Details of thymocyte proliferation and maturation are important for understanding the variability of T cell functions. The following aspects need to be clarified in this context: 1) Ascertaining why hematopoietic committed pre-T cells migrating in the blood stream are trapped and proliferate in the subcapsular region of the thymus. Further morphological and functional studies of stromal cells in situ in the thymus should be conducted. The factors responsible for thymocyte proliferation should be also analysed. 2) How phenotypic maturation of thymocytes takes place and how T-cell receptor (TCR) expression is processed and controlled. 3) Subsequently, how does the selection of thymocytes take place? Is negative selection in fact responsible for the unresponsiveness of the body to self antigens? Further morphological analyses of thymic epithelial cells, thymocytes themselves and macrophages need to be conducted, together with immunohistochemical analyses using many monoclonal antibodies directed against these cells and the extracellular matrix, in order to analyse the cellular dynamics and cell-to-cell interactions occurring in thymus tissues in situ. 4) How mature thymocytes migrate to the periphery: Perivascular structures and the cells accumulating in this space should be intensively analysed morphologically and phenotypically. Lymphatic structures related to the thymus tissues also remain to be studied. To understand these questions, several useful experimental systems can also be employed: 1) Phylogenetical analyses, 2) ontogenical analyses, 3) thymus tissues regenerating from radiation or thymotoxic drugs, 4) thymomas, and 5) thymus tissues from transgenic animals. These important problems could be further studied in such experimental models both morphologically and immunohistochemically, since many useful tools are now available for studies of phenotypic and other markers of thymocytes and thymic epithelial cell. Subsequently, further detariled analyses of thymic structures may shed more light on the mechanisms underliying thymocyte proliferation and maturation.

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Recent advances in tissue culture technology and molecular biology have extended our understanding of the functional morphology of the thymus. The importance of a crosstalk between lymphoid cells and stroma has been appreciated as a prerequisite for the normal development of both. The network of direct cellular interactions and soluble factors comprising part of the microenvironment is far from being elucidated but the highly ordered thymic architecture clearly plays a pivotal role in normal thymic function. Insight into the genetic control of stroma development is only emerging while knowledge on the genetic control of the various steps in T cell development is already advanced and rapidly expanding.
The present paper gives an overview on the cellular components and matrix molecules of the human thymic microenvironment and their development during ontogeny. The intrathymic cytokine network is shortly reviewed. Special emphasis is put on molecules mediating lymphoepithelial interactions that are necessary for the expansion and early selection of immature thymocytes from precursor cells and for the generation of an MHC restricted and self tolerant T cell repertoire by positive and negative selection. Considering these physiological mechanisms we summarize the molecular pathology of the microenvironment and lymphocyte/stroma interactions in thymic epithelial tumors (thymomas). Finally, a pathogenetic model for paraneoplastic myasthenia gravis is given. We suggest abnormal autoantigen-specific positive selection of naive T cells as the essential molecular mechanism by which thymomas contribute to the autoimmunization against the acetylcholine receptor and other muscle proteins.

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This paper reviews the mechanism of sex hormone actions on the thymus, presenting mainly our data obtained at the cellular and molecular levels. First, data supporting the “genomic” action via the nuclear sex hormone receptor complexes are as follows: 1) sex hormone receptors and the thymic factor (thymulin) are co-localized in thymic epithelial cells, but not in T cells; 2) production / expression of thymic factors (thymulin, thymosin α₁₎ are remarkably inhibited by sex hormone treatment; 3) sex hormones cause changes in T cell subpopulations in the thymus; and 4) sex hormones strongly influence the development of thymus tumors in spontaneous thymoma BUF/Mna rats through their receptor within the tumor cells. Secondly, data indicating the “non-genomic” action of sex hormones via a membrane signal-generating mechanism are as follows: 1) the proliferation/maturation of thymic epithelial cells is mediated through protein kinase C activity introduced by sex hormones; 2) sex hormones directly influence DNA synthesis and cdc2 kinase (cell cyclepromoting factor) activity.

