Lymphocyte subpopulation in neoplastic and non-neoplastic thymus and in blood of patients with Myasthenia gravis.

The lymphocyte subpopulations in the thymus and in the blood were investigated in ten myasthenic patients who had been thymectomized. Histologically, the thymuses tested comprised three cases of thymoma including two cases with malignant characteristics, five cases of hyperplastic thymus with lymph follicles and germinal centres, and two cases of persistent thymus without lymph follicles. Virtually all lymphoid cells in the three thymomas formed spontaneous rosettes with sheep red blood cells as did normal thymocytes from non-myasthenic patients. There was no significant proportion of immunoglobulin Ig-bearing lymphocytes. While the majority consisted of cells forming spontaneous rosettes with sheep red blood cells, there was a certain proportion (2-17%) of Ig-bearing lymphocytes in four of five hyperplastic thymuses, in one of two persistent thymuses, and in a residual atrophic thymus of a thymoma. The myasthenic patients tested were for the most part normal, as compared with healthy individuals, in the proportion of rosette-forming lymphocytes and Ig-bearing lymphocytes in the blood collected immediately before and one to three months after thymectomy. The presence of Ig-bearing lymphocytes in the thymus was not necessarily related to the appearance of circulating antibody to striated muscle. The antibody to striated muscle was demonstrated in all myasthenic patients with thymoma.     

Immunopathological study related to myoglobin in myasthenic and non-myasthenic thymuses

Twelve biopsied thymuses taken from 4 cases with myasthenia gravis (MG group) and 8 cases without myasthenia gravis (control group) including 2 thymoma cases in each group were immunopathologically investigated in relation to myoglobin (Mb). Mb positive cells of various degrees were detected in all thymuses of both groups, and immunoelectron microscopical examination disclosed that Mb positive cells corresponded to interdigitating reticulum cells and myoid cells in non-neoplastic thymuses, and neoplastic epithelial reticular cells in thymomas. Anti-Mb antibody staining by direct immunoperoxidase technique revealed positive localization to the lymphoid cells in the thymuses of 2 cases of MG group with thymoma. In addition, indirect immunofluorescent study with the serum of each case which was applied to the normal human skeletal muscle, showed positive staining of the sarcoplasm in 3 cases of MG group, including 2 thymoma cases, and using peroxidase labeled serum IgG F (ab')2 of the same patients this anti-muscle antibodies were proved to be against both postsynaptic cytosol and sarcoplasm of the extraocular muscle of the guinea pig. From these results, it was suggested that Mb may conduct itself as a homologous antigen between the thymus and the skeletal muscle in the myasthenic patient with thymoma, and in the thymus the interdigitating reticulum cell, the myoid cell, or the neoplastic epithelial reticular cell may retain or produce Mb as an antigen-presenting cell.     

IMMUNOPATHOLOGICAL STUDY RELATED TO MYOGLOBIN IN MYASTHENIC AND NON-MYASTHENIC THYMUSES

Twelve biopsied thymuses taken from 4 cases with myasthenia gravis (MG group) and 8 cases without myasthenia gravis (control group) including 2 thymoma cases in each group were immunopathologically investgated in relation to myoglobin (Mb). Mb positive cells of various degrees were detected in all thymuses of both groups, and immunoelectron microscopical examination disclosed that Mb positive cells corresponded to interdigitating reticulum cells and myoid cells in non-neoplastic thymuses, and neoplastic epithelial reticular cells in thymomas. Anti-Mb antibody staining by direct immunoperoxidase technique revealed positive localization to the lymphoid cells in the thymuses of 2 cases of MG group with thymoma. In addition, indirect immunofluorescent study with the serum of each case which was applied to the normal human skeletal muscle, showed positive staining of the sarcoplasm in 3 cases of MG group, including 2 thymoma cases, and using peroxidase labeled serum IgG F(ab')₂ of the same patients this anti-muscle antibodies were proved to be against both postsynaptic cytosol and sarcoplasm of the extraocular muscle of the guinea pig. From these results, it was suggested that Mb may conduct itself as a homologous antigen between the thymus and the skeletal muscle in the myasthenic patient with thymoma, and in the thymus the interdigitating reticulum cell, the myoid cell, or the neoplastic epithelial reticular cell may retain or produce Mb as an antigen-presenting cell.     

