Implantation of cultured thymic fragments in congenitally athymic (nude) rats

Summary Cultured thymic fragments correspond to the thymic microenvironment depleted of lymphocytes and dendritic cells. When these fragments are implanted under the kidney capsule of congenitally athymic rats, lymphocytes and dendritic cells of host origin enter the graft and induce thymus-dependent immunity in the recipient. This paper describes the ultrastructure of the fragments and the changes that occur during the restoration of normal thymic architecture. At the end of the culture period of 6–9 days and in the early stages after implantation, the grafts consist of keratin-containing epithelial cells of unusual morphology that can be labelled with antibodies raised against the epithelium of the mid/deep cortex and the subcapsule/medulla. Normal thymic architecture develops, including nerves and blood vessels, as lymphocytes populate the environment, and by 4–6 weeks the epithelial cells are the same phenotypically and ultrastructurally as those found in normal rat thymus. However, some areas without lymphocytes still contain the atypical epithelial cells seen before implantation. Large multinucleated giant cells are also present with a few associated epithelial cells of subcapsular/medullary phenotype. In conclusion, the cultured thymic fragments contain a hitherto unknown precursor epithelial cell with an atypical ultrastructure and phenotype that is not seen in normal development.     

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

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

Normal thymic cortical epithelial cells developmentally regulate the expression of a B-lineage transformation-associated antigen

Nonlymphoid, stromal cells in the mouse thymus are believed to be important in T cell maturation and have been proposed to play a central role in the acquisition of major histocompatibility complex (MHC) restriction and self-tolerance by maturing thymocytes. Both cortical and medullary epithelial cells in the thymus express high levels of class II (A) major histocompatibility antigens (MHC Ags). We show here that a specific subset of these A^– epithelial cells express a transformation-associated antigen (6C3Ag) found previously on the surfaces of Abelson murine leukemia virus-transformed pre-B cells and on those bone marrow-derived stromal cell clones which support normal and preneoplastic pre-B cell proliferation. Among solid lymphoid organs, only the thymus contains 6C3Ag¹ cells and within the thymus, this antigen is found exclusively on A^– epithelial cells in cortical regions. It is striking that the expression of the 6C3Ag on thymic epithelium is developmentally regulated, suggesting a role for this lymphostromal antigen in the maturation of the thymic microenvironment.     

Ontogenesis of rat immune system: Proteasome expression in different cell populations of the developing thymus

Immune proteasomes in thymus are involved in processing of self-antigens, which are presented by MHC class I molecules for rejection of autoreactive thymocytes in adults and probably in perinatal rats. The distribution of immune proteasome subunits LMP7 and LMP2 in thymic cells have been investigated during rat perinatal ontogenesis. Double immunofluorescent labeling revealed LMP7 and LMP2 in thymic epithelial and dendritic cells, as well as in CD68 positive cells - macrophages, monocytes - at all developmental stages. LMP2 and LMP7 were also detected by flow cytometry in almost all thymic CD90 lymphocytes through pre- and postnatal ontogenesis. Our results demonstrate that the immune proteasomes are expressed in all types of thymic antigen presenting cells during perinatal ontogenesis, suggesting the establishment of the negative selection in the thymus at the end of fetal life. The observation of the immune proteasome expression in T lymphocytes suggests their role in thymocyte differentiation besides antigen processing in thymus.     

Thymic cell populations in amphibia: Quantitative study of the growth,stability, and regression of the cell populations in the thymus of the newtPleurodeles waltlii Michah

Summary Twenty days after fertilization (stage 40) the thymus ofPleurodeles waltlii consists of two main cell types: epithelial reticular cells (71%) and lymphoid stem-cells (24%).Between day 20 and day 72 (stage 53) the lymphoid stem-cells differentiate into lymphocytes, via the lymphoblast state. Commencing at day 20, epithelial reticular cells are transformed into epithelial reticular dense cells. Following day 65, other epithelial reticular cells begin to differentiate into epithelial hypertrophic cells, and these subsequently form thymic cysts. During this whole period intense proliferation takes place.The three types of polynuclear cells (neutrophil, eosinophil, and basophil), the macrophages, and the plasmocytes differentiate outside the thymus then migrate into it through the vascular system.Around day 72 (stage 53), the mature thymus consists of two parts: the first is visible as a background or cortex-like area, the second comprises medulla like spots, formed by small numbers of cysts.Around metamorphosis the cell populations reach a stable state.After metamorphosis the relative frequency of the lymphoid cell population progressively decreases, while the proportion of epithelial hypertrophic cells, together with cyst surface area, is increased. Consequently the ratio of cysts/background area increases with age.     

C-type virus-lymphocyte interactions in developing mouse thymus.

