Keratan sulfate proteoglycan during embryonic development of the chicken cornea

Antibodies to corneal keratan sulfate proteoglycan (KSPG) were used to characterize the pattern of KSPG accumulation during differentiation of neural crest cells in the stroma of embryonic chick cornea. Immunohistochemistry with monoclonal antibody I22 to keratan sulfate found this KSPG antigen localized inside stromal cells at stage 29 (Day 6), ca. 12 hr after migration into the primary stroma. A 2- to 3-day lag then occurred before appearance of extracellular keratan sulfate, first seen on Day 9 (Stage 35) in the posterior stroma. Keratan sulfate antigen accumulated in a posterior to anterior direction during subsequent development. Uniform staining of the stroma for keratan sulfate did not occur until after Day 16. Among several tissues, only corneal stroma contained an extracellular matrix which stained for keratan sulfate, though intracellular staining of some cartilage cells was observed. Accumulation of KSPG antigens in developing cornea was measured in unfractionated guanidine extracts with a quantitative ELISA using three different antibodies against KSPG. Increases were first detected after Day 9 using monoclonal I22, and somewhat later with the other two antibodies. Assays with all three antibodies detected a sustained, exponential increase of KSPG throughout the 5 days prior to hatching. Keratan sulfate continued to accumulate after hatching, but an antibody with specificity to KSPG core protein, detected no relative increase in antigen after hatching. This suggests a modulation of KSPG primary structure late in development and after hatching. Overt differentiation of individual neural crest cells thus appears to begin ca. 12 hr after their arrival in the primary stroma; a lag of 2-3 days precedes active secretion of KSPG.     

The development of the lymphoid organs of flounder, Paralichthys olivaceus, from hatching to 13 months

The growth of the lymphoid organs, such as head kidney, spleen and thymus were studied in flounder, Paralichthys olivaceus Temminck & Schlegel, from hatching to 13 months of age. Except for the thymus, all organs grew as the fish grew. By 2 months of age the lymphoid organs attained their maximum relative weight. The organ weight showed a closer correlation to body weight than they did to age. The total number of leucocytes in the lymphoid organs increased with age, but the number per milligram of lymphoid organ remained constant. A micro and ultrastructural study of the lymphoid organs showed that the full development of the lymphoid organs was not achieved until the juvenile stage. The spleen and head kidney had mixed populations of "red" and "white" cells. The head kidney was more lymphoid than the spleen. The thymus involuted quickly during the first 6 months. The blood components had no obvious relationship with age or season during the period studied.     

Quantitative evaluation of the total number and distribution of lymphocytes in young pigs.

In young pigs, the spleen, thymus and all lymph nodes were dissected out and weighed. The relative content of lymphoid cells was determined from histological sections. The number of nucleated cells was evaluated by two different methods: firstly, by measuring the DNA content of samples of lymphoid tissue and dividing by the DNA content of a single nucleus; and, secondly, by counting all lymphoid cells in histological sections of defined volumes of these organs. The number of lymphoid cells in tonsils, gut, bone marrow and lung were determined using histological evaluations and the volumes or weights of these organs. The resulting average number of lymphocytes was 321 times 10 (9) for a pig of 26 kg body weight. The lymphocytes showed the following distribution in lymphoid and non-lymphoid organs: thymus 44%, spleen 9%, mesenteric lymph nodes 17%, cervical lymph nodes 9%, other peripheral lymph nodes 3%, gut-associated lymphocytes 5%, tonsils 2%, bone marrow 5%, blood 3%, lung 0.2% and an estimated figure of 3% for all other tissues.     

Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants

We have tested the elicitation of innate defence-related responses in two stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss) after they were exposed to different concentrations of lipopolysaccharide (LPS), levamisole, or polyinosinic polycytidylic acid (poly-I:C). For comparison, cultures of rainbow trout head kidney macrophages were also included in the study, and the effect of the immunostimulants on the phagocytic activity, the intracellular and extracellular reactive oxygen species and nitric oxide production were assayed. Although the responses varied depending upon the concentration of the immunostimulants and the particular cell line, our results demonstrate that those activities were enhanced in the TSS and TPS-2 cell lines after exposure to any of the immunostimulants. These results indicate that the stromal cells of the main lympho-haemopoietic organs of O. mykiss develop innate defence responses, which are enhanced by well-known immunostimulants. In addition, such enhancement of the defence responses in the TSS and TPS-2 cell lines could be also elicited when they were exposed to conditioned supernatants from levamisole- or poly I:C-stimulated HK macrophage cultures, thus demonstrating that the haemopoietic stromal cells respond to macrophage-derived factors. Moreover, we demonstrate that the stromal cell lines constitutively expressed the Toll-like receptors TLR3, TLR5 and TLR9 genes. The results are discussed considering the role of the lympho-haemopoietic stromal cells in the innate immune responses, and the possibility of using histiotypic cell cultures of non-leucocyte cells of the haemopoietic organs to develop in vitro methods to select new immunostimulant candidates for aquaculture.     