Subpopulations of lymphocytes in human thymomas.

Lymphocyte populations in six normal thymuses and ten thymomas were examined. The majority of lymphocytes from both thymus and thymoma differ from peripheral T lymphocytes in their capacity to form E-rosettes resistant to incubation at 37 degrees C. Low percentages of T lymphocytes bearing receptors for the Fc portion of IgG (TG) and IgM (TM) were found in normal thymus. In contrast, lymphocytes from five out of nine thymomas showed remarkable percentages of TM cells. Compared with normal thymocytes, lymphocytes from seven out of ten thymomas responded vigorously to mitogens. The possible origin and nature of thymoma lymphocytes are discussed.     

Surface immunoglobulin on cultured foetal mouse thymocytes

Organ cultures of 14–15 day foetal mouse thymus were used as a source of non-neoplastic differentiating T cells, free of contaminating B cells. Viable cells obtained from such cultured thymuses were radio-iodinated and immunoglobulins (Ig) were isolated by co-precipitation from the ¹²⁵I-labelled cell-surface proteins released during 1 h of incubation at 37°. The precipitates, both reduced and unreduced, were then analysed by polyacrylamide gel electrophoresis. The unreduced material migrated in a 5% gel as a single peak with a mobility slightly faster than that of mouse IgG. After reduction, however, two peaks were obtained (in a 10% gel), one corresponding in migration to mouse light chain and the other which moved slightly faster than mouse μ chain. This pattern was identical with that previously seen for both surface Ig of normal mouse thymocytes and neoplastic T lymphoma cells. Uncultured, 15 day foetal thymocytes did not produce any detectable co-precipitated cell surface material. Ig detected in these experiments was therefore produced during in vitro culture by non-neoplastic T cells in a system free of contaminating B cells and mouse serum proteins.     

新生儿胸腺的透射电镜观察

应用透射电镜观察了11例人新生儿胸腺的超微结构,其中基本正常胸腺5例,萎缩胸腺6例.结果显示正常新生儿胸腺小叶已基本完好形成,皮、髓质分界清楚,胸腺外皮质区胸腺细胞分裂增殖旺盛;胸腺实质上皮细胞至少可分为四种类型,上皮细胞与胸腺细胞紧密直接接触,两者共生发育.萎缩胸腺见巨噬细胞吞噬大量坏死的胸腺细胞,重度萎缩胸腺基本结构紊乱,上皮细胞变性、崩解,上皮网架受损.     

Microscopic thymoma: histological evidence of multifocal cortical and medullary origin

Twenty cases of macroscopically non-neoplastic thymuses obtained from patients with myasthenia gravis have been studied histologically. Seven cases were characterized by lymphoid follicular hyperplasia and 13 by involutional changes of variable degree. In three cases (15%), one with lymphoid follicular hyperplasia and two with involutional changes, multiple microscopic epithelial lesions, 0.2-0.4 mm in diameter and consistent with foci of microscopic thymoma, were observed. Most of them were related to the thymic cortex and one, displaying a different histological pattern, was located in a medullary area. These observations provide morphological evidence of a possible multifocal origin of thymoma from distinct epithelial clones present in the different topographic areas of the human thymus.     

In vitro behavior of thymic nurse cell-like complexes from mechanically and enzymatically dissociated frog tadpole thymuses

Cellular complexes, analogous by virtue of their external appearance, size, and number of seemingly internalized thymocytes to thymic nurse cells (TNCs) of endothermic vertebrates, were seen in short-term cultures (6–8 days) of mechanically and enzymatically dissociated thymuses of leopard frog tadpoles. Most TNC-like complexes from mechanically disrupted thymuses were covered with many thymocytes that morphologically resembled the “internalized” thymocytes. With time in culture, most complexes remained spherical and lost their externally adherent and “internalized” thymocytes. Some complexes, however, adhered to the glass substratum by means of macrophage-like cells. After one typically appearing TNC from a mechanically dissociated thymus had released its “internalized” thymocytes and spread completely over the glass substratum, it could be seen to consist actually of 9–10 stromal cells with the appearance of epithelial cells, macrophages, and dendritic cells. TNC-like structures from enzymatically dissociated thymuses had few, if any, attached thymocytes. Although these structures closely resembled murine TNCs initially, they displayed abnormal transformations within a few days of culture. Our observations led us to question the assumption that all TNCs from mechanically as well as enzymatically isolated TNCs from vertebrate thymuses are single cells. Rather, some if not all of the so-called TNC may actually be entities composed of several stromal cell types that enclose thymocytes. We suggest that this configuration seen in vitro may reflect the architecture of the compartmentalized reticular stromal cell meshwork that characterizes the intact thymus.     

