Cell kinetics in the fetal mouse thymus: precursor cell input, proliferation, and emigration

The entry and differentiation of lymphoid precursor cells (LPC) in grafted mouse fetal thymuses and the emigration of explants thymocytes has been followed in a system in which donor and host lymphocytes could be distinguished on the basis of Thy-1 expression. It appears that LPC that invade the fetal mouse thymus between 10 and 13 days rapidly differentiate into Thy-1 positive thymocytes, giving rise to all of the lymphoid populations of both cortical and medullary locations until approximately the end of the first week after birth. Lymphoid precursor cells that enter the fetal thymus after 13 days of fetal life only differentiate into Thy-1 positive lymphocytes 6 or 7 days after birth, when they give rise to a second generation of thymocytes that grows exponentially and completely replaces the first generation in approximately 8 days. All cells leaving the thymus during the first 2 wk of life appear to be derived from the first wave of precursors.     

Cytolytic effects of glucocorticoid on thymus-medullary lymphocytes incubated in vitro

Thymus cortical and medullary lymphocytes (TCL and TML) were incubated in vitro with or without cortisol. The cytolytic effects of the steroid on both thymocytes were examined in the microscope and by measurements of protein and RNA synthetic activities. There did not appear to be significant differences in the extent of damage by glucocorticoid exposure between both thymocytes, although the reduction of RNA synthesis in TML was less marked as compared with that in TCL. The facts that lymphocytes in thymus medulla were able to survive after cortisone injection and that the cells incubated in vitro were affected by the steroid presented the hypothesis that there might be some particular mechanisms in thymus medulla to protect lymphocytes from the cytolytic action of glucocorticoid.     

Ontogeny and organization of the stationary non-lymphoid cells in the human thymus

Summary Ontogenetic differentiation of the human thymus was investigated in 50 embryos by means of light and electron microscopic methods in an attempt to clarify the morphogenesis of the complicated microecology of thymic tissue. At the 8th gestational week (g.w.), the primordium of the thymus contains almost exclusively undifferentiated epithelial cells. At the 10th g.w., the epithelial cells in the central part are spindle-shaped. During the subsequent weeks the cortical region of the thymus becomes separated into lobes by mesenchymal septa containing hemopoietic precursor cells and large electronlucent cells with irregularly shaped nuclei. The latter cells are also found in the deeper presumptive medullary regions of the thymus; they differentiate into interdigitating reticulum cells (IDC). The permeation of the medulla of the thymus by non-epithelial IDC occurs concurrently with the formation of cortical and medullary epithelial cells. Between the 12th and 14th g.w. the cortical and medullary differentiation is completed. At this time-stage cortical small lymphocytes differ in morphological shape from medullary lymphocytes, the latter acquiring the appearance of immunocompetent T cells and establishing intimate contact with the IDC.These findings indicate that the thymic cortex and medulla contain different epithelial cells. In addition, the thymic medulla displays cells characterized by the morphology of typical interdigitating reticulum cells of peripheral lymphoid tissue. The structural pattern of the thymus is correlated to morphologically differing lymphoid cell populations in the cortical and medullary regions.This investigation was supported by grants from the Deutsche Forschungsgemeinschaft and by the Sonderforschungsbereich 111The authors dedicate this paper to Professor Helmut Leonhardt on the occasion of his 60th birthday. The authors also appreciate the excellent technical assistance of Mrs. I. Knauer, Mrs. H. Waluk and Mrs. H. Siebke     

On the Role of the Reticular-epithelial Complex in Transplantation of the Thymus

Transplantation of the neonatal thymus, into young, adult hosts, resulted in massive cell death of graft cortical lymphoid tissue with apparent selective survival of the reticular-epithelial cells. The central area of the graft was progressively cleared of cell debris and the characteristic thymic architecture restored within fourteen days of grafting. Evidence obtained from the regeneration of different-sized transplants suggested that the size and shape attained by the regenerated graft was closely related to the size and shape of the donor tissue.
When donor rat thymuses were transplanted in Millipore chambers, the lymphocyte population did not reappear and after seven days only reticular-epithelial cells remained, retaining their normal appearance. However, when these thymic remnants were removed from the chambers and transplanted into secondary hosts, the thymus regenerated normally, suggesting that the lymphocytes in the regenerated gland were derived from the host. Thymic remnants after cortisol treatment of donors also formed distinct organs after grafting despite the fact that they contained few donor lymphocytes. From the differential effects of cortisol on host and transplanted thymus and the different growth characteristics of transplants it appears that transplants differ in their growth/involution control system from the host thymus.     

