Phenotypic and functional characterization of human thymic stromal cell lines.

To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.     

Proliferation of murine thymic lymphocytes in vitro is mediated by the concanavalin A-induced release of a lymphokine (costimulator).

Mitogen-induced proliferation of lymphocytes may in theory result directly from the interaction of mitogen with the cells, or indirectly as a result of the mitogen-stimulated release of lymphokines. In the case of murine thymic lymphocytes exposed to concanavalin A (Con A) in tissue culture, we have determined that mitogenesis depends upon a lymphokine. Interaction of the thymic lymphocytes with lectin is necessary, but not sufficient, for mitogenesis. A lymphokine, or costimulator for mitogenesis, is released by normal spleen or thymus cells during the first 16 hr of their exposure to Con A, and in the presence of a phytomitogen it stimulates thymic mitogenesis. Under conditions of low costimulator levels, no mitogenesis follows the interaction of Con A with cells. The response of adult CBA/J mouse thymocytes to phytohemagglutinin (PHA) is very low, compared to their response to Con A. When costimulator is added to PHA, the cells respond as well as they do to Con A. Costimulator does not act through Con A-binding sites on thymus cells. Its production is dependent on both cells carrying omega surface antigen (T lymphocytes) and adherent cells of the macrophage-monocyte series. The adherent population, but not the T cells, may be heavily irradiated without affecting production of costimulator. Costimulator is not a mitogen on its own.     

Quantitative study of regression and regeneration of the thymic cell population after X-irradiation in the newtPleurodeles waltlii Michah

Summary The changes in cell numbers of different thymic cell populations and the conditions governing the regeneration of these populations and the thymus itself were examined after X-irradiation (700 rads) of different parts of the body. The general effects of the irradiation were studied in each experimental group in terms of mortality and growth rate. The particular effects on each thymic cell population were studied by the measurement of mitotic activity and of evaluation of the changes in numbers among these populations in the thymus itself, and were compared with the effects in the granulopoietic layer of the liver and in the spleen. The great reduction in the number of lymphocytes after irradiation demonstrates that they are more radiosensitive than other cell types; this reduction can be compensated for by the arrival of new lymphoid cells originating from other lymphoid organs (if they have been protected from irradiation) and by allowing thymic regeneration. Thus, irradiation has indirect effects on non-irradiated areas, and demonstrates that the lymphoid cell population has a high potential for multidirectional migration.     

Interferons mediate terminal differentiation of human cortical thymic epithelial cells

In the thymus, epithelial cells comprise a heterogeneous population required for the generation of functional T lymphocytes, suggesting that thymic epithelium disruption by viruses may compromise T-cell lymphopoiesis in this organ. In a previous report, we demonstrated that in vitro, measles virus induced differentiation of cortical thymic epithelial cells as characterized by (i) cell growth arrest, (ii) morphological and phenotypic changes, and (iii) apoptotis as a final step of this process. In the present report, we have analyzed the mechanisms involved. First, measles virus-induced differentiation of thymic epithelial cells is shown to be strictly dependent on beta interferon (IFN-beta) secretion. In addition, transfection with double-stranded RNA, a common intermediate of replication for a broad spectrum of viruses, is reported to similarly mediate thymic epithelial cell differentiation through IFN-beta induction. Finally, we demonstrated that recombinant IFN-alpha, IFN-beta, or IFN-gamma was sufficient to induce differentiation and apoptosis of uninfected thymic epithelial cells. These observations suggested that interferon secretion by either infected cells or activated leukocytes, such as plasmacytoid dendritic cells or lymphocytes, may induce thymic epithelium disruption in a pathological context. Thus, we have identified a new mechanism that may contribute to thymic atrophy and altered T-cell lymphopoiesis associated with many infections.     