Alterations in thymocyte subpopulations in Down's syndrome (trisomy 21)

To correlate the histologically observed thymic abnormalities with the cellular immunodeficiency found in Down's syndrome (DS), thymus fragments and thymocyte suspensions from 14 noninstitutionalized DS subjects were studied. Histologic examination and immunohistologic studies using an anticluster of differentiation (CD) 1 monoclonal antibody showed a contracted cortex due to cortical thymocyte depletion. When DS unselected thymocytes were phenotyped, a significant reduction of CD3-, CD1-, CD4-, and CD8-positive cells was found as compared to controls. To evaluate if the deficient expression of these markers was due to the reduction of thymocyte subsets identifiable on the basis of their physical properties, we separated DS unselected thymocytes into 10 fractions by continuous Percoll density gradient centrifugation. DS thymuses were almost completely devoid of high density thymocytes. Since in normal thymus, these cells correspond to small CD1+, CD4+, CD8+, and 50% CD3+ cortical thymocytes, their absence may explain the unrestricted reduction of markers on DS unfractionated thymocytes. Furthermore DS thymuses appeared to be enriched in CD1+ first fraction (Fr1) low density thymocytes of the Percoll gradient. Fr1 CD1+ cells constitute the main spontaneously proliferating pool in normal human thymus. When the spontaneous proliferating activity of DS Fr1 was compared to that of the control, a significant reduction was observed. This reduction associated with the absence of high density thymocytes, with the reduction of cells expressing alpha- and beta-chains of the T cell receptor and in conclusion with the lymphocyte depletion, suggests that in DS thymuses there is a deficient expansion of immature T cells resulting in a reduction of the various thymocyte subpopulations, including the thymocyte pool able to differentiate into functionally mature T cells.     

Thymocyte precursors in early-thymectomized Xenopus: migration into and differentiation in allogenic thymus grafts

In an anuran amphibian, Xenopus laevis, thymectomy of 4-day-old larvae abrogates T-cell dependent immune responsiveness. When such early-thymectomized (TX) diploid frogs were implanted with histocompatible triploid thymuses and grafted 8 weeks later with skin from third-party donors, the grafts were rejected relatively normally in 20-27 days. Microspectrophotometric determination of ploidy 3-5 months after thymus reconstitution revealed that most thymocytes were donor-derived. In contrast, when TX frogs received allogeneic triploid thymuses, they rejected skin grafts from a third-party donor relatively slowly (48-92 days) but did not reject skin from the thymus donor. Most thymocytes in such animals were of host origin. Host thymocytes were present 4 weeks after thymus implantation and became dominant population by 12 weeks. Few thymus implant-derived donor cells were detectable in the host spleen. These data suggest that existence of precursor cells in TX Xenopus that can functionally differentiate along a T-cell pathway as a result of microenvironment provided by the thymus implant.     

Immunohistochemical Evidence of Active Thymocyte Proliferation in Thymoma: Its Possible Role in the Pathogenesis of Autoimmune Diseases

Eight cases of human thymoma have been analyzed on cryostat sections with the monoclonal antibody Ki67, which reacts with cells in the proliferative phases of the cell cycle. The aim was to assess the proportion of proliferating thymocytes among lymphoid cells in the thymoma samples. In all cases a large number of cells (mean, 58.75%; range, 35-80%), recognized as thymocytes by morphology and lack of cytokeratin expression in a combined immunohistochemical assay, exhibited nuclear Ki67 staining. These findings differ from the reactivity pattern observed in age-matched nonneoplastic thymuses where lower growth activity of cortical thymocytes was observed (15-20% Ki67+ cells). Intensive thymocyte proliferation in thymomas may represent one of the factors which lead to autoimmunity in myasthenia gravis and thymomas.     

