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.     

A Common Stem Cell for Murine Cortical and Medullary Thymic Epithelial Cells?

We have addressed the question whether the epithelial stroma in the thymus is derived from a common stem cell or whether cortical and medullary epithelial cells are derived from different embryonic stem cells emerging, for example, from endoderm and ectoderm. By the use of rapidly expanding cultures of thymic epithelial cells (TEC) from 14 to 16 day-old murine fetuses and by specific antibodies against cortical and medullary epithelium, respectively, we were able to demonstrate a small subpopulation of double-labeled TEC in the cultures. These cells were not present in TEC cultures initiated from thymuses of neonatal mice. Double-labeled TEC were also found in tissue sections from fetal thymuses. These findings may indicate that TEC populations of the cortex and the medulla are derived from a common stem cell, with potential for differentiation toward both cortical and medullary TEC.     

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 .     

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.     

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.     

Comparative ultrastructural characteristics of epithelial cells of the parenchyma of the thymus gland and thymoma

Comparative ultrastructural investigation of the parenchyma was performed for human thymus (7 specimens) and thymoma (34 specimens). For thymic parenchyma eight types of epithelial cells were identified. These may be represented by three different histogenetic lines: 1) spindle-shaped appendiceal cortical cells (type 1-4) 2) medullary cells showing signs of squamous-epithelium differentiation (type 5-6) 3) medullary cells with signs of glandular differentiation (type 7-8). Thymomas demonstrated cells ultrastructurally similar to thymic parenchyma and exhibiting features of either cortical or medullary epithelial cells. These findings served the basis for dividing thymomas into 3 major groups: 1) with cortical cell differentiation 2) with cortical and medullary cell differentiation 3) with medullary cell differentiation.     

Dynamic spatio‐temporal contribution of single β5t+ cortical epithelial precursors to the thymus medulla

Intrathymic T‐cell development is critically dependent on cortical and medullary thymic epithelial cells (TECs). Both epithelial subsets originate during early thymus organogenesis from progenitor cells that express the thymoproteasome subunit β5t, a typical feature of cortical TECs. Using in vivo lineage fate mapping, we demonstrate in mice that β5t⁺ TEC progenitors give rise to the medullary TEC compartment early in life but significantly limit their contribution once the medulla has completely formed. Lineage‐tracing studies at single cell resolution demonstrate for young mice that the postnatal medulla is expanded from individual β5t⁺ cortical progenitors located at the cortico‐medullary junction. These results therefore not only define a developmental window during which the expansion of medulla is efficiently enabled by progenitors resident in the thymic cortex, but also reveal the spatio‐temporal dynamics that control the growth of the thymic medulla.     

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.     

Modest cortex and promiscuous medulla for thymic repertoire formation

Recent advances in the understanding of medullary thymic epithelial cell biology, and especially of their promiscuous gene expression, have highlighted the indispensable role of thymic medulla in shaping a self-tolerant T-cell repertoire. Additionally, our recent results have shown that cortical thymic epithelial cells possess a unique proteasome (the so-called thymoproteasome), which seems to possess limited protein degradation capability for generating peptides that are loaded onto class I major histocompatibility complex molecules. We discuss here the unique role of thymoproteasomes in the development and repertoire formation of CD8+ T cells, focusing on the stepwise and contrasting roles of cortical epithelial cells and medullary epithelial cells. These results could offer fundamental new insights into the molecular mechanisms of T-cell repertoire selection.     

Thymoma and thymic carcinoma

Summary Based on the light microscopical features of normal thymic epithelial cells, human thymoma was divided in different types, namely cortical, medullary, and mixed ones, according to the epithelial cell (EC) type. Lymphoid cell populations with morphological features of either cortical or medullary thymocytes were found according to different types of EC in thymoma. The histological variation of the different types of thymoma are demonstrated. In a retrospective study of 58 thymomas and 13 thymic carcinomas, malignant invasive character as well as the occurrence of myasthenia gravis were both found to be related to the neoplastic proliferation of the cortical epithelial cells, whereas in the usual mixed type of thymoma and the medullary type no gross invasion or metastases were noticed.These results are discussed in view of recent concepts and immunological findings of thymus microarchitecture.This work has been supported by the Deutsche Forschungsgemeinschaft, SFB Project CN 2 and A 8. Part of the data were presented at the 68. Tagung der Deutschen Gesellschaft für PathologieSupported by the Deutscher Akademischer Austauschdienst     

