Immunohistochemical demonstration of H antigen, peanut agglutinin receptor, and Saphora japonica receptor expression in infant thymuses and thymic neoplasias.

Ten infant thymuses and 13 primary thymic tumors obtained from archived paraffin-embedded tissue were examined for the presence of tissue blood group O antigen (H), peanut agglutinin receptor antigen (PNA-r), Saphora japonica agglutinin receptor antigen (SJA-r), carcinoembryonic antigen (CEA), cytokeratin (CK), and epithelial membrane antigen (EMA). In the thymuses studied, Hassall's corpuscles contained abundant immunoreactive CK, PNA-r, and H antigens, whereas CEA, SJA-r, and EMA were present focally in Hassall's corpuscles. Immunoreactive CK, PNA-r, and CEA were demonstrated focally in the subcapsular region, cortical nurse cells, and subcapsular-perivascular monocytic cells, respectively. PNA-r was present in all 12 epithelial type tumors, including all eight thymomas. CEA was present in nine tumors, including six thymomas. Six thymomas contained H antigen and SJA-r; five continued CK and EMA. SJA-r and EMA were also present in one carcinoid tumor of thymic origin. In epithelial thymomas, the antigens stained nests of epithelial cells resembling the pattern of staining in Hassall's corpuscles. Membrane staining of spindle cells of both spindle cell and epithelial thymomas was less intense than staining of epithelial type cells.     

Analysis of the fine distribution of thymic epithelial microenvironmental molecules by immuno-electron microscopy

Normal T cell development depends upon an interaction between progenitor cells and their microenvironment. Previously raised monoclonal antibodies to subtypes of human thymic epithelium were used with gold-coupled reagents in immuno-electron microscopy to study the fine cellular distribution of the molecules to which these Mabs bind. Mab MR6, thought to recognize the human IL-4 receptor, shows strong surface labeling of cortical epithelial cells and thymic nurse cells, and very weak staining of thymocytes, medullary macrophages and interdigitating cells. Mab 1st 8.18 labels the surface of Hassall's corpuscles and associated medullary epithelial cells. The molecules detected by these two antibodies are therefore located in a position where they are available to interact with external cellular and soluble signals within the thymus. In contrast Mabs MR10 and 19 recognize Intracellular molecules within subcapsular, perivascular and some medullary epithelium. These molecules may represent soluble material awaiting secretion from the cell; alternatively, they may be internal structural proteins.     

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 .     

Epithelial cell heterogeneity in the guinea pig thymus: immunohistochemical characterization of four thymic epithelial subsets defined by monoclonal anti-keratin antibodies

Keratins are a family of related polypeptides constitutive of the cytoskeleton of epithelial cells and are never found in nonepithelial tissues. Thymic epithelial cells (TEC), known to induce T cell differentiation, are the keratin-containing cells within the thymus. Using four monoclonal anti-keratin antibodies (KL1, KL4, AE2, AE3) directed against keratins of different molecular weight, we have investigated the guinea pig thymic epithelium. The immunohistochemical analysis of thymic cryostatic sections revealed that the keratin expression of TEC varied according to their location in the thymic lobula; the thymic cortex was specifically stained by AE3 whereas the thymic medulla and the subcapsular cortex were recognized by KL4. In addition, KL1 and AE2 exclusively labeled Hassall's corpuscles. The biochemical analysis of keratins extracted from the thymus showed that each TEC subset was characterized by an unique pattern of keratin polypeptides. This study extends the concept of thymic epithelium heterogeneity and suggests that anti-keratin antibodies which allow the typing of TEC subsets may be valuable tools for studying the differentiation of thymic epithelium and its in vitro function on T lymphocytes.     