The ontogeny of MHC class I expression in rainbow trout (Oncorhynchus mykiss)

In the present study, clonal rainbow trout (Oncorhynchus mykiss) embryos and larvae were assayed for the expression of key molecules involved in specific cell-mediated cytotoxicity using an anti-MHC class I monoclonal Ab and by RT-PCR using specific primers derived from classical MHC class I (class Ia), TCR and CD8. Whereas RT-PCR revealed that MHC class Ia and CD8 were expressed from at least 1 week after fertilisation (p.f.) on, TCR expression was detectable from 2 weeks p.f. Immunohistochemistry indicated an early and distinct expression of MHC class I protein in the thymus. Positive lymphoid, epithelial and endothelial cells were found in the pronephros, in the spleen and in the inner and outer epithelia at later stages. Whereas in older rainbow trout the intestine is counted among the organs of the highest class I expression, during ontogeny it was the last site (39 days after hatching) where such expression was detectable. Knowledge on the appearance of the assayed key molecules during fish development is relevant for the pathogenesis of infections as well as for early vaccine delivery. Besides such information regarding the development of the adaptive immune system, immunohistochemistry revealed that in early larvae MHC class I was expressed in neurons whereas in older rainbow trout this was not observed.     

BEN, a novel surface molecule of the immunoglobulin superfamily on avian hemopoietic progenitor cells shared with neural cells.

BEN is a novel molecule of the immunoglobulin superfamily that we previously identified by means of a monoclonal antibody on neural cell populations during avian development and epithelial cells of the bursa of Fabricius. In this paper, we describe the expression of BEN by hemopoietic cells during ontogeny. In the thymus, BEN is expressed as early as E9, and from E12 until just after hatching 30-60% of thymocytes are BEN positive. Thus the cells expressing BEN are immature thymocytes and not yet differentiated T cells. In the spleen, BEN expression parallels the myelopoietic activity. It is present on 75% of splenocytes during embryonic development and falls rapidly to 20% of cells during the first week after hatching when the spleen is becoming a secondary lymphoid organ. BEN is also found on a large proportion (about 80% positive cells) of bone marrow cells during ontogeny. Post hatching, BEN is present on 40-50% of bone marrow cells. The population of BEN-positive cells in the bone marrow includes myeloid and erythroid progenitor cells, identified by their ability to form colonies in vitro. BEN expression is lost as progenitor cells proliferate and differentiate to develop mature colonies in the clonal assay. Mature myeloid cells, such as macrophages, granulocytes, thrombocytes, and erythrocytes do not express the BEN antigen. Taken together, these data demonstrated that BEN is a stage-specific rather than a lineage-specific differentiation antigen expressed by immature hemopoietic cells.     

Ontogeny of lymphoid organs and development of IgM-bearing cells in Atlantic halibut (Hippoglossus hippoglossus L.)

In teleost fish, the head kidney, thymus, and spleen are generally regarded as important immune organs. In this study, the ontogeny of these organs was studied in Atlantic halibut (Hippoglossus hippoglossus), larvae at various stages of development. We observed that the kidney was present at hatching, the thymus at 33 days post hatch (dph), while the spleen was the last organ to be detected at 49 dph. All three lymphoid organs were morphologically well developed during late metamorphic stages. Real time RT-PCR analysis showed that IgM mRNA expression could be observed at 66 dph and later, which correlates well with in situ hybridisation data showing that a few IgM positive cells could be detected in the anterior kidney and spleen from 66 dph. Our data also showed that the highest levels of IgM mRNA could be detected in halibut spleen. Immunostaining using a monoclonal antibody against halibut IgM detected IgM positive cells at 94 dph in both the head kidney and the spleen, which is much later than the IgM mRNA. Numerous cells expressing both IgM mRNA and protein could be detected in the spleen and anterior kidney and also to some extent in thymus specimens from adult halibut.     

