T-lymphocyte differentiation antigens (CD1a, CD3, T-cell antigen receptor). Only four of the 14 thymomas could be classified into either medullary or cortical type thymoma based on the immunophenotype of epithelial cells. Ten cases escaped immunophenotypical classification due to co-expression of medullary and cortical antigens by the tumour cells. This aberration from the normal phenotype might indicate the failure of differentiation of such tumours. The immunophenotype of the associated lymphocytes, on the other hand, made it possible to classify the tumours as cortical (5 cases), mixed (2) and medullary (3) thymomas. Four thymomas escaped this classification scheme due to the absence of lymphocytes (2) or to a hybrid immunophenotype (2). Nevertheless, thymocytes of cortical type clearly predominated and were seen in all thymomas with associated lymphocytes. This feature may constitute a good diagnostic tool in differential diagnosis since, in 28 mediastinal or extramediastinal metastasis of tumours not derived from thymic epithelium and associated with various numbers of lymphocytes, none of them were found to contain CD1a positive lymphocytes.     

Activation of cortical and inhibited differentiation of medullary epithelial cells in the thymus of lymphotoxin-beta receptor-deficient mice: an ultrastructural study

The reciprocal influences of thymic lymphocyte and nonlymphocyte populations, i.e. thymic cross-talk, are necessary for the proper maturation of thymocytes and the development/maintenance of thymic stromal microenvironments. Although the molecular influences exerted by thymic stromal cells on maturing thymocytes have been extensively studied, the identity of signalling molecules used by thymocytes to influence the thymic stromal cells is still largely unknown. Our study provides the first ultrastructural evidence that the functional lymphotoxin-beta receptor (LTbetaR) signalling pathway is engaged in the cross-talk between thymocytes and the thymic stromal cell population. We show that LTbetaR signalling is of the utmost significance for the preservation of the subcellular integrity of all thymic epithelial cells. In the absence of LTbetaR there is (1) hypertrophy and activation of cortical thymic epithelial cells, (2) the complete loss of fully differentiated medullary thymic epithelial cells, and (3) the inhibited differentiation of remaining medullary thymic epithelial cells with the appearance of prominent intercellular cysts in the thymic medulla.     

Histopatholgical spectrum of thymic neoplasms: twelve-year experience at a referral hospital in north India

This study was undertaken to determine the histopathological spectrum and clinical profile of thymic neoplasms at a tertiary referral care centre. A total of 96 thymectomy specimens were received during the study period (1992-2004), which consisted of 54 neoplasms and 42 benign lesions. Among the neoplasms there were 48 thymic epithelial tumors, 3 thymolipomas and 3 thymic carcinoids. The former comprised of 36 male (75%) and 12 female patients (25%) ranging in age from 2-70 years (mean 37 years). Among paraneoplastic syndromes in thymic epithelial tumours, 27 out of 48 (56.25%) cases were associated with myasthenia gravis and one case was associated with pure red cell aplasia. The most frequent histological subtype was cortical thymoma (43.24%) followed by predominantly cortical (24.32%) and well-differentiated thymic carcinoma (18.92%). On staging, all cases of mixed and predominantly cortical subtype were stage 1 whereas one medullary and 2 cortical thymomas and 4 well differentiated thymic carcinoma (WDTC) showed pleural and pericardial invasion (stage III). This study has revealed that half of thymic epithelial tumours presented as myasthenia gravis. The cortical thymoma was the most frequently encountered histologic subtype and most commonly associated with myasthenia gravis.     

Effects of cyclosporin A on mouse thymus: immunochemical and ultrastructural studies.

The in vivo effect of cyclosporin A (CsA) on the murine thymus was investigated by studying the ultrastructural cellular alterations, which are described for the first time, and the immunohistochemical modifications of thymocytes and thymic reticulo-epithelial cells (TREC). A marked reduction of the thymus size became apparent after 6 days of CsA treatment (10 mg/kg/day). Light microscopy studies using polyclonal antibodies (Ab) or monoclonal Ab specific to lymphoid sub-populations (anti-CD4, anti-CD5, anti-CD8), and specific to epithelial cells (anti-keratin: AKs, K8), to cortical TREC (TR4) and to subcapsular/medullary TREC (TR5, 3H9) showed that the number of CD4- 8+ or CD4+ 8- medullary thymocytes dramatically decreased and that the cortical TREC are affected, after 10 days of CsA treatment. These observations were confirmed by electron microscopy studies demonstrated that the medullary lymphoid population disappeared almost entirely. TREC, principally cortical type, presented signs of cellular lysis. A few cortical thymocytes showed some damage. At day 8 the medullary thymic tissue was disorganized, but no change was noted in the subcapsular area right up to the final day (day 10) of CsA treatment. These results suggest that CsA has a harmful effect on cortical TREC, which affects the development of immature thymocytes.     

Human thymic epithelium and T cell development: current issues and future directions

The human thymus develops early in fetal gestation with morphologic maturity reached by the beginning of the second trimester. TE3+ cortical thymic epithelium is most likely derived from endodermal third pharyngeal pouch, while A2B5/TE4+ medullary and subcapsular cortical thymic epithelium is likely derived from third pharyngeal cleft ectoderm. Fetal liver and yolk sac CD7+, CD4-, CD8-, surface(s) CD3- T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression of other T lineage surface molecules on developing thymocytes. CD7+, CD4-, CD8-, sCD3- thymocytes give rise to T cells of both the TCR alpha beta and TCR gamma delta lineages. Human thymic epithelial cells produce numerous cytokines including IL1, IL6, TGF alpha, leukemia inhibitory factor (LIF), M-CSF, G-CSF and GM-CSF- molecules that likely play important roles in multiple stages of thymocyte selection, activation and differentiation. Important areas for future research on human thymic epithelium include study of lymphoid and non-lineage differentiation potentials of CD7+, CD4-, CD8-, sCD3- T cell precursors in response to TE-cell produced cytokines, study of the triggering signals of cytokine release within the thymic microenvironment, and study of TCR-MHC mediated TE-thymocyte interactions.     