Bone marrow-derived progenitor T cells convey the origin of maturational arrest from CD4+CD8+ to CD4-CD8+ thymocytes in LEC mutant rats.

A mutant strain of rats, LEC, shows a novel arrest of T cell maturation from CD4+CD8+ to CD4+CD8- but not to CD4-CD8+ cells in the thymus. Transplantation of LEC rat fetal thymuses into the subcapsule of the kidney of athymic nude rats resulted in a normal maturation of thymocytes in the thymus graft. Furthermore, both single-positive thymocytes and peripheral lymph node T cells expressed T cell receptor alpha/beta antigen, and lymph node T cells acquired the ability to produce interleukin 2 upon mitogen stimulation. Transplantation of fetal thymuses from LEA rats, which express the same major histocompatibility complex haplotype as LEC rats, into LEC rat kidney subcapsule resulted in the maturational arrest from CD4+CD8+ to CD4+CD8- cells in the thymus graft. These data strongly suggest that bone marrow-derived progenitor T cells carry the cause of maturational arrest and that the thymic stroma of LEC rats has a normal potential to nurse thymocytes.     

儿童胸腺退化与疾病的关系

对７０例儿童胸腺进行组织学，８种淋巴细胞分化抗原及角蛋白和Ｓ－１００蛋白免疫组织化学及超微结构观察。胸腺退化可发生于各种疾病。胸腺奶穹不改变胸腺细胞（Ｔｈｙ－Ｃ）的分化顺序和亚群分布。在胸腺退化的主要改变中，树突状细胞数量减少及上皮细胞受损退变化比Ｔｈｙ－Ｃ数量减少具有更重要意义。巨噬细胞的吞噬作用可能只是排除死亡Ｔｈｙ－Ｃ的次要方式。     

Nonneoplastic and nonhyperplastic thymus in myasthenia gravis. An immunohistochemical study with double immunoenzymatic labeling of basement membrane and cellular components

Histologically normal thymus (type A) in patients with myasthenia gravis (MG) was immunohistochemically compared with hyperplastic MG thymus (type B) and normal non-MG thymus. In formalin-fixed, paraffin-embedded sections of ten type A, ten type B, and eight non-MG cases, the thymic epithelium and other cellular components were stained in conjunction with the basement membrane by a double immunoenzymatic method. This technique demonstrated a moderate architectural disturbance in type A thymus, with distended perivascular space (PVS), elongated medullary epithelium, and disrupted basement membrane. These changes were more prominent in type B thymus but were minimal to lacking in non-MG thymus. Compared with those in non-MG thymus, the myoid cells in MG thymuses of both types tended to cluster around the Hassall's corpuscles, with a slight decrease in number in type B but not in type A. B-lymphocytes were present in type B, type A, and non-MG thymuses in that order of abundance; the cells were confined to the medullary parenchyma in the non-MG group but were numerous both in the PVS and medulla in the MG groups. T-lymphocytes were increased in the expanded PVS of type A and B MG thymuses. The number of interdigitating reticulum cells was similar in the three groups, but the cellular distribution was more dispersed in MG thymuses of both types. These findings, although previously described in type B thymus, have not been well recognized in type A thymus. They support the view that a common abnormality (presumably chronic thymitis), differing in degree only, underlies MG thymuses regardless of the presence of follicular hyperplasia.     

T-cell development in human thymoma.

Human thymoma is derived from thymic epithelial cells and often associated with a large number of cortical thymocytes. Since thymic epithelial cells play key roles in T-cell development in the normal thymus, we hypothesized that the neoplastic epithelial cells of thymoma may support T-cell differentiation. We attempted to reconstitute the T-cell development in vitro by using neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on a proportion of CD4-CD8- cells in thymoma. These CD34+CD4-CD8- cells also expressed both IL-7R alpha-chain and common gamma-chain. Purified CD4-CD8- cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4+CD8+ cells via CD4 single positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. The CD34+CD4-CD8- cells purified from a normal thymus also differentiated to CD4+CD8+ cells in an allogeneic co-culture with the neoplastic epithelial cells of thymoma. In addition, a pleural dissemination from thymoma contained a large amount of cortical thymocytes. These results suggest that the neoplastic epithelial cells retain the function of thymic epithelium and can support T-cell development in thymomas.     