Brain-associated-theta antiserum. Differential effects on lymphocyte subpopulations

Subpopulations of lymphocytes were delineated, both in vitro and in vivo, using brain associated-θ antisera. Lymphocytes were depleted from the cortical areas of the thymus and the T-dependent regions of the spleen. No depletion occurred in the medullary area of the thymus or in the deep cortical areas of the lymph node. These histological findings correlated well with the effects of brain associated-θ antisera using an in vitro cytotoxicity assay. Greater concentrations of antisera were required to cause cell death of lymphocytes from lymph node than lymphocytes from either spleen or thymus. These data would suggest that the θ antigen may be either lost or modulated during the process of lymphocyte differentiation.     

Cytoarchitecture of the Xenopus thymus following gamma-irradiation

This paper describes in vitro and in vivo attempts to deplete the 4- to 8-month-old Xenopus laevis (J strain) thymus of its lymphocyte compartment. Gamma irradiation (2-3000 rad) of the excised thymus, followed by two weeks in organ culture, is effective in removing lymphocytes, but causes drastic reduction in size and loss of normal architecture. In contrast, in vivo whole-body irradiation (3000 rad) and subsequent in situ residence for 8-14 days proves successful in providing a lymphocyte-depleted froglet thymus without loss of cortical and medullary zones. In vivo-irradiated thymuses are about half normal size, lack cortical lymphocytes, but still retain some medullary thymocytes; they show no signs of lymphocyte regeneration when subsequently organ cultured for 2 weeks. Light microscopy of 1 micron, plastic-embedded sections and electron microscopy reveal that a range of thymic stromal cell types are retained and that increased numbers of cysts, mucous and myoid cells are found in the thymus following whole-body irradiation. In vivo-irradiated thymuses are therefore suitable for implantation studies exploring the role of thymic stromal cells in tolerance induction of differentiating T lymphocytes.     

Carbonic anhydrase activity in peripheral T-lymphocytes and appearance of the activity during their maturation in the thymus. A histochemical demonstration

Carbonic anhydrase (CA) activity is demonstrated in lymphoid tissue for the first time using the histochemical (Hansson's) method. A CA-positive reaction was seen in lymphocytes present in T-lymphocyte areas in both the lymph node and the spleen. The most intense staining was seen in the small T-lymphocytes, whereas the medium-sized T-lymphocytes were less markedly stained. The cortical lymphocytes in the thymus were completely devoid of staining, but the small medullary T-lymphocytes stained intensely. The results suggest that peripheral and thymic medullary T-lymphocytes contain CA activity, which appears in these cells during their maturation in the thymus.     

Density-dependent cell division after cortisol treatment of rat thymus in relation to age involution.

Cellular proliferation, in relation to cell density was investigated in the thymus of control and cortisol treated animals at 6 and 18 weeks of age. It was found that there was very little difference in the response of the two age groups to cortisol treatment. Cell density and cellular proliferation were markedly reduced 2 days after cortisol administration. From 4 days there was a rapid increase in cellular proliferation to triple the control rate. The mitotic index remained above normal until 12days then decreased to control values at 14 days. During this time the cell density of the thymus was being progressively restored. At all stages of regeneration, the mitotic index at first increased to a maximum at the mean cell density then decreased at the highest cell concentrations. A model system is discussed to account for this density dependent control of cellular proliferation in the thymus.     

Carbonic anhydrase activity in peripheral T-lymphocytes and appearance of the activity during their maturation in the thymus

Summary Carbonic anhydrase (CA) activity is demonstrated in lymphoid tissue for the first time using the histochemical (Hansson's) method. A CA-positive reaction was seen in lymphocytes present in T-lymphocyte areas in both the lymph node and the spleen. The most intense staining was seen in the small T-lymphocytes, whereas the mediumsized T-lymphocytes were less markedly stained. The cortical lymphocytes in the thymus were completely devoid of staining, but the small medullary T-lymphocytes stained intensely. The results suggest that peripheral and thymic medullary T-lymphocytes contain CA activity, which appears in these cells during their maturation in the thymus.     

Fine structure of small lymphocytes in the thymus of the mouse: Qualitative and quantitative analysis by electron microscopy