Relationship between the expression of class I antigen and reactivity of chicken thymocytes

The expression of major histocompatibility complex (MHC) class I antigens in ontogenesis and the distribution of B-F⁺ cells, defined by means of a monoclonal antibody, were studied by indirect membrane immunofluorescence tests on suspensions of thymus, bursa, spleen, peripheral blood lymphocytes (PBL) and red blood cells (RBC) from 18-day-old chicken embryos and chickens from 1–90 days after hatching. At 18 days of incubation and at the first day after hatching, RBC, PBL, and the cells from bursa and thymus are negative. The percentage of positive PBL and bursal cells increases up to 9 days after hatching. By 2 weeks after hatching almost 100 % of the RBC, PBL, bursa, and spleen cells were positive whereas the thymus showed only 20% positive cells. Analysis on 4-m-thick, frozen acetone-fixed tissue sections of thymus showed that medullary cells are positive, while the cortical area is negative. The graft-versus-host (GvH) competence of these thymus subpopulations was compared after sorting by the fluorescence-activated cell sorter and injection into MHC incompatible embryos. GvH reactivity was associated primarily with the B-F⁺ population. Double staining studies with peanut agglutinin (PNA)-fluorescein isothiocyanate and a rabbit-anti-Ig tetramethyl isothiocyanate-conjugate proved that the PNA^– thymocytes are identical with B-F⁺ thymocytes.Abbreviations used in this paper: FACS fluorescence-activated cell sorter - FCS fetal calf serum - FITC-Ig fluorescein isothiocyanate-conjugated immunoglobulin - GvH graft-versus-host - HAT hypoxanthineaminopterin-thymidine - HBSS Hanks' balanced salt solution - IIF indirect immunofluorescence - MCA monoclonal antibody - MHC major histocompatibility complex - NWL normal white Leghorn - OS Obese strain - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline - PNA peanut agglutinin - RBC red blood cells - TRITC-Ig tetramethyl isothiocyanate-conjugated immunoglobulin     

Distribution of cells bearing the Tac antigen during ontogeny of human lymphoid tissue

The monoclonal antibody anti-Tac, which binds to the interleukin 2 (IL 2) receptor, was used to identify this antigen in human fetal and adult lymphoid tissue. Liver, spleen, thymus, lymph node, and peripheral blood were examined for Tac-positive cells with the use of frozen sections or cytocentrifuge preparations. The results show that cells in the fetal and neonatal thymus express the Tac antigen; these cells are predominantly located in the medulla. The liver and spleen of both fetus and adult exhibit very few Tac-positive cells. Double staining demonstrates that cells bearing the Tac-antigen stain with Leu-4, an anti-T cell antibody. In adult lymph node tissue, the Tac-bearing cells are predominantly distributed in the interfollicular area, with positive cells also present in the germinal center and mantle zone. The Tac antigen is present on both T and B cells. Few Tac-positive cells are present in the circulating peripheral blood.     

Dendritic cells in the rat spleen follicles

Follicles of peripheral lymphoid organs (rat) contain a type of non-lymphoid cell which is capable of arresting antigen-antibody complexes at the cell surface. These so-called dendritic cells can be visualized in immunized rats by staining antigen-antibody complexes with immunohistoperoxidase techniques. The present study concerns a classification of these cells and comparison with known non-lymphoid cell types such as macrophages, marginal metallophils and tingible body macrophages in the rat spleen follicles. Immunoenzyme histochemical and (enzyme) histochemical techniques have been combined in the same tissue sections to correlate the functional capacity of binding immune complexes with morphological characteristics or phagocytic capacity. Dendritic cells show silver affinity but do not demonstrate a characteristic pattern of hydrolytic enzymes or phagocytosis.     

Tissue distribution and appearance in ontogeny of alpha/beta T cell receptor (TCR2) in chicken

We have performed immunoperoxidase staining on cryostat tissue sections and immunofluorescence analysis on cell suspensions to identify cells expressing the alpha/beta T cell antigen receptor during ontogeny and adult life in chickens. We used the mouse monoclonal antibody, TCR2, which was previously shown to recognize the alpha/beta TCR in chickens. TCR2+ cells were observed in thymic cortex and medulla and in T-dependent areas of spleen, intestine, and cecal tonsils of young adult chickens. Some TCR2+ cells were found in the cortex of bursal follicles and in liver. The first TCR2+ cells appear in thymus on Day 13 of the embryonic life and it is only after hatching that TCR2+ cells begin to migrate to the periphery.     