Cervical thymuses exist, but no cervical thymomas develop in thoracic thymoma-prone BUF rats

To confirm the existence of the cervical thymus and the development of cervical thymoma in thymoma‐prone BUF/Mna (BUF) rats, we examined cervical organs and adjacent tissues, and thoracic thymic tissues of the three inbred strains, BUF, ACI/NMna (ACI), and WKY/NCrj (WKY), and 11 congenic strains, in which genetic regions of rat nude (Rnu), thymus enlargement‐1 and thymus enlargement‐2 (Ten1 and Ten2), thymoma susceptibility of rat‐1 (Tsr1), atrophy of fast‐twitch muscles‐1 (Aftm1) and proteinuria of rat‐1 (Pur1) were transferred into BUF, ACI or WKY rats. These organs and tissues were fixed en block in 10% formalin and cut coronally into four to six slices of 3‐mm thickness, depending on the age of the rat, and embedded together in one block for each rat. Sections were cut and stained with haematoxylin and eosin and examined microscopically. Cervical thymuses were detected in 12–21% of rats from these inbred and congenic strains. No cervical thymuses were found in BUF‐Rnu/Rnu rats, which were athymic. All of 42 BUF, 2 of 55 BUF‐Rnu/+ and 28 of 33 ACI‐Tsr1/Tsr1 rats survived more than 52 weeks, and developed thoracic thymoma, but no cervical thymomas did. It is therefore clear that cervical thymuses behave differently from thoracic thymuses in spontaneous thymomagenesis in BUF rats.     

Maturation of fetal thymocytes in organ culture: secretion of interleukin 2, interferon and colony stimulating factors

This paper describes the differentiation of fetal thymocytes in organ cultures of embryonic thymuses. After one week in organ culture, fetal thymocytes from 14 d old mouse embryos could secrete IL-2, CSF and IFN upon stimulation with Con A. No constitutive secretion of lymphokines was observed. The only type of CSF produced was granulocyte-macrophage (GM) CSF. In contrast to thymocytes from adult mice, organ-cultured embryonic thymocytes did not secrete IFN-gamma, but IFN alpha/beta, in response to Con A. This is the first indication that secretion of IFN alpha/beta, but not IFN-gamma, can be induced in cells of the T cell lineage by a T cell specific mitogen. These results show that the embryonic thymus provides a sufficient environment for the development of some of the secretory functions of T cells and suggest that differentiating T cells acquire the ability to secrete IFN alpha/beta before IFN-gamma.     

Cytokeratin profiles of the thymus and thymomas: histogenetic correlations and proposal for a histological classification of thymomas

AIMS: Since cytokeratins (CKs) are useful as differentiation markers for histogenetic and classification studies, we investigated the CK profiles of the thymus and thymomas in an attempt to understand the histogenetic correlation and to propose a histological classification. METHODS AND RESULTS: Nine thymuses and 34 thymomas were immunostained for various CKs of different molecular weights and involucrin. Based on cytomorphology and histoarchitecture, thymomas were classified into spindle cell (SC), small polygonal cell (SPC), mixed, organoid, large polygonal cell (LPC) and squamoid (SQ) thymomas for compiling CK profiles. The thymus was shown to comprise four epithelial compartments, each expressing a different CK profile. Different histological types of thymoma expressed different CK profiles. By correlating the CK profiles of the thymus and thymoma, SPC, SC and LPC thymomas appeared to be related to subcapsular, medullary and cortical cells, respectively. Organoid thymoma recapitulated the structure and CK profile of the normal thymus, while SQ thymoma acquired additional squamous type CK. The applicability and usefulness of the proposed histological classification were evaluated on 147 thymomas by correlating the results with their invasive behaviour. One hundred and thirty-nine cases (95%) could be classified and different histological types correlated strongly with their invasive behaviour. CONCLUSIONS: The thymus is a complex epithelial organ composed of heterogeneous cell types giving rise to various related histological types of thymoma. The results of the CK profile study supports the proposed histological classification, which is pathologically applicable and clinically useful in correlating with invasiveness. This cytomorphological classification, supported by the CK expression patterns, is comparable to Müller-Hermelink classification and the new WHO histological classification except that a separate group of SPC thymoma expressing only CK14 and CK19 was identified and separated from mixed thymoma.