Summary Thymic small lymphocytes of dd-mice were qualitatively and quantitatively studied by electron microscopy. Differences in fine structure were revealed between cortical and medullary small lymphocytes. Cortical small lymphocytes are rounded in cell outline with a round nucleus. The cytoplasm surrounding the nucleus as a narrow rim is scanty and appears relatively dense due to an abundance of free ribosomes. The cell organelles are not well developed. On the other hand, medullary small lymphocytes are more irregular in shape with uneven cell membranes. Their nuclei are also more irregular in outline with frequent infoldings of the nuclear membrane. The cytoplasm is more abundant and paler with less numerous ribosomes. The cell organelles are better developed.Quantitative analysis was made of both cortical and medullary small lymphocytes by means of the point counting method. The nuclei of both cortical and medullary small lymphocytes are almost the same in size (a diameter of 4.9 ). The cell sizes are different between cortical and medullary lymphocytes: cortical small lymphocytes with a diameter of 5.5 were smaller than medullary ones with a diameter of 6.4 .Cortical small lymphocytes are very sensitive to the destructive effects of hydrocortisone, whereas the medullary ones are resistant.Periarteriolar lymphoid sheath in the splenic white pulp, which is known to be a thymus-dependent area, contains small lymphocytes that were similar in cytological details to medullary small lymphocytes of the thymus.In the light of the recent knowledge about a recirculating long-lived small lymphocyte pool, it appears probable that medullary small lymphocytes represent a contribution to the pool and that small lymphocytes with a long life span can be cytologically identified by electron microscopy.     

A morphometrical study of human fetal thymus

A morphometrical study was made of the thymus lobules in the cortical and medullary layers of male and female fetuses, 16 to 31 weeks old, divided into the age groups 16 to 19, 20 to 23, 24 to 27 and 28 to 31 weeks of intrauterine life. Results were correlated with body and thymus weights; the lobular, cortical and medullary volumes, as obtained using a histometrical method, were proportional to thymus, but not body weight increase. No differences were noted in the volumes and weights investigated as a function of fetal sex.     

Maturational impairment of thymic lymphocytes in streptozotocin-induced diabetes in rats

Streptozotocin (STZ)-induced diabetes in rats was associated with marked decreases in thymus weight and the number of thymic lymphocytes. Histologically, the cortical lymphocytes which were present near the cortico-medullary junction in the thymus seemed to be reduced selectively in the STZ-induced diabetes. Rosette-forming cells, which bind to guinea pig erythrocytes in the presence of fetal calf serum, were also significantly decreased. Insulin treatment allayed these intrathymic changes. Preincubation of thymic lymphocytes from diabetic rats with thymosin fraction 5 significantly enhanced the percentage of rosette-forming cells to near the control level. These results suggest that a maturational impairment of thymus cortical lymphocytes may be caused in STZ-induced diabetes with hypoinsulinemia and it may be intimately related to reductions in thymus weight and the number of thymic lymphocytes.     

Immunoendocrine interrelationship--effect of ovariectomy on spleen and thymus in mouse.

Increase in the weights of spleen were observed 15 days after ovariectomy in adult as well as immature mice. However, no corresponding change was recorded in the splenocyte count. Morphological alterations in the shape of germinal centres was noted after ovariectomy in adult and immature mice. Increase in weight of the thymus could be demonstrated in adult mice only, with a concomitant enlargement of cortical and medullary region.     

Viral infection of the thymus.

We have examined infection of the thymus during congenitally acquired chronic lymphocytic choriomeningitis virus (LCMV) infection of mice, a classic model of antigen-specific T-cell tolerance. Our results show that (i) infection starts at the fetal stage and is maintained throughout adulthood, and (ii) this chronic infection of the thymus can be eliminated by transfer of virus-specific cytotoxic T lymphocytes (CTL) that infiltrate the thymus and clear all viral products from both medullary and cortical regions. Elimination of virus from the thymus results in abrogation of tolerance. During the fetal stage, the predominant cell type infected is the earliest precursor of T cells with a surface phenotype of Thy1+ CD4- CD8- J11d+. In the adult thymus, infection is confined primarily to the cortisone-resistant thymocytes present in the medullary region. The infected cells are CD4+ and J11d+. The presence of J11d, a marker usually associated with immature thymocytes, on infected single positive CD4+ "mature" thymocytes is intriguing and suggests that infection by this noncytolytic virus may affect development of T cells. There is minimal infection of the CD8+ medullary thymocytes or of the double positive (CD4+ CD8+) cells present in the cortex. Infection within the cortex is confined to the stromal cells. Interestingly, there is infection of the double negative (CD4- CD8-) thymocytes in the adult thymus, showing that even during adulthood the newly developing T cells are susceptible to infection by LCMV. Virus can be eliminated from the thymuses of these carrier mice by adoptive transfer of medullary region first and then from the thymic cortex. This result clearly shows the need to reevaluate the widely held notion that mature T cells are unable to reenter the thymus. In fact, in our experiments the donor T cells made up to 20 to 30% of the total cells in the thymus at 5 to 7 days after the transfer. The number of donor T cells declined as virus was eliminated from the thymus, and at 1 month posttransfer, the donor T cells were hardly detectable. The results of this study examining the dynamics of viral infection and clearance from the thymus, the primary site of T-cell development, have implications for understanding tolerance induction in chronic viral infections.     

The distribution of adenosine deaminase among lymphocyte populations in the rat.