The thymus gland in secondary immunodeficiency

The microenvironment within the thymus gland and various thymic hormones facilitates the maturation of prothymocytes to functional T lymphocytes. Abnormal thymic morphology is a hallmark feature of several primary and secondary immunodeficiencies. Thymus glands from patients with the acquired immunodeficiency syndrome or graft-vs-host disease and from patients receiving cyclosporine A therapy are depleted of thymocytes, have a striking reduction in thymic epithelial cell mass, and are virtually devoid of Hassall's corpuscles. In malnourished individuals, similar thymic atrophy is present, although the Hassall's corpuscles are present and cystically dilated. Absence of differentiation antigens or anomalous expression of major histocompatibility antigens occurs on thymic epithelial cells in these conditions, and resembles immunologic abnormalities of the thymus in severe combined immunodeficiency. In acquired immunodeficiency syndrome, graft-vs-host disease, and cyclosporine therapy, there is an expansion of cytotoxic/suppressor (CD8) lymphocytes. Experimental evidence suggests that in many situations, such cells may cause damage to the thymic epithelium. The damage to the thymic epithelium may alter the thymic microenvironment and contribute to the immune dysfunction observed in these patients. In addition, a damaged microenvironment may hinder therapeutic efforts to reconstitute immunity.     

Monoclonal antibody against carcinoembryonic antigen (CEA) identifies two new forms of crossreacting antigens of molecular weight 90,000 and 160,000 in normal granulocytes

Two new forms of non-specific crossreacting antigens (NCAs) were identified in the Nonidet P40 (NP-40) extracts of normal granulocytes by precipitation with the monoclonal antibody (MAb) 192 directed against carcinoembryonic antigen (CEA) and already known to crossreact with the perchloric acid soluble NCA-55. The NP-40 soluble NCAs recognized by MAb 192 have apparent mol. wts of 90,000 and 160,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both NCAs appear to consist of a single monomeric polypeptide chain, since they have the same electrophoretic mobility in SDS-PAGE under reduced and non-reduced conditions. When granulocytes were extracted with perchloric acid instead of NP-40, only the 55,000 mol. wt antigen, corresponding to the previously described NCA-55, was precipitated by MAb 192. Furthermore, it was shown that NCA-55 is not a degradation product of NCA-90 or NCA-160 due to the perchloric acid treatment because exposure to perchloric acid of NCA preparations purified from NP-40 extracts did not change their apparent mol. wts in SDS-PAGE. It was also shown that NCA-160 is not a granulocytic form of CEA because it was not precipitated by the MAb 35 reacting exclusively with CEA. Immunocytochemical studies of granulocytes and macrophages showed that MAb 192 stained both types of cells whereas MAb 47 stained only the granulocytes and MAb 35 none of these cells. In granulocytes both MAbs reacted with antigens associated with granules and also present at the periphery of the nucleus as well as in the Golgi apparatus. The NCA-90 identified by MAb 192 was found by sequential immunodepletion to be antigenically distinct from the NCA-95 precipitated by MAb 47. The epitope recognized by MAb 192 on CEA and NCA molecules appears to be on the peptidic moiety because the antigens deglycosylated by the enzyme Endo F were still precipitated by this MAb. Taken together, the results indicate that MAb 192 identifies two novel forms of NCA (NCA-90 and NCA-160) in NP-40 extracts of granulocytes, which are distinct from CEA and the previously described NCA-55 and NCA-95 identified by MAbs 192 and 47, respectively, in perchloric acid extracts of granulocytes.     

Localization of HIV-1 in human thymic implant in SCID-hu mice after intravenous inoculation

Human immunodeficiency virus type 1 (HIV-1) was immunohistochemically and ultrastructurally localized in human thymus implants in SCID-hu mice 3 weeks after intravenous (i.v.) inoculation of the virus. A viral antigen (gp120) was predominantly distributed in and around the epithelial cells in Hassall's corpuscles as demonstrated by fluorescence immunohistochemistry. Occasional solitary round cells positive for the viral antigen but negative for cytokeratin were detected in the perivascular areas. Ultrastructural examinations clearly revealed a number of mature viral particles in the intercellular spaces of the Hassall's corpuscles. Thus the present study indicates the possibility that thymic epithelial cells in Hassall's corpuscles act as a target and/or reservoir in an early stage of HIV infection.     

A unified theory of central tolerance in the thymus

Non-deletional tolerance resulting in the generation of regulatory T cells within the thymus is a key mechanism for the establishment of immunological self-tolerance. How the high-affinity self-reactive regulatory T cells escape negative selection and what type of antigen-presenting cells positively select them within the thymus are unsolved questions. Previous studies suggest that thymic epithelial cells are crucial for the positive selection of regulatory T cells in thymus. A recent study from my group shows that a subset of dendritic cells (that have been "educated" by the thymic stromal lymphopoietin molecule produced by a thymic cell type known as Hassall's corpuscles) positively select regulatory T cells within the medulla of human thymus. Here, I discuss the implications and historical context of this new result and suggest that a subset of mature dendritic cells within the thymic medulla protects the medium- to high-affinity self-reactive T cells from negative deletion and induces their differentiation into regulatory T cells in the thymus.     