The appearance of C-type virus particles in thymus cells of Swiss mouse embryos, 11.5 to 15.5 days post-conception age (PCA), was studied with the electron microscope. In thymic rudiments of all specimens examined, virus particles were seen in epithelial cytoplasm, budding from epithelial cell surfaces and in extracellular spaces. Lymphoid cells were first seen in thymic rudiments of 13.5 days PCA, and did not display virus particles at this stage. At 14.5 days PCA, thymic lymphocytes had localized plasmalemmal thickenings of high electron-density which were adjacent to extracellular virus particles. Viruses appeared to be penetrating thymic lymphocytes by viropexis in embryos of 15.5 days PCA. At this stage, many lymphocytes also had cytoplasmic virus-containing vesicles and viral buds at their surfaces. These observations suggest the possibility that, in embryos, C-type viruses are transmitted horizontally from thymic epithelium to early populations of thymic lymphocytes.     

The thymus microenvironment in regulating thymocyte differentiation

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

Ontogeny of the thymus in an antarctic teleost,Harpagifer sp. (Notothenioidei: Perciformes)

Summary The development of the thymus was examined in different stages of Harpagifer sp. from Signy Island (South Orkney Islands; 60°43S, 45°38W). The thymus was typical, both in position and structural development, of that observed in warmer-water teleosts. The infiltration of the thymic epithelia was not observed until 4 weeks post-hatch. Full development of the lymphoid organs was not achieved until the juvenile stage. Although an increased infiltration of the thymus, by sub-epithelial connective tissues and epithelial mucous cells, occurred in the juvenile and adult stages, there was no evidence of an advanced stage of thymic regression or involution in the adult Harpagifer. Thus a suppressive influence of the low temperature environment, on the onset and degree of thymic development and involution, was indicated in this species.The signy Island population of Harpagifer has been given the species name H. antarcticus (Prof. J.C. Hureau; personal communication)     

The thymus microenvironment in regulating thymocyte differentiation

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

The fine structure of the thymus of the fetal and neonatal monkey (Macaca mulatta)

Summary The morphologic features of the fetal and neonatal thymus were investigated by light and electron microscopy to determine developmental changes. Primitive epithelial cells differentiate into reticular epithelial cells, medullary epithelial cells, elongated epithelial cells, Hassall's corpuscles and cysts. Thymocytes first appear at 50 days fetal age and the number of thymocytes is amplified from 75–150 days fetal age. Minor differences between the fetal thymus of the monkey and that of other species were observed. Possible functions for the various cellular components of the fetal monkey thymus are discussed.This research was supported in part by grants 5-F3-CA-28, 793-02 and FR-0167 from the National Institutes of Health.     

Thymic microenvironment and cultures derived from mouse thymic explants

Summary Cultures derived from thymus fragments of embryonic (18–19 day old), newborn or one month old C57 BL mice have been characterized functionally (phagocytic and nonspecific esterase activities) and morphologically by means of light, scanning (SEM) and transmission (TEM) electron microscopy. The observations show the heterogeneity of the cell populations composing the monolayers. After a few days incubation macrophages appear as the predominating cell type, while epithelial cells usually constitute no more than 30% of the cells. Experiments designed to determine the fate of lymphocytes adhering to the monolayers lead us to believe (on the basis of SEM morphometric analysis) that the survival of lymphocytes attached either to thymic macrophages or to epithelial cells is improved during the first days of coculture. This survival enhancement does not, however, appear to be a specific inductive effect since a similar survival increase is found when lymphocytes adhere to non-thymic cells. In contrast with the monolayer, the expiant provides a three-dimensional culture system able to preserve intact thymic microenvironmental conditions since numerous lymphocytes are found even in five week old cultures which were not overlaid with thymocytes or spleen cells.     

The human thymic microenvironment: Thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation

Normal T-cell development is dependent on interactions with the thymic microenvironment; thymic epithelial cells are thought to play a key role in the induction of thymocyte maturation, both through direct contact and, indirectly, via thymic hormone secretion. It has been postulated that thymic epithelial cells progress through an antigenically defined pathway of differentiation similar to that of epidermal keratinocytes. As keratins vary according to epithelial cell type and the stage of epithelial cell maturation, we used a panel of monoclonal antibodies against keratins to study specific types of keratin intermediate filaments within human thymic epithelium. The demonstration in human thymus of keratins previously shown to be associated with distinct stages of epidermal keratinocytic maturation would support the hypothesis that thymic epithelial cells undergo sequential stages of differentiation. Two-dimensional immunoblot analysis of cytoskeletal extracts from human thymus revealed that thymic epithelium contains the following keratins: 1-2, 5, 6, 7, 8, 10, 13, 14, 15, 16, and 17 (molecular masses, 65-67, 58, 56, 54, 52, 56.5, 51, 50, 50', 48, and 46 kilodaltons, respectively). Thus, in thymic epithelium, we found keratins previously observed in epidermal basal cells (5, 14, 15), as well as keratins specific for terminally differentiated keratinocytes in supra-basal epidermis (1-2, 10). Indirect immunofluorescence (IF) performed on fetal and postnatal human thymus demonstrated that keratin epitopes recognized by antibodies AE-3, 35 beta H11, and RTE-23 are present on epithelial cells of the subcapsular cortex, the cortex, the medulla, and Hassall's bodies. In contrast, antibodies AE-1 and RTE-22 reacted primarily with neuroendocrine thymic epithelium (subcapsular cortex, medulla, Hassall's bodies). The epithelial reactivity of antibody AE-2 was limited to epithelial cells in Hassall's bodies and did not appear until 16 weeks of fetal gestation i.e., when Hassall's bodies first formed. Two-dimensional gel analysis of thymic keratins demonstrated that antibody AE-2 identified only the keratins with molecular masses of 56.6 and 65-67 kilodaltons (10 and 1-2 respectively) in thymus. These data, together with the selective reactivity of AE-2 with Hassall's bodies in fluorescence assays, demonstrate the localization in Hassall's bodies of the high-molecular-weight keratins associated with the late stages of epidermal cell maturation. In summary, we demonstrated that human thymic epithelium contains specific keratins found in multiple epithelial types as well as keratins associated with both early and late stages of epidermal cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Establishment of functional epithelial cell lines from a rat thymoma and rat thymus

Summary Seven clonal epithelial cell lines from a thymoma of an (ACI/NMs×BUF/Mna)F1 rat and seven clonal epithelial cell lines from an ACI/NMs rat thymus were established in a medium containing 1 μM dexamethasome (DM) and were characterized cytologically. Long-term treatment of DM stabilized the epithelial nature of these epithelial cells irreversibly. The established cell lines showed a polygonal shape, were positively stained with antikeratin antiserum and had tonofilaments and desmosomes. Species of their keratin paptides were the same as those of normal thymic epithelial cells in primary cultures. The cell lines were positively stained with Th-4 monoclonal antibody which preferentially stains the medullary epithelial cells of the thymus, but not with Th-3 which preferentially stains the subcapsular and cortical epithelial cells of the thymus. The cells from the rat thymoma were much large than those from the normal thymus, as reflected in their primary cultures. No transformed phenotypes, such as high growth rate, high saturation density anchorage independency, low serum dependency and so on, were found on the cell lines from the thymoma as in the cell lines from the normal thymus by in vitro assays. DNA synthesis of the thymic lymphocytes was stimulated by culturing with a line of rat thymoma with no lectins. Thymic lymphocytes strongly bound on the cell lines from the thymoma and changed the shape of the cells. These cell lines may be useful to investigate the mechanism of thymomegenesis and the interactions between epithelial cells and thymocytes in the rat thymoma.     

Histological and cytological studies on the developing thymus of mandarin fish Siniperca chuatsi (Perciformes: Teleostei)

The thymus of the mandarin fish, Siniperca chuatsi, was examined by light and transmission electron microscopy to understand its formation and cellular composition. Larvae of the mandarin fish were collected and sectioned from 1 to 35 days post‐hatching (dph). On dph 7 the thymus was packed with lymphocytes. From 12 dph onward, mucous cells were observed on the epithelial layer; from 23 dph, three zones could be differentiated in the thymic parenchyma. The thymus was connected with the extension of the third, fourth and fifth branchial pouches throughout early development, remaining in a superficial position in the adult S. chuatsi. In the thymus of the adult fish, thymic epithelial cells (TECs) characteristic of tonofilaments were observed, with limiting TECs (LECs) found in subcapsular, subseptal, perivascular and nurse‐like TECs containing viable intact lymphocytes inside their vacuoles. In addition, three kinds of granulocytes were observed throughout the thymus, and an incomplete blood–thymus barrier was found in the inner zone. Other cell components such as cystic cells, macrophages and plasma cells, were also described in the thymus of the adult S. chuatsi. The thymus development in mandarin fish agrees, to some extent, with the ontogenetic patterns observed in other fish species.     

Lymphocyte depletion in thymic nurse cells: a tool to identify in situ lympho-epithelial complexes having thymic nurse cell characteristics

Summary In situ pre-existing complexes of epithelial cells and thymocytes having thymic nurse cell characteristics were visualized in the murine thymus cortex using dexamethasone as a potent killer of cortisone-sensitive thymocytes. The degradation and subsequent depletion of cortisone-sensitive thymocytes enclosed within cortical epithelial cells appeared to be paralleled by thymocyte degradation and depletion in thymic nurse cells isolated from thymic tissue fragments from dexamethasone-treated animals. This suggests that thymic nurse cells are derived from pre-existing sealed complexes of cortical epithelial cells and thymocytes. Not all thymocytes situated within in situ epithelial or thymic nurse cells complexes appear to be cortisone-sensitive: a minority of 1–2 thymocytes per complex survives the dexamethasone-treatment, thus constituting a minor subset of cortical cortisone-resistant thymocytes predominantly localized within cortical epithelial cells in situ and within thymic nurse cells derived from such structures. Cortisone resistance in thymocytes thus seems to be acquired within the cortical epithelial cell microenvironment. Cortisone-resistant thymocytes in thymic nurse cells express the phenotype of mature precursors of the T helper lineage, indicating that the in situ correlates of thymic nurse cells may play an important role in T cell maturation and selection.     