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。     

Characterization of T lymphocyte colony-forming cells in the mouse. The colony-forming cell is confined to Lyt-1,2,3+ cell subsets in the thymus and the lymph nodes

When cell populations from the thymus were studied with FACS, it was found consistently that the brightly labeled Thy-1.2+ populations contained very few T colony-forming cells (CFC), while these latter cells were numerous in the cell populations showing lower Thy-1.2 antigen density. This was paralleled by findings after peanut agglutinin (PNA) separation that showed enrichment of CFC in the PNA-negative medullary population, and by sorting based on TL, T-200, and H-2 determinants or light scatter properties of the cells. By FACS sorting of Lyt-labeled thymic cells, it was also shown that CFC were predominantly present in cell populations that were brightly Lyt-1+, and exclusively in populations that were Lyt-2+ and Lyt-3+. After FACS sorting of lymph node cells, no major differences in colony formation were found between dully- and brightly-labeled Thy-1.2+ or Lyt-1+ populations, or between lymphoid cells showing different light scatter characteristics. In addition, it was shown that CFC--like thymic CFC--were of the Lyt-1,2,3+ phenotype. It is concluded that the CFC may be present in several differentiation steps of Lyt-1,2,3+ cell lines, and that the frequency of these cells increases from the thymic cortex via the medulla and to peripheral lymphoid tissues.     

The stem cell antigens Sca-1 and Sca-2 subdivide thymic and peripheral T lymphocytes into unique subsets

Stem cell Ag 1 and 2 (Sca-1 and Sca-2), so named due to their expression by mouse bone marrow stem cells, were evaluated for expression by populations of cells within the thymus. Immunohistochemical analysis demonstrated that Sca-1 was expressed by cells in the thymic medulla and by some subcapsular blast cells, as well as by the thymic blood vessels and capsule. Sca-2 expression, which was limited to the thymic cortex, could be associated with large cycling thymic blast cells. Both Sca-1 and Sca-2 were expressed on a sub-population of CD4-CD8- thymocytes, and this subpopulation was entirely contained within the Ly-1lo progenitor fraction of cells. Sca-1 expression by a phenotypically mature subset of CD4+CD8- thymocytes was also noted. Conversely, Sca-2 expression was observed on a phenotypically immature or nonmature subpopulation of CD4-CD8- thymocytes. MEL-14, an antibody that defines functional expression of a lymphocyte homing molecule, identified a small population of thymocytes that contained all four major thymic subsets. Sca-2 split the MEL-14hi thymocyte subset into two Sca-2+ non-mature/immature phenotype fractions and two Sca-2- mature phenotype fractions. In peripheral lymphoid organs, Sca-1 identified a sub-population of mature T lymphocytes that is predominantly CD4+CD8-, in agreement with the thymic distribution of Sca-1. Peripheral T cells of the CD4-CD8+ phenotype were predominantly Sca-1-. In contrast, Sca-2 did not appear to stain peripheral T lymphocytes, but recognized only a subset of B lymphocytes which could be localized by immunohistochemistry to germinal centers. Thus, expression of Sca-1 is observed throughout T cell ontogeny, whereas Sca-2 is expressed by some subsets of thymocytes, including at least one half of thymic blasts, but not by mature peripheral T lymphocytes.     

Acid phosphatase cytochemistry of mitogen-transformed normal and chronic lymphocytic leukemia lymphocytes

Acid phosphatase cytochemistry was performed on lymphocytes stimulated in vitro with phytohemagglutinin, pokeweed mitogen, or concanavalin A. These electron microscopic studies demonstrated that activated lymphocytes from both normals and patients with chronic lymphocytic leukemia (CLL) had an increased number of lysosomes relative to resting cells. At the time of maximum thymidine incorporation, a reduced number of lysosomes was present in many transformed CLL lymphocytes, mainly medium-sized blast cells, in comparison to transformed normal cells. The findings demonstrate a lysosomal abnormality in phytomitogen transformed CLL lymphocytes which may be related to functional defects of these cells or to an incomplete transformation of a residual population of normal lymphocytes.     

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.     