The human thymus microenvironment: heterogeneity detected by monoclonal anti-epithelial cell antibodies.

Monoclonal antibodies were raised against human thymus stromal cells and their specificity for the epithelial component of thymus stroma assessed by double immunofluorescence using anti-keratin antibodies to identify epithelium. Our monoclonal antibodies identify six distinct patterns of epithelial cell antigen expression within the thymus: pan epithelial (antibody IP1); cortex (MR3 and MR6); cortical/medullary junction (IP2); subcapsule and subpopulation of medulla (MR10/MR14); Hassall's corpuscles and adjacent subpopulation of medulla (IP3); Hassall's corpuscles only (MR13/IP4). This heterogeneity of antigen expression suggests that many different epithelial microenvironments exist within the human thymus.     

Characterization of a monoclonal antibody, RTE-21, that binds to keratohyalin granule-associated proteins in epithelial cells of human skin and thymus

The role of skin and thymic epithelium in the promotion of T-cell activation and maturation is currently an area of great interest. In thymus, epithelium is located in the cortex, medulla, and in medullary epithelial swirls called Hassall's bodies. During studies of antigens shared by skin and thymic epithelial cells, we produced a murine monoclonal antibody (RTE-21) raised against the rat thymic epithelial cell line, IT26R21, that identified an antigen present in terminally differentiated epithelium of normal human skin and thymus. In indirect immunofluorescence studies, antibody RTE-21 identified cytoplasmic granules located in the stratum granulosum of normal human skin, Hassall's bodies of human thymus, and in a subset of cells of the IT26R21 rat thymic epithelial cell line. Moreover, the granular reactivity pattern of antibody RTE-21 in the stratum granulosum could be extracted by 1 M potassium phosphate (1 M KPO4). A 1 M KPO4 extract of epidermis containing keratohyalin granule proteins was dialyzed against distilled water, solubilized in reduced sample buffer and subjected to polyacrylamide gel electrophoresis and Western immunoblot analysis. In this assay, antibody RTE-21 recognized proteins of 70, 36, 34, and 30 kDa. Using antibody RTE-21 and indirect immunofluorescence, we demonstrated that keratohyalin granules in human thymus were localized exclusively to Hassall's bodies. These data support the notion that human thymic Hassall's bodies result from terminal differentiation of medullary thymic epithelium. Thus, antibody RTE-21 should be an important probe for the study of skin and thymic epithelial cell maturation in vitro and in vivo.     

Immunohistochemical study of tyrosine phosphorylation signaling in Hassall's corpuscles of the human thymus

Tyrosine phosphorylation signaling has been reported to play a key role in thymocyte development. However, the physiological role of signaling in thymus stroma is poorly understood, and there is lack of information on the in situ localization of elements of the signaling pathway in thymus stroma. In the present study, we have found by immunohistochemical analysis that tyrosine-phosphorylated proteins are present in high amounts in Hassall's corpuscles of the thymus medulla. Hassall's corpuscles represent end stages of maturation of thymic medullary epithelium. We have also investigated the localization of the src family that is involved in tyrosine phosphorylation signaling in Hassall's corpuscles. A member of the src family protein tyrosine kinases, p59fyn, was shown to be abundantly expressed in the outer layer of Hassall's corpuscles. Another member of the family, p60c-src, was highly expressed in the entire Hassall's corpuscles. Furthermore, p50csk and p130cas, both of which are involved in the pathway, were shown to be preferably expressed in the outer layer of Hassall's corpuscles. These findings suggest that tyrosine phosphorylation signaling may play a role in thymic medullary epithelial maturation and that the src family is involved in the process.     