Ontogeny of the immune system of fish

Information on the ontogeny of the fish immune system is largely restricted to a few species of teleosts (e.g., rainbow trout, catfish, zebrafish, sea bass) and has previously focused on morphological features. However, basic questions including the identification of the first lympho-hematopoietic sites, the origin of T- and B-lymphocytes and the acquisition of full immunological capacities remain to be resolved. We review these three main topics with special emphasis on recent results obtained from the zebrafish, a new experimental model particularly suitable for study of the ontogeny of the immune system because of its rapid development and easy manipulation. This species also provides an easy way of creating mutations that can be detected by various types of screens. In some teleosts (i.e., angelfish) the first blood cells are formed in the yolk sac. In others, such as zebrafish, the first hematopoietic site is an intraembryonic locus, the intermediate cell mass (ICM), whereas in both killifish and rainbow trout the first blood cells appear for a short time in the yolk sac but later the ICM becomes the main hematopoietic area. Erythrocytes and macrophages are the first blood cells to be identified in zebrafish embryos. They occur in the ICM, the duct of Cuvier and the peripheral circulation. Between 24 and 30 hour post-fertilization (hpf) at a temperature of 28 degrees C a few myeloblasts and myelocytes appear between the yolk sac and the body walls, and the ventral region of the tail of 1-2 day-old zebrafish also contains developing blood cells. The thymus, kidney and spleen are the major lymphoid organs of teleosts. The thymus is the first organ to become lymphoid, although earlier the kidney can contain hematopoietic precursors but not lymphocytes. In freshwater, but not in marine, teleosts the spleen is the last organ to acquire that condition. We and other authors have demonstrated an early expression of Rag-1 in the zebrafish thymus that correlates well with the morphological identification of lymphoid cells. On the other hand, the origins and time of appearance of B lymphocytes in teleosts are a matter of discussion and recent results are summarized here. The functioning rather than the mere morphological evidence of lymphocytes determines when the full immunocompetence in fish is attained. Information on the histogenesis of fish lymphoid organs can also be obtained by analysing zebrafish mutants with defects in the development of immune progenitors and/or in the maturation of non-lymphoid stromal elements of the lymphoid organs. The main characteristics of some of these mutants will also be described.     

Accumulation of eosinophils and monocytes in lymphoid organs of chick-embryos. I. Effect of antigenic stimulation.

The effect of antigenic stimulation on the migration pattern of eosinophils and monocytes was studied during the embryonic stage in chickens. On the 13th embryonic day, chickens were injected with sheep red blood cells as antigen into the allantoic cavity and the relative frequency of oxidase positive cells (OPC) was determined as the total number of eosinophils and monocytes in the bursa of Fabricius, spleen, and thymus. Three and five days after the antigenic stimulation, the frequencies of OPC increased in both the spleen and thymus and then decreased to the normal level just before hatching. However, bursal frequencies of OPC were always low in both the cortex and medulla when compared with the controls. These events indicated that eosinophils and monocytes accumulated in the spleen and thymus after the antigenic stimulation. Furthermore, different frequencies of OPC among the embryonic lymphoid organs showed different responses in the migration of eosinophils and monocytes.     

Production and characterization of monoclonal antibodies to rabbit lymphocyte subpopulations. I. Tissue immunofluorescence and flow cytometric analysis

Nine monoclonal antibodies to rabbit T cells and B subpopulations have been generated from three separate fusions of spleen cells from mice immunized with fractionated populations of rabbit lymphocytes. These monoclonal antibodies, as well as a previously described rabbit T cell monoclonal antibody, 9AE10, have been analyzed by immunofluorescence staining on frozen tissue sections of rabbit thymus, spleen, and appendix. This screening method permits rapid identification of the lymphocyte subdomains in each tissue which is not possible by other screening methods. Each monoclonal antibody selected has a unique tissue staining pattern. Flow cytometric analysis of these monoclonal antibodies, using indirect immunofluorescence techniques on thymocytes, splenocytes, and PBL, revealed varying percentages of positive cells and individual mean fluorescence intensities indicating different epitope densities for each antigen. These monoclonal antibodies are now being used to characterize normal lymphocyte function and the role of specific lymphocyte subpopulations in experimental disease models in the rabbit.     