Thymic epithelium in vitro. III. Cytokine production by a thymic epithelial subset

We have previously shown that two populations of thymic epithelium can be separated in culture on the basis of their differential growth rates and their adherence to the culture substratum, and maintained as long-term, morphologically distinct cell cultures, TECs and TECL. We have also described the effects of supernatants from the small epithelial cell (TESs) on the proliferative responses of thymocytes cocultured with mitogen and TESs over a 72-hr period. We now describe the effects in thymic epithelial supernatants (TESL) of soluble factors produced by TECL (the large epithelial cell) on thymocytes costimulated with mitogen and compare their effects to those derived from TECs. Both TESL and TESs suppress optimally stimulated thymocytes and enhance the proliferative responses of suboptimally stimulated thymocytes over a 72-hr period. The suppressive activities produced by TECL and TECs appear distinct, based upon markedly different molecular weights, but have similar sensitivities to heat treatment. The enhancing activities are of similar molecular weight, but have different sensitivities to heat treatment. In addition, TECL synthesize four- to fivefold less PGE2 than TECs. These results provide additional distinctions between the two cell types, and taken in conjunction with data on the anatomic distribution of similar cells, suggest that although they have similar functional effects in vitro, they may prove to have separable roles in vivo.     

Measles Virus Infection Induces Terminal Differentiation of Human Thymic Epithelial Cells

Measles virus infection induces a profound immunosuppression that may lead to serious secondary infections and mortality. In this report, we show that the human cortical thymic epithelial cell line is highly susceptible to measles virus infection in vitro, resulting in infectious viral particle production and syncytium formation. Measles virus inhibits thymic epithelial cell growth and induces an arrest in the G₀/G₁ phases of the cell cycle. Moreover, we show that measles virus induces a progressive thymic epithelial cell differentiation process: attached measles virus-infected epithelial cells correspond to an intermediate state of differentiation while floating cells, recovered from cell culture supernatants, are fully differentiated. Measles virus-induced thymic epithelial cell differentiation is characterized by morphological and phenotypic changes. Measles virus-infected attached cells present fusiform and stellate shapes followed by a loss of cell-cell contacts and a shift from low- to high-molecular-weight keratin expression. Measles virus infection induces thymic epithelial cell apoptosis in terminally differentiated cells, revealed by the condensation and degradation of DNA in measles virus-infected floating thymic epithelial cells. Because thymic epithelial cells are required for the generation of immunocompetent T lymphocytes, our results suggest that measles virus-induced terminal differentiation of thymic epithelial cells may contribute to immunosuppression, particularly in children, in whom the thymic microenvironment is of critical importance for the development and maturation of a functional immune system.     

Thymic accessory cell complexes in vitro and in vivo: morphological study

Summary Murine thymic macrophages and interdigitating cells, also called thymic accessory cells, were characterized by means of light- and electron microscopy. The cells were studied in suspension, during isolation by enzymatic digestion and in vivo. They were observed as isolated cells or as components of multicellular complexes, some of which were rosettes and were composed of lymphoid cells centered on each type of accessory cell. We also noted other cell complexes including macrophages that resembled classical epithelial nurse cells. We consider that multicellular complexes represent lymphostromal associations already existing in vivo, because we observed them at the periphery of thymic pieces undergoing enzymatic treatment. The heterogeneity of macrophages that we observed in vitro was also noted in vivo. In vivo macrophages were of three types: classical phagocytic cells distributed throughout the gland, cortical elongated cells in close contact with lymphoid blast cells, and atypical nurse cells containing mitotic cells and located in the inner cortex. The morphological aspects of the latter two cell types suggest that cortical macrophages in vivo have other roles: they can be interpreted as images of positive or negative cell selection. We also believe that rosettes are formed by elongated cortical macrophages when they are enzymatically isolated from the thymus.Part of this work was presented at the Second Thymus Workshop, Rolduc, The Netherlands, April 1989     

Relationship between structure and T-lymphocyte maturation in human thymomas. Enzyme histochemical and immunohistological studies