The cytoarchitecture of the human thymus detected by monoclonal antibodies

Seven monoclonal antibodies were produced against human thymic stromal cells. The monoclonal antibodies were put into two groups depending on whether the cells they detected were keratin-positive or -negative. Demonstrated in the keratin-negative group were a granular reticular meshwork, a stellate population predominantly in the medulla, and secretory cells associated with Hassall's corpuscles. In the keratin-positive group we showed two extensive epithelial networks, a trabecular and rare medullary epithelial population, and thymus-specific epithelium restricted to the subcapsule and the medulla. A novel finding was that some of the monoclonal antibodies and also OKM1 identified keratin-negative cells within Hassall's corpuscles, which implies that there are macrophages associated with these structures. The monoclonal antibodies should prove useful for separating and classifying subpopulations of stromal cells and also for monitoring changes in the thymic architecture in different thymic pathologies.     

Human thymus contains amnion epithelial antigens

Antibodies produced in rabbits to detergent-solubilized human amnion were found to react with Hassall's corpuscles in human thymus. Following nomenclature for placental antigens, the immunogenic group responsible for these antibodies has been tentatively designated as amnion antigens 1 (AA1). The anti-AA1 antisera did not react with other thymic components, nor did they react with any other extra-embryonic tissues than amniotic epithelium. Some adult ectodermally derived tissues, such as breast ductal and corneal epithelium, reacted with anti-AA1, but others such as skin and vagina did not. These findings link an antigenic relationship between amniotic epithelium and certain ectodermal derivatives. Amnion exists long before these tissues are formed, raising the possibility that amniotic epithelium may play an inductive role in their development.     

Thymic epithelial cells expressed unusual follicular dendritic cell markers: thymic extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue

Described herein is a case of thymic extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue. Using immunohistochemical double staining it was found that most of the thymic lymphoid follicles in this case possessed cytokeratin-positive and follicular dendritic cell (FDC) marker-positive cells. Moreover, using immunoelectron microscopy it was confirmed that some of the double-positive cells were thymic epithelial cells. The candidate of cytokeratin subtype expressed on the double-positive cells was cytokeratin 1 (CK1), which was expressed only by the epithelium of Hassall's corpuscles in thymuses from age-matched patients with myasthenia gravis. The present case indicates a possibility that some thymic epithelial cells become FDC, although it was uncertain whether they were derived from the epithelia of Hassall's corpuscles or whether they were at the same differentiation stage as Hassall's corpuscles.     

Pathologic appraisal of the thymus gland in acquired immunodeficiency syndrome in children. A study of four cases and a review of the literature

We studied the entire thymus gland by step-serial sections in four fatal cases of acquired immunodeficiency syndrome (AIDS) in children. The location, configuration, and blood vessels of the thymus gland were normal. The weight was reduced. There was severe depletion of both lymphocytes and Hassall's corpuscles. Because of the normal location, configuration, and blood vessels and the seemingly less severe lymphocytic depletion than is seen in congenital immunodeficiency syndromes and because of the clinical and epidemiologic features, we concluded that this syndrome in children represents AIDS. We postulated that the epithelial cells of the Hassall's corpuscles may be the primary site of thymic injury in AIDS.     

Immunolocalization of the mitogen-activated protein kinase signaling pathway in Hassall's corpuscles of the human thymus

In the present study, we investigated the immunohistochemical localization of mitogen-activated protein kinase (MAPK) signaling pathway in the human thymus. Three members of MAPK, the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK) and the p38 kinase, showed differential expression patterns in the thymus medulla. The phosphorylated form of ERK (p-ERK) was abundantly present in the outer layer of Hassall's corpuscles, and the phosphorylated form of p38 kinase (p-p38 kinase) was present in the entire Hassall's corpuscles. The phosphorylated form of JNK (p-JNK) was expressed in medullary thymocytes. We also examined localization of MAPK kinases (MAPKK or MEK) which specifically activate MAPK. MEK1, an activator of ERK, was found in the outer layer of Hassall's corpuscles where p-ERK was expressed. MEK3, an activator of p38 kinase, was also expressed in the outer layer. MEK4 and MEK7, which are activators of JNK, were present in the entire Hassall's corpuscles. Thus, differential expression of MAPK in the thymus supports the concept that the MAPK signaling pathway controls the specificity of functional thymic responses to extracellular stimuli. Furthermore, the abundant expression of various elements of the pathway in Hassall's corpuscles suggests that the pathway is involved in thymic medullary epithelial maturation.     

Reptilian thymus gland: an ultrastructural overview.