Changes in the distribution of the 34-kdalton tyrosine kinase substrate during differentiation and maturation of chicken tissues

We examined the distribution of the 34-kilodalton (34-kD) tyrosine kinase substrate in tissues of adult and embryonic chicken using both a mouse monoclonal antibody and a rabbit polyclonal antibody raised against the affinity purified 34 kD protein. We analyzed the localization by immunoblotting of tissue extracts, by immunofluorescence staining of frozen tissue sections, and by staining sections of paraffin-embedded organs by the peroxidase antiperoxidase method. The 34-kD protein was present in a variety of cells, including epithelial cells of the skin, gastrointestinal, and respiratory tracts, as well as in fibroblasts and chondrocytes of connective tissue and mature cartilage, and endothelial cells of blood vessels. The 34-kD protein was also found in subpopulations of cells in thymus, spleen, bone marrow, and bursa. The protein was not detected in cardiac, skeletal, or smooth muscle cells, nor in epithelial cells of liver, kidney, pancreas, and several other glands. Although most neuronal cells did not contain the 34-kD protein, some localized brain regions did contain detectable amounts of this protein. The 34-kD protein was not detected in actively dividing cells of a number of tissues. Changes in the distribution of the 34-kD protein were observed during the differentiation or maturation of cells in several tissues including epithelial cells of the skin and gastrointestinal tract, fibroblasts of connective tissue, and chondroblasts.     

鲤鱼早期发育过程中免疫相关器官的发生(英文)

参与鱼类免疫应答的主要组织和器官有肾（特别是头肾）、脾、胸腺、血液和淋巴等。近３０年来,随着鱼类免疫学的迅速发展,国内外许多学者在鱼类免疫相关组织和器官的结构、功能及免疫机理方面作了不少工作,但在鱼类免疫相关器官的发生方面所作工作很少。本文以正常鲤鱼的受精卵及不同发育阶段的幼鱼为材料,以Ｂｏｕｉｎ’ｓ液固定,常规石蜡包埋,连续切片,然后利用酸性条件下的阿新兰染色和过碘酸─—雪夫氏试剂反应相接合的方法（ＡＢ－ＰＡＳ染色）处理切片,对鲤鱼早期发育过程中免疫相关器官的发生作了初步研究。结果表明,肾脏是出现最早的免疫器官;孵化前第一天（受精后第五天）就已经发现肾小管（Ｆｉｇ．１）,孵化后第二天,在两个大的心窦之上出现网状的头肾组织,两条主要的,肾小管向后延伸在接近肛门处联合。在这个时期,肾小管之间分布有许多未分化的造血于细胞（Ｆｉｇ．２）和成熟的红细胞（Ｆｉｇ．３）。孵化后第五天,肾小管盘绕卷曲,肾小管之间的血细胞数目也有增加,出现体积较小的淋巴细胞（Ｆｉｇ．４）。孵化后第九天,造血组织间发育中的淋巴细胞数量大大增加,至孵化后３０天,头肾、中肾、小管已很少,大部分被造血组织所充满。脾脏于孵化当天出现,位于肝胰脏之左     

Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.     

Effect of selenium and vitamin E dietary deficiencies on chick lymphoid organ development

Diets specifically deficient in selenium (Se) and/or vitamin E or adequate in both nutrients were fed to chicks from the time of hatching. Lymphoid organs (bursa, thymus, and in some instances, spleen) were collected from chicks 7-35 days of age. Growth of the chicks fed these diets was monitored over the experimental period as was lymphoid organ growth. The development of the primary lymphoid organs was further assessed by histological techniques and the organ contents of vitamin E (alpha-tocopherol) and Se were determined. Specific deficiencies of either Se or vitamin E were found to significantly impair bursal growth as did a combined deficiency. Thymic growth was impaired only by the combined deficiency diet. Severe histopathological changes in the bursa resulted from the combined deficiency and these were detectable by 10-14 days after hatching. These changes were characterized by a gradual degeneration of the epithelium and an accompanying depletion of lymphocytes. Similar changes, although slower to develop and less severe, were observed in the thymus as a result of the combined deficiency. When both serum and tissue levels of vitamin E and Se were monitored, it was observed that these were rapidly and independently depleted by the specific deficiency diets. These data suggest that the primary lymphoid organs are major targets of Se and vitamin E dietary deficiencies and provide a possible mechanism by which immune function may be impaired.     