Twenty-six human thymomas were studied in an attempt to correlate their morphological appearance with the type and degree of T-lymphocyte maturation, as determined by acid alpha-naphthyl-acetate esterase (ANAE) activity and immunological analysis. Four normal human thymuses were used for purposes of comparison. Two morphological patterns were identified in the thymomas. The distinction was based largely on similarities between the neoplastic epithelial cells and normal cortical and medullary epithelial cells, and on the relative proportions of epithelial cells and lymphocytes. By these criteria "medullary" and "cortical" patterns were identified. In several thymomas both patterns were present in the same tumor ("mixed-type pattern"), producing alternating dark cortical-like areas and lighter foci of medullary differentiation. A good correlation was found between the two patterns and the phenotype of the T-associated lymphoid component. ANAE activity, which was completely lacking in normal cortical thymocytes, was almost absent in the phenotypically immature T-cells of cortical-type thymomas. By contrast, in the medullary-type thymomas, T-cells showed immunological features in common with medullary thymocytes. This was characterized by strong ANAE activity in the majority of cells with a staining pattern corresponding to that of peripheral T-lymphocytes. In addition, most of the proliferating epithelial cells in medullary-type thymomas stained strongly with anti-cytokeratin and anti-epidermal-type keratin antisera. In the mixed-type thymomas the epithelial cell morphology and the immunohistochemical and enzymic features of the T-cells were found to be closely related to the respective cortical--or medullary-like areas. It was concluded that the various characteristics of normal thymic cortex and medulla studied are also present in thymomas. In particular, in medullar-type thymomas the presence of many of the features of normal thymic medulla, such as a squamous cell component, macrophages and interdigitating reticulum cells, may constitute a microenvironment which operates actively in T-cell education. This may account for the functional activities, characteristic of peripheral T-lymphocytes, which T-lymphocytes attain in these thymomas.     

Study of dipeptidyl peptidase IV as a surface marker of human natural killer cells

The enriched populations of natural killer (NK) cells have been obtained using two different approaches. The first one was based on the separation in a density gradient of Percoll and further formation of rosettes with sheep erythrocytes. This method allowed to isolate the population containing approximately 70% of CD16(+)-cells. The second approach consisted in separating lymphocytes on a flow cytofluorometer FACS II. Using this method the population with 80% CD16(+)-cells was isolated from PBMC. Studies on morphological, phenotypical and functional characteristics of the first population revealed that NK cells constituted 70% of total number of cells; T-lymphocytes, 8-lymphocytes and monocytes constituted the minor population (10%, 8% and 1% respectively). Activity of DPIV was determined on both cell populations obtained. As it was shown, approximately 27% of the cells isolated using percoll density gradient and 22% of the cells after the separation on a flow cytofluorometer carried the enzyme molecules on the cell surface. The results of the present study apparently indicate that part of NK cells (about 10%) is characterised by the presence of DPIV on the cell surface.     

Mouse thymic virus infection: ultrastructural and immunocytochemical studies of infected thymus cells.

Only limited attention has been paid to the cell population that is affected in the course of Mouse Thymic Virus (MTV) infection. In the present study, thymic cells of newborn mice infected with MTV were examined for general ultrastructural and immunocytochemical characteristics. The earliest sign of infection was detected 5 days after inoculation. Lymphocytes, epithelial reticular cells, macrophages, and lymphoepithelial cell complexes (thymic nurse cells) were affected. Viral particles and filamentous structures were present in both the nucleus and the cytoplasm of these cells. At more advanced stages of cellular necrosis, 6 to 7 days post-infection, cytoplasmic granulation and loss in definition of cytoplasmic organelles became apparent. This was followed by nuclear degradation and cellular aggregation. The selective effect of MTV on lymphocyte subpopulations was also observed. Two populations of infected lymphocytes were identified by single and double immunogold labelling employing monoclonal antibodies and different sizes of gold particles. CD4+8+ and CD4+8- lymphocytes were found to be selectively lysed by MTV.     

Cellular basis of B cell clonal populations in old mice.