Adenosine deaminase (ADA) activity was determined in young rat lymphocyte populations. The ADA-specific activity (per 10(8) cells and per milligram protein) was 3- to 10-fold higher in thymocytes than in lymphocytes from thoracic duct, lymph node, spleen, and bone marrow. The high ADA activity in thymocytes appeared to be preferentially associated with cortical thymocytes. Enrichment or depletion of cortical thymocytes by density gradient centrifugation, cortisone treatment, or selective lysis with anti-Thy-1 plus complement resulted in parallel increases or decreases in ADA levles. These results also suggested that medullary thymocytes have ADA levels similar to those of peripheral lymphocytes. "Immature" cortical thymocytes and thymocyte progenitors appeared to have low ADA activity; low enzyme levels were found in fetal thymus at 16 days of embryonic life, in the early phases of thymus regeneration, and in a "null" cell population isolated from bone marrow. This study demonstrates that ADA activity varies markedly during T lymphocyte differentiation and suggests that fundamental differences in nucleotide metabolism may exist in T cells at different stages of development.     

Precursor immigration and thymocyte succession during larval development and metamorphosis in Xenopus

The developing thymus in Xenopus was examined at four different levels: 1) precursor immigration of cytogenetically distinct embryonic stem cells; 2) waves of colonization during tadpole life and metamorphosis; 3) inter-thymic exchange of cells between separate lobes; and 4) development of cortical and medullary thymocytes. Based on the flow cytometric analysis of cytogenetically distinct thymocytes, there were at least two periods of stem cell immigration into the thymus, one during early larval life and the second before or during metamorphosis. Within the thymus, cohorts of cells derived from the first wave of immigration expanded at different times. The initial expansion occurred before 35 days of development. Cells involved in the second period of expansion were also derived from the initial immigrants, expanded after 35 days, and resulted in a turnover of thymocytes during the larval period. Precursor cells entering the thymus during metamorphosis expanded and resulted in an additional replacement of thymocytes. Cortical and medullary thymocytes were isolated from animals that received embryonic stem cell grafts. No differences in the presence or absence, or in the percentages, of donor thymocytes in these different fractions were observed. When limiting numbers of stem cells were transplanted, several cases of asymmetrical thymic lobe colonization were observed. These data suggested that an inter-thymic exchange of cells did not occur during larval life.     

A homing receptor-bearing cortical thymocyte subset: implications for thymus cell migration and the nature of cortisone-resistant thymocytes

The thymus exports a selected subset of virgin T lymphocytes to the peripheral lymphoid organs. The mature phenotype of these thymus emigrants is similar to that of medullary thymocytes and has been cited as supporting a medullary rather than cortical exit site. Using the monoclonal antibody MEL-14, we identify a 1%-3% subpopulation of thymocytes that expresses high levels of a receptor molecule involved in lymphocyte homing to peripheral lymph nodes. We present evidence that these rare MEL-14hi thymocytes are predominantly of mature phenotype and represent the major source of thymus emigrants. Surprisingly, MEL-14hi thymocytes are exclusively cortical in location, although their mature phenotype may allow them to masquerade as medullary cells in conventional studies. We also demonstrate that unlike medullary thymocytes, many cortisone-resistant thymocytes (CRT) are MEL-14hi. Thus, in contrast to current dogma, CRT do not represent a sample of medullary thymocytes as they are found in situ and their level of immunocompetence does not necessarily reflect that of the medullary population. Our findings refute the hypothesis that phenotypically and functionally mature cells are restricted to the medulla, and support our proposition that most thymus emigrants are derived from the MEL-14hi cortical subset.     

Enzymatic and morphological response of the thymus to drugs in normal and zinc-deficient pregnant rats and their fetuses

In the thymus of normally fed pregnant rats the plasma membrane enzymes dipeptidyl peptidase IV (DPP IV) and alkaline phosphatase (alP) were found in cortical and medullary lymphocytes (thymocytes). Plasma membrane aminopeptidase A (APA) and adenosine monophosphate hydrolysing phosphatase (AMPP) were present in cortical reticular cells. In medullary reticular cells, aminopeptidase M (APM), gamma-glutamyl transferase (GGT), adenosine triphosphate (ATPP) and thiamine pyrophosphate (TPPP) cleaving phosphatases were detected. Medullary reticular cells did not contain APA. Lysosomal DPP I and II, acid phosphatase, acid beta-D-galactosidase, beta-D-N-acetyl-glucosaminidase, beta-D-glucuronidase and non-specific esterases occurred especially in macrophages at the corticomedullary junction. The 21-day-old fetal thymus showed a similar reaction pattern as the maternal organ except for APA which was absent before birth. After treatment of the pregnant rats with valproic acid (VPA), salicylic acid (SA), streptozotocin