Development of T lymphocytes in Xenopus laevis: appearance of the antigen recognized by an anti-thymocyte mouse monoclonal antibody

Development of T lymphocytes in Xenopus laevis was studied using a mouse monoclonal antibody (mAb), XT-1, that was produced against surface determinants on thymocytes of J strain frog. Ontogenic studies, employing immunofluorescence, showed that cells positive for the determinant recognized by XT-1 mAb (XT-1⁺ cells) were first detected in the thymus of J strain Xenopus by Nieuwkoop and Faber stage 48 (7 days postfertilization) and then in the spleen, liver and kidney by stage 52 (20 days postfertilization). Percentages of XT-1⁺ cells in the thymus increased rapidly by stage 49 (10 days postfertilization) and reached adult levels by stage 52, and those in the spleen, liver, and kidney reached adult levels by stage 56 (40 days postfertilization). Electron microscopic immunohistochemistry revealed that most XT-1⁺ cells in thymuses from stage 56 larvae were typical small lymphocytes (4–7 μm in diameter). In contrast, many XT-1⁺ cells in larval thymuses at stage 49 are large (8–10 μm in diameter) lymphoblastoid cells. Thymectomy at stage 46 (5 days postfertilization) depleted XT-1⁺ cells in larval and adult lymphoid organs to background levels. These results suggest that the XT-1⁺ cells are differentiated from the lymphoid precursor cells in the thymus before the appearance of small lymphocytes and migrate into peripheral lymphoid organs. The cell surface determinant recognized by the XT-1 mAb may provide an important marker for the differentiation of T lymphocytes in Xenopus.     

Immunohistology of lymphoid and non-lymphoid cells in the thymus in relation to T lymphocyte differentiation

The present paper reports the distribution of lymphoid and non-lymphoid cell types in the thymus of mice. To this purpose, we employed scanning electron microscopy and immunohistology. For immunohistology we used the immunoperoxidase method and incubated frozen sections of the thymus with 1) monoclonal antibodies detecting cell-surface-differentiation antigens on lymphoid cells, such as Thy-1, T-200, Lyt-1, Lyt-2, and MEL-14; 2) monoclonal antibodies detecting the major histocompatibility (MHC) antigens, H-2K, I-A, I-E, and H-2D; and 3) monoclonal antibodies directed against cell-surface antigens associated with cells of the mononuclear phagocyte system, such as Mac-1, Mac-2, and Mac-3. The results of this study indicate that subsets of T lymphocytes are not randomly distributed throughout the thymic parenchyma; rather they are localized in discrete domains. Two major and four minor subpopulations of thymocytes can be detected in frozen sections of the thymus: 1) the majority of cortical thymocytes are strongly Thy-1+ (positive), strongly T-200+, variable in Lyt-1 expression, and strongly Lyt-2+; 2) the majority of medullary thymocytes are weakly Thy-1+, strongly T-200+, strongly Lyt-1+, and Lyt-2- (negative); 3) a minority of medullary cells are weakly Thy-1+, T-200+, strongly Lyt-1+, and strongly Lyt-2+; 4) a small subpopulation of subcapsular lymphoblasts is Thy-1+, T-200+, and negative for the expression of Lyt-1 and Lyt-2 antigens; 5) a small subpopulation of subcapsular lymphoblasts is only Thy-1+ but T-200- and Lyt-; and 6) a small subpopulation of subcapsular lymphoblasts is negative for all antisera tested. Surprisingly, a few individual cells in the thymic cortex, but not in the medulla, react with antibodies directed to MEL-14, a receptor involved in the homing of lymphocytes in peripheral lymphoid organs. MHC antigens (I-A, I-E, H-2K) are mainly expressed on stromal cells in the thymus, as well as on medullary thymocytes. H-2D is also expressed at a low density on cortical thymocytes. In general, anti-MHC antibodies reveal epithelial-reticular cells in the thymic cortex, in a fine dendritic staining pattern. In the medulla, the labeling pattern is more confluent and most probably associated with bone-marrow-derived interdigitating reticular cells and medullary thymocytes. We discuss the distribution of the various lymphoid and non-lymphoid subpopulations within the thymic parenchyma in relation to recently published data on the differentiation of T lymphocytes.