Previous studies from this laboratory have shown that >85% of old mice have stable B cell clonal populations detectable by Ig heavy chain complementary-determining region 3 mRNA size analysis and confirmed by sequence analysis. B cells from the same clone are frequently detected in several lymphoid compartments of the same mouse. We now report the phenotype of all ten stable B cell clonal populations detected in five 20-month-old C57BL/6 mice. These clonal B cells appear to develop in the periphery and nine of the ten B cell clonal populations expressed the CD5 cell surface marker. Stable B cell expansions may be dominated by cells at two stages of differentiation. Some B cell populations were detected with DNA as well as RNA and represent large clonal populations of B cells, detectable in several lymphoid compartments. These populations are found predominantly in B cell populations expressing CD45R/B220 and the mRNA coding for the membrane-bound form of the mu Ig heavy chain, which suggests a predominance of B lymphocytes in these populations. In other cases, smaller clonal populations were detected only in splenic RNA samples. These clonal populations were found predominantly among CD45R/B220- B cells and did not express the membrane-bound form of the micro Ig heavy chain. We offer the hypothesis that the B cell clonal populations present in old mice may be precursors of the two types of B cell neoplasms which are dominated by CD5+ B cells (B cell chronic lymphocytic leukemia) or plasma cells (multiple myeloma).     

Macrophages and epithelial cells of the thymus gland. An ultrastructural study in the natterjack, Bufo calamita

In the present study, the ultrastructure of the stromal components, basically epithelial elements and macrophages, of the thymus of adult natterjacks, Bufo calamita has been analyzed. A network of stellate epithelial-reticular cells joined together by desmosomes, constitutes the main component of the thymic parenchyma in both cortex and medulla. In the medulla pale, electron-lucent epithelial cells, sometimes showing surface interdigitations, are striking elements. Moreover, uni- and multicellular epithelial cysts appear in the thymic medulla as well as granulated cells of possible endocrine significance. Remarkably, isolated or grouped gland cells whose morphology and cytoplasmic content resemble that of the skin glands, were occasionally found. Finally, macrophages, multinucleated giant cells and dendritic-like cells, the latter intimately associated to lymphocytes, occur in the thymus of Bufo calamita. The most remarkable morphologic characteristics of all those non-lymphoid cell types, as well as their possible functional significance are comparatively discussed with available information on the amphibian and higher vertebrate thymic cytoarchitecture.     

Studies of the immunological capacity of germ-free mouse radiation chimeras. IV. Cell-mediated immunity

The cell-mediated immune (CMI) response of germ-free mouse radiation chimeras was compared with that of conventional mice. Spleen or thymus cells from chimeric or normal mice were injected intravenously into lethally irradiated, allogeneic hosts. Spleens of the irradiated hosts were assayed for effector cells using the ⁵¹Cr release assay. Spleen cells from syngeneic and allogeneic chimeras and normal mice were equally active in giving rise to effector cells. However, thymus cells from allogeneic chimeras were completely inactive within 9 months post-bone marrow transplant while thymus cells from syngeneic chimeras and normal mice still remained functional. Although allogeneic chimeras contain cells potentially reactive toward host antigens, cells cytotoxic to host antigens were not detectable. In addition, these studies indicate the helper cell and effector cell, both associated with T-derived lymphocytes, represent two different populations.     

Separation and characterization of human T G and T M lymphocyte populations

During the last few years a number of experimental evidences have shown the presence of Fc receptors for IgG or IgM on the membrane of human T cells. These two different receptors are detectable and mutually exclusive on distinct cell populations named respectively TG, TM and T "null" (which lack detectable receptors). Studies on the functional activities of these cells have shown that TM and TG lymphocytes play an antitetical role in regulating B cell response, TM exerting an "helper" activity on the differentiation of B lymphocytes while TG having a "suppressor" one. The aim of this study has been to determine the values of these two subpopulations in a group of twenty control subjects. Our results have shown that TG constitute 10%, whereas TM represent 40% of the total T cells. After EA-G rosetting, the purification of this subpopulation on a density gradient has shown an enrichment of more than 90% in TG cells, while TM contaminate this fraction for less than 4%. The purity of the fraction containing TM has been evaluated using the localization of alpha-naphthyl acetate esterase activity, which has shown that more than 88% of the cells in this fraction are positive for this enzyme.