Like in higher vertebrates, the thymus gland of reptiles consists of lymphoid cells within epithelial framework and characteristic myoid cells. Mammalian-like Hassall's corpuscles are absent. Secretory cells, secretory and degenerative cysts as well as phagocytic cells, and plasma cells can be observed. Interdigitating cells and some characteristic features of thymic innervation and vascular system are also described in the reptilian thymus gland.     

Immunohistochemical characterization of rat thymic non-lymphoid cells. I. Epithelial and mesenchymal components defined by monoclonal antibodies.

Molecular microenvironmental heterogeneity within rat thymus was studied by a panel of 13 monoclonal antibodies (mAbs) raised to thymic epithelial (TE) cells and mesenchymal stroma. Based on their anatomical distribution patterns observed with immunohistological techniques on frozen sections and double immunostaining using anti-keratin antibodies to identify epithelium, they were subdivided into five groups: (i) pan TE cells antibodies (R-MC 2, 3 and 8); (ii) cortical TE cells antibodies (R-MC 13-17); (iii) antibodies detecting subcapsular and subtrabecular TE cells and most medullary TE cells (R-MC 18-20); (iv) antibody to Hassall's corpuscles (HC) and a small subpopulation of medullary TE cells (R-MC 22); (v) mesenchymal stroma antibody (R-MC 23). The obtained results show phenotypic heterogeneity of rat thymic epithelium and its distinction compared to mesodermal-derived compartment.     

Temporal lymphoreticular changes caused by ts1, a paralytogenic mutant of Moloney murine leukemia virus TB.

Inoculation of newborn FVB/N mice with ts1, a mutant of Moloney murine leukemia virus TB, induced severe thymic atrophy, spongiform polioencephalomyelopathy, and fatal posterior paralysis of the affected mice 35-40 days after inoculation. During the early course of infection viral replication was found in the spleen and, more importantly, within the thymus. Of these organs, the thymus was affected most severely by ts1-infection. Thymic weights of infected mice decreased markedly during disease progression, culminating in severe atrophy at the time of paralysis. During the first 10 days after inoculation, the virus replicated within the endothelial lining of splenic and thymic capillaries and was released albuminally into the basement membrane before spreading outwardly into perithelial, epithelial, and reticuloendothelial cells. Within these cells there was productive viral replication and subsequent dissemination of the virus to the thymic T cell population. Early infection (up to 10 days after inoculation) of the thymus induced an increase in thymocytic mitosis, followed by a progressive increase in thymocytic death between 15 and 35 days after inoculation. Thymuses from paralyzed mice killed 30-39 days after inoculation, demonstrated pronounced involution, characterized by loss of lobular architecture, effacement of the cortex and medulla, severe depletion of thymocytes, and partial or complete loss of Hassall's corpuscles. Immunohistochemistry for viral antigens showed positive labeling of splenic megakaryocytes, reticuloendothelial cells, and thymocytes in mitosis, and reticulo-epithelial-endothelial cells of the thymus. The thymic phase of viral replication appeared to be crucial for development of neurological lesions and posterior paralysis.     

Nicotinic acetylcholine receptor-like protein in the human myasthenic thymus

The localization of the nicotinic acetylcholine receptor-like protein (nAChR-LP) in seven human thymi with lymphofollicular hyperplasia and four thymomas from patients with myasthenia gravis (MG) were studied using the polyclonal antibody against nAChR-LP from a fetal calf thymus. By immunoelectron microscopy in both normal thymi and thymi from patients with MG, nAChR-LP was found in the tonofilaments and the plasma membrane of mainly the secretory-type cells of Hassall's corpuscles and some medullary epithelial cells. This finding is the first confirmation of the epithelial nature of the nAChR-LP positive cells in the outer rim of Hassall's corpuscles by immunoelectron microscopy. The positive cells also showed reactivity with anti-major histocompatibility complex (MHC)-class II antibody. These findings suggest that the nAChR-LP positive cells in Hassall's corpuscle and the thymic medulla are responsible for the induction of autoimmunity in MG. A part of this study was presented at the 31st Annual Meeting of The Japanese Society of Neurology, Kanagawa, May 23–25, 1990 and the 15th World Congress of Neurology, Vancouver, Canada, September 5–10, 1993.