Evaluation of prognostic features in thymic epithelial tumors

Based on the original proposals of Müller-Hermelink [3,8] and the study of 95 tumor specimens from the files of our institute we have established a new concept for the classification of thymic epithelial tumors. Thymomas are related to the structural components of normal thymus and divided in medullary, mixed, predominantly cortical and cortical types. In addition a well-differentiated thymic carcinoma with partial loss of organotypic differentiation is characterized and distinguished from other carcinoma types with total lack of specific thymic features. Prognostic evaluation showed, that medullary and mixed thymomas are always benign tumors, whereas predominantly cortical thymomas, cortical thymomas and well-differentiated thymic carcinomas are low-grade malignant tumors with increasing invasiveness and even metastatic capacity. Moreover the proliferation rate of neoplastic epithelial cells in vitro, which was studied in 12 cases, correlated to the different tumor types and their growth behaviour in vivo.     

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.     

Epithelial cells and macrophages in myasthenia gravis thymus culture

Monolayer culture of thymic nonlymphoid cells derived from female patients with myasthenia gravis (MG) and individuals who underwent heart surgery was established to investigate the cellular composition of the thymic microenvironment and the interaction of nonlymphoid cells with autologous thymocytes. Thymic epithelial cells were identified by immunoperoxidase staining using monoclonal antibodies (mAbs) specific for cytokeratin and MR6 and MR19 antigens expressed on cortical and medullary epithelial cells, respectively. Macrophages were characterized by determination of alpha-naphthyl acetate esterase activity and detection of M1 antigen by mAb. It was demonstrated that in MG thymus cultures the number of cortical MR6+ epithelial cells is significantly reduced, and the ability of the remaining MR6+ cells to bind autologous thymocytes is markedly affected. On the other hand, the number of macrophages and the interaction of those cells with thymocytes were similar in MG and control thymus cultures. Since MR6+ epithelial cells are numerically and functionally affected in MG, maturational events of T cells occurring in the inner cortex may be altered. The mechanisms underlying the induction and expansion of T helper clones in MG are discussed.     

Culture of thymic epithelial cells from mice and age-related studies on the growing cells

Culture of epithelial cells from the thymus of mice was achieved in a medium modified to favor epithelial growth while inhibiting proliferation of fibroblasts. Epithelial cells were identified by the presence of desmosomes in electron microscopic preparations and by antibody to intermediate filaments containing keratin. Morphologically, the cells thus positively identified displayed two main patterns: carpets of large flat cells resembling paving stones which are confluent along the length of their membranes, and networks of cells interconnected by long cytoplasmic processes. These two types of cells were dominant in cultures derived from mice of all ages tested (newborn to nine months) but the relative proportion of each type appeared to change with the age of the donor mice and also with the concentrations of cortisone in the culture medium. Autoradiography revealed that the cultured cells were dividing, and that (in the presence of cortisone) the rate of DNA synthesis was decreased in a portion of the epithelial cells derived from mice in which thymic involution was already underway.     

Epithelial cell proliferation in thymic hyperplasia induced by triiodothyronine.

A significant hyperplasia of the thymus was induced in mice, treated with triiodothyronine during the first month of life. Stereological data showed that, in both treated and control mice, mononucleate epithelial cells were four times more numerous in the medulla that in the cortex. After triiodothyronine treatment, their absolute number in both cortex and medulla increased two-fold. The number of thymic epithelial cells could thus be regulated by thyroid hormones. The cortical volume in treated mice was also twice that of controls but medullar volume showed and increase of only fifty percent. Cortical epithelial cells increased at the same rate of the cortex volume by medullary epithelial cells grew more rapidly. In fact the medullary volume enlargement could be be explained mainly by the growth of the epithelium. Medullary lymphocytes did thus not preliferate in the same way as cortical lymphocytes after thyroid hormone administration. The recently described multinucleate epithelial cells were not modified in number and were thus insensitive to thyroid hormones.     