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Thymic nurse cells (TNCs) were studied using an animal model, BUF/Mna rats, which spontaneously develop benign thymomas of epithelial origin with age. The unusual increment and high availability of TNCs in this thymus enabled us to analyze TNCs directly either in tissue sections or on smears after enzymatic isolation. No structural or phenotypical abnormality in these TNCs was detected as assessed by electron microscopy and immunohistochemistry.
Typical TNCs were widely distributed in the cortical areas but not in the medullary areas. They showed characteristic euchromatic bright nuclei and enclosed intra-TNC cells with an investment of relatively light cytoplasm with abundant small vesicles and roughendoplasmic reticulum. The intra-TNC cells were mostly double positive (CD4⁺CD8⁺) cortical thymocytes, though macrophage populations could also be distinguished by their content of membrane-bounded phagosomes, multivesicular bodies and other inclusion bodies, and by their lack of cytoskeletal keratin filaments. High voltage electron microscopy revealed that intra-TNC cells were separated into several compartments by extremely thin internal veils of the TNC processes. The outer veils of the TNCs were continuous with occasional small gaps through which intra-TNC cells could migrate in and out of the compartments. Immunohistochemical analyses revealed that the TNCs per se were positive for MHC class I and class II, keratin and thymulin, but lacked both lymphocyte and macrophage markers. Among all adhesion molecules tested, ICAM-1 was strongly expressed on almost all TNCs. A minority of TNCs also contained either LFA-1α or LFA-1β positive cells.
These results suggest that TNCs may form a rather dynamic microenvironment for T cell development where either nursing or clearance of thymocytes take place, depending on the cellular components of intra-TNC cells. Macrophage populations may also play crucial roles as the third component within TNCs.

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As the first step toward understanding the identity and functions of thymic macrophages in situ, we examined the phenotypic heterogeneity of mouse thymic macrophages in tissue sections by the immunohistochemical double staining method with four monoclonal antibodies (F4/80, Mac-2, anti-CD32/16 and anti-I-A antibodies) as macrophage markers. Morphologically, three types of macrophages were identified: dendritic, round and flat-shaped. Dendritic macrophages were scattered throughout the thymus, and most of them were stained by all four markers. Among these macrophages, those at the cortico-medullary region (CMR) expressed a high intensity of CD32/16 antigen. Round macrophages were also distributed throughout the thymus; most of them, however, were localized in the cortico-medullary region to the medulla. These cells were F4/80-negative, Mac-2-positive, CD32/16-negative and I-A-positive. In contrast, round macrophages located at the cortex expressed F4/80. Flatshaped macrophages were localized at the subcapsular region of the cortex where active lymphopoiesis was observed. This type was positive for F4/80 and CD32/16, but negative for Mac-2. Furthermore, most of the three types of thymic macrophages showed intense reactions of the I-A antigen within the cytoplasm in addition to the expression of I-A antigen on the cell membrane. These results indicate that morphological characteristics of thymic macrophages at different locations reflect phenotypic variations detected in immunohistochemistry, and suggest that these different type macrophages may play distinct roles at various locations in thymocyte development in the thymus.

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Although glucocorticoid has been considered to cause thymocyte apoptosis in vitro, few studies have presented its in vivo effect. We report here on kinetics of glucocorticoid-induced murine thymocyte death in vivo by the TUNEL method.
TUNEL-positive cells were observed as early as at 2h after intraperitoneal injection of glucocorticoid. Most TUNEL-positive thymocytes were phagocytosed by acid phosphatase positive macrophages. “Free” (not phagocytosed) TUNEL-positive cells were not detected at early stages (by 4h). At 6 to 8h after the injection, the number of phagocytosed thymocytes per individual macrophage had reached its maximum, and at 8 to 12h many ruptured macrophages ingesting too many dying thymocytes became noticeable. During the process, no additional macrophages appeared to be mobilized to the thymus. At 6 to 8h after the injection, however, coincidentally with the fact that macrophages had become unable to further ingest dying lymphocytes, dead cells were left unphagocytosed, and ultimately became “free” positive cells, probably due to some proteolytic process ongoing within the thymus.
As late as at 12h, morphological examination revealed that epithelial cells seemed to begin engulfing thymocytes, almost simultaneously with the start of rupture of the macrophages due to the ingestion of too many thymocytes. Epithelial cells were readily identified by desmosomes and tonofilaments, in addition to euchromatic nuclei. Altogether, these results suggest that: 1) even though thymocytes were exposed to glucocorticoid in vivo, most of them were not TUNEL-positive unless they were phagocytosed; 2) even after most macrophages had ingested too many cells at later stages, macrophages in other locations did not migrate to the thymus; and finally, 3) deletion of damaged-thymocytes was also carried out by thymic epithelial cells, though not frequently, at around 12h and later.