斜带石斑鱼胸腺的显微和超微结构

本文通过解剖及组织切片技术、光学显微镜、透射和扫描电子显微镜技术,对斜带石斑鱼(Epinephelus coioides)胸腺器官组织进行了观察研究。结果表明:斜带石斑鱼胸腺实质主要由胸腺细胞(淋巴细胞)和网状上皮细胞构成。鱼体从Ⅰ龄之后,其胸腺发生明显的变化,与幼鱼有所不同,主要是胸腺可明显区分为三个区域:胸腺外皮质区、内皮质区和髓质区。外皮质区主要由网状上皮细胞、黏液细胞、成纤维细胞和少量淋巴细胞构成,细胞排列疏松;内皮质区主要由密集的淋巴细胞和网状上皮细胞组成,以含有大量的淋巴细胞为特征;髓质区主要由淋巴细胞和较多的网状上皮细胞构成,总体特征是淋巴细胞数量比内皮质区的少,且细胞排列较疏松。外皮质区、内皮质区相当于高等脊椎动物的皮质;髓质区相当于高等脊椎动物的髓质。髓质区之下有结缔组织,在Ⅱ龄以上的成体出现胸腺小体(Hassall's corpuscles)或类似胸腺小体的结构,而且随着年龄的增加,胸腺外皮质区增厚,结缔组织增加,还表现在内皮质区和髓质区组织逐渐萎缩变薄,胸腺的细胞组成类型和淋巴细胞数量上有所变化等等。这些现象在Ⅱ龄鱼开始出现,即胸腺呈现退化迹象,在Ⅲ龄以上鱼体呈现明显的退化和萎缩。胸腺表面扫描电镜结果表明:其上皮细胞表面具有微嵴以及由微嵴组成的指纹状结构,有一些微孔分布。透射和断面扫描电镜的结果进一步表明:胸腺组织内的细胞成分复杂,除了淋巴细胞和网状上皮细胞外,还具有巨噬细胞、肥大细胞、肌样细胞、浆细胞、指状镶嵌细胞和纤维细胞等。     

中华鳖胸腺显微和亚显微结构及其在进化上的意义

应用透射电子显微镜观察了中华鳖胸腺的显微和亚显微结构。发现中华鳖胸腺从结构上可分为皮质和髓质,皮质富含淋巴细胞,髓质富含上皮性网状细胞。在各发育期中华鳖胸腺皮质、髓质交界处和髓质区有明显的囊状胸腺小体和交错突细胞。在２００ｇ以上的成年鳖胸腺内发现有同心圆状胸腺小体。电镜下,胸腺内淋巴细胞分为大、中、小３型。上皮性网状细胞从亚显微结构上分为支持型和分泌型。胸腺囊包括细胞内囊和细胞间囊,以细胞内囊居多     

Acid phosphatase activity and cell death in mouse thymus

Summary The distribution of acid phosphatase activity in the thymus of young (8 week) and old (42 week) mice is presented. In 8 week old mice acid phosphatase positive cells represent 1.27±0.13% of the total population whereas in 42 week old mice, showing involution of the thymus, acid phosphatase positive cells represent 2.40±0.17% of the total population. Loci of free acid phosphatase activity have been interpreted as sites of cell lysis and death. This has been confirmed at electron microscope level where free acid phosphatase has been demonstrated in the cytoplasm of lysing thymic lymphocytes. Vacuolar sites of acid phosphatase activity have been demonstrated in macrophages which appear to dispose of the lymphocytes. Extensive autophagic activity occurs in the epithelial reticular cells. The role of acid phosphatase in thymic lymphocyte deletion and in the tissue dynamics of the thymus is discussed.     

Ultrastructure of thymic myoid cells

Mature myoid cells in the parenchyma of reptilian thymus contain all the organelles typical of striated muscle. The presence of both immature and degenerating stages indicates a turnover of myoid cells in the adult thymus. In the earlier stages of differentiation myoid cells resemble thymic epithelial cells. A close parallel exists between developing myoid cells, skeletal muscle differentiating in vitro, rhabdomyoma and rhabdomyosarcoma. Elaborate lattice-like structures are formed by transverse tubules. These structures are compared with similar configurations which have been described in muscle and mitochondrial cristae.