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.     

Thymic vasculature: organizer of the medullary epithelial compartment?

The epithelial component of the thymic environment is organized into discrete cortical and medullary compartments that mediate different aspects of thymocyte differentiation. The processes controlling the growth and organization of these epithelial compartments are poorly defined. In this study we have used a novel approach to define the three-dimensional organization of thymic epithelial (TE) compartments to demonstrate that the organization of the medullary TE compartment is very complex. A spatial relationship of medullary thymic epithelium with vascular elements of the thymus was demonstrated by simultaneous immunohistochemical labeling of vascular elements and medullary TE. Medullary TE was often arranged as perivascular cuffs surrounding intermediate-sized vessels, but was not associated with either the capillary network or large centrally located vessels. Similar analyses of RAG-2(-/-) thymi revealed a striking physical association of medullary TE with vascular elements. Ultrastructural analysis of the RAG-2(-/-) thymus indicated a preferential association of focal accumulations of medullary TE with post-capillary venules. These data suggest that discrete segments of the thymic vasculature provide cues that act in concert with thymocyte-derived stimuli to effect normal development of the thymic environment.     

Restricted expression of transgenic HLA-DRA gene in thymic epithelial cells and its role in acquisition of T cell tolerance to self-superantigens and processed DRα-derived peptide

We have established a set of transgenic mouse lines in which the HLA-DRA gene was expressed in different cell types. In one line (DRα-24), DRαEβ^b molecules were expressed on thymic medullary and cortical epithelial cells and all lineages of bone marrow-derived antigen-presenting cells (APC) except for thymic macrophages. By contrast, expression of the molecules in another line (DRα-30) was found on thymic medullary and cortical epithelial cells but not on bone marrow-derived APC in the thymus and periphery. To evaluate the role of thymic epithelial cells in acquisition of T cell tolerance, comparative analysis of DRα-24 and DRα-30 was performed. In DRα-30, T cells expressing TcR Vβ5 and Vβ11 were eliminated to comparable levels to those in DRα-24, suggesting that expression of the DRαEβ^b molecules on thymic epithelial cells are sufficient for clonal deletion of the self-superantigen-reactive T cells. In addition, CD4⁺ T cells from DRa-30 as well as those from DRα-24 were tolerant to DRα-derived peptide/I-A^b complex expressed on spleen cells from DRα-24 even in the presence of exogenous interleukin-2. These observations suggest that expression of the DRα chain in thymic epithelial cells could induce T cell tolerance directed toward naturally processed DRα-derived peptide bound to I-A^b molecules, probably via clonal deletion of the self-reactive T cells.     

Thymocytes and RelB-dependent medullary epithelial cells provide growth-promoting and organization signals,respectively, to thymic medullary stromal cells

The thymic medulla is composed of distinct epithelial cell subsets, defined in this report by the reactivity of two novel antibodies, 95 and 29, raised against mouse thymic epithelial cell lines. These antibodies were used to probe the development of medulla in wild-type or mutant thymuses. In CD3σ-deficient mice where thymocyte maturation is arrested at the CD4^? CD8^? stage, few scattered 95⁺ and 29⁺ epithelial cells are found. When few mature thymocytes develop as in CD3-ζ/η mice, expansion and organization of 95⁺ but not 29⁺ cells, becomes detectable. In RelB-deficient mice, T cell maturation proceeds normally but negative selection is inefficient due to the lack of thymic medulla and dendritic cells. Strikingly, 29⁺ epithelial cells are absent and 95⁺ medullary epithelial cells are scattered throughout the thymus, intermingling with CDR1⁺ cortical epithelium. In chimeric mice lacking only dendritic cells, the corticomedullary junction persists and both 95⁺ and 29⁺ epithelial cells are localized in the medulla. These results suggest that two types of signals are required for development of thymic medulla. A growth signal depends upon the presence of maturing thymocytes, but organization of the thymic medulla requires the presence of activated 29⁺ medullary epithelial cells.     

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.