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Reconstituted rat thymuses were studied by immunohistochemistry, transmission electron microscopy (TEM) and flow cytofluorometry on days 0, 1, 2, 3, 5, and 7 after whole-body sublethal irradiation (6Gy). One day after irradiation, numerous apoptotic cells were seen in the cortical thymus; the percentage of the sub-G1 peak representing apoptotic cells was 8.9% in the DNA content histogram of cytofluorometry. On day 3, the thymic structure had been destroyed and no distinction was drawn between the cortex and medulla. In this stage, few thymocytes but many macrophages were present, and the percentage of the sub-G1 peak reached a peak at 13.0%. Bromodeoxyuridine (BrdU) incorporated cells gradually increased after irradiation, and immunohistochemically numerous apoptotic cells were found primarily in the cortex on day 7. These thymocytes showed some levels of electron density of the nucleus as revealed by TEM. The percentage of S phase cells did not change markedly (20-30%) based on one-color DNA content histograms, but the percentage of early S and S phase cells was extremely high on day 7 (70%). These data indicate that a part of DNA synthetic cells may result in apoptosis. The combination of immunohistochemistry, TEM and flow cytofluorometry to analyze DNA content and BrdU incorporation proved a useful tool for investigating the reconstituted thymus.

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The overall architecture and structure of the perivascular space in the rat thymus were studied by light microscopy using silver-impregnated sections and sections stained immunohistochemically with anti-cytokeratin antibody, and by transmission and scanning electron microscopy (TEM and SEM). In silver-impregnated sections, the perivascular space was delimited by a thin sheath of delicate argyrophilic fibers from the thymic parenchyma in the cortico-medullary region and medulla. This space was continuous with the septal connective tissue, indicating that this was the connective tissue compartment rather than with the epithelial compartment of the parenchyma. In the medulla, the perivascular space widened at places, where the argyrophilic sheath was often discontinuous and the boundary between the perivascular space and parenchyma was indistinct. Lymphatics were located in the perivascular space of the corticomedullary region and sometimes in the wide perivascular space of the medulla.
The presence of a thymic epithelial sheath surrounding the perivascular space was confirmed by light microscopy of anti-cytokeratin antibody immunostained sections and by TEM. SEM observations revealed three-dimensionally that the epithelial sheath lined by collagen fibrillar (i. e., argyrophilic) layer form a rather continuous tubular structure in the cortico-medullary region, while it often interrupted in the medulla. These findings indicated that the perivascular space (i. e., the connective tissue compartment) is extensively open to the parenchyma (i. e., the epithelial compartment) in some portions of the medulla, where medullary lymphocytes are probably freely exposed to blood borne substances similar to the peripheral lymphoid tissues.

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The spatial distribution and migration of Kurloff cells containing PAS-positive large inclusion bodies in the thymus of estradiol-treated guinea pigs were histochemically studied by a combination of light and electron microscopy. Male guinea pigs were examined at various intervals from 7 days to 3 months after a single subcutaneous injection of estradiol. Differentiation of lymphatics from blood capillaries was performed by a 5′-nucleotidase (5′-Nase) staining method and the occurrence of Kurloff cells within 5′-Nasepositive lymphatics was confirmed by ultrastructural histochemistry. Several Kurloff cells first appeared at 7 days within lymphatics in the thymic capsule or interlobular connective tissues. At 12-15 days after estradiol administration, a lymphatic accumulation, a so-called “lymphatic center”, was seen in the thymic septa even though few Kurloff cells were present within the thymic parenchyma. The “lymphatic center” contained many Kurloff cells located in its periphery and in the surrounding marginal sinus which communicated with the thymic interlobular lymphatics. At 21 days after estradiol, Kurloff cells were preferentially accumulated along the corticomedullary junction extravascularly. Later the distribution was more diffuse. The conspicuous accumulation of Kurloff cells in the corticomedullary region could reflect an inability of Kurloff cells to use blood vessels as a route for migration. These findings strongly suggest the afferent migration of Kurloff cells into the thymus via lymphatics.

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The monoclonal antibody YeT-2, generated in mice hyper-immunized with thymic lymphocytes of the yellowtail, Seriola quinqueradiata, reacts with the major population of peripheral blood lymphocytes, which might be putative T cells. In this study, we examined the cross-reactivity of YeT-2 with lymphocytes from various fish species. Flow cytometric analysis showed that YeT-2 reacts with 69.8% lymphocytes in the thymus, 89.7% in the peripheral blood, 87.5% in the spleen, and 59.7% in the head-kidney. Among the six fish species examined, only the red sea bream, Pagrus major, which is included in the same suborder Percoidei with the yellowtail, showed the presence of YeT-2 positive cells. Electron microscopic studies revealed that YeT-2 positive cells in the peripheral blood of the red sea bream were lymphocytes or unidentified leucocytes. Thymic lymphocytes of the red sea bream were also immunocytochemically stained with YeT-2. The molecular weight of the YeT-2 cross-reacting antigen on blood cells from the red sea bream was identical with that from the yellowtail, which was identified at approximately 115 kDa. These results suggest that the monoclonal antibody YeT-2 recognizes a conserved antigen on lymphocytes common to the red sea bream and yellowtail.

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