Monoclonal antibodies to stromal cell types of the mouse thymus

Seven hybridoma cell lines secreting monoclonal antibodies (mAb) to nonlymphoid cells of the mouse thymus have been prepared. These mAb clearly demonstrate the heterogeneity of the thymic stroma. Based on their anatomical distribution patterns observed with the immunoperoxidase technique on frozen tissue sections, they were subdivided into four groups. The first group of mAb, ER-TR1, 2 and 3, detects antigens encoded for by the I region of the major histocompatibility complex. These antigens are expressed on both stromal and lymphoid cells in lymphoid organs. mAb of the second category, ER-TR4, react with epithelial cells in the thymic cortex. mAb of the third group detect stromal cells of the thymic medulla. One antibody of this group, ER-TR5, exclusively reacts with medullary epithelial cells. ER-TR6, the other antibody of this group, reacts with medullary interdigitating cells and macrophages. The fourth type of antibodies, ER-TR7, detects the reticular fibroblasts of the thymus. The possible role of the thymic cell types detected by the present antibodies in T cell differentiation is discussed.     

Thymic epithelial cells of severe combined immunodeficiency (SCID) mice.

To characterize thymic epithelial cells of SCID (severe combined immunodeficiency) mice in comparison with those of Balb C mice, we did an immunohistochemical study using cortical and medullary epithelial cell specific monoclonal antibodies (MoAbs), Th-3 and Th-4, as well as gel electrophoresis and immunoblotting. The thymi of SCID mice were composed of epithelial cells and a few lymphocytes. Most epithelial cells were immunostained diffusely with Th-3, which indicated that they might be "cortical-type" epithelial cells. There were a few clusters of stellate cells with dendritic processes which were negative with Th-3 but stained strongly with Th-4. Cortical type epithelial cells and most of the Th-4 reacting cells were strongly immunostained with cytokeratin antibody MNF116. By immunoblotting, cytokeratin polypeptides No. 10 and 18 were detected in both SCID and Balb C mice; however, the relative amounts of each cytokeratin polypeptides were different. With immunohistochemical and immunoblotting results, we conclude; 1) Th-3 and Th-4 are reliable markers for cortical and medullary thymic epithelial cells in SCID mice; 2) disorganization of cells thymic structure is mostly due to maldevelopment of medullary epithelial and T lymphocytes; and 3) the composition of cytokeratin subfamilies of SCID mice thymi may represent a phenotypic marker of the maldevelopment of medullary epithelial cells.     

Thymic microenvironment reconstitution after postnatal human thymus transplantation

A functional thymus develops after cultured thymus tissue is transplanted into subjects with complete DiGeorge anomaly. To gain insight into how the process occurs, 7 post-transplantation thymus biopsy tissues were evaluated. In 5 of 7 biopsies, the thymus appeared to be predominantly cortex with thymocytes expressing cortical markers. Unexpectedly, the epithelium expressed both cortical [cortical dendritic reticulum antigen 2 (CDR2)] and medullary [cytokeratin (CK) 14] markers. Early medullary development was suggested by epithelial cell adhesion molecule (EpCAM) reactivity in small areas of biopsies. Two other biopsies had distinct mature cortex and medulla with normal restriction of CK14 to the medulla and subcapsular cortex, and of CDR2 to cortex. These data are consistent with a model in which thymic epithelium contains CK14+ "progenitor epithelial cells". After transplantation these cells proliferate as CK14+CDR2+ thymic epithelial cells that are associated with cortical thymocytes. Later these cells differentiate into distinct cortical and medullary epithelia.     

The predominant lymphocyte in most thymomas and in nonneoplastic thymus from patients with myasthenia gravis is the cortical thymocyte

Cell suspensions prepared from 12 specimens of nonneoplastic thymus (6 normal and 6 from patients with myasthenia gravis) and from 17 thymomas were investigated with a panel of monoclonal antibodies. The great preponderance of thymocytes from the 12 nonneoplastic specimens and from 13 of the 17 thymomas (2 of 3 predominantly lymphocytic tumors and 11 of 12 mixed tumors) displayed the surface phenotype of cortical or common thymocytes. These cells formed rosettes with unsensitized sheep erythrocytes (E-rosettes) at both 4 and 37 degrees C, and reacted with the following monoclonal antibodies: OKT1 (thymic and peripheral T cells), OKT6 (common thymocytes), OKT10 (replicating lymphoid cells), OKT11 (sheep cell receptor), and both OKT4 (inducer-helper T cells) and OKT8 (cytotoxic-suppressor T cells). Few B cells (lymphocytes with either immunoglobulin or Ia-like antigen on the cell surface), and few cells with receptors for transferrin and interleukin 2 were detected. Thymocytes from 3 of the 4 remaining thymomas (2 predominantly epithelial tumors and 1 mixed tumor) displayed surface marker characteristics of medullary thymocytes or peripheral T cells; i.e., they were reactive with OKT1, OKT3 (peripheral T cells), OKT11, and either OKT4 or OKT8, and were also E-rosette positive only at 4 degrees C and TdT negative. Thymocytes from the final tumor, a lymphocytic thymoma, exhibited an intermediate phenotype. Thus, almost all mixed (11 of 12) and lymphocytic (2 of 3) thymomas were composed predominantly of cortical thymocytes, while the medullary cell was the rule in the two tumors that were predominantly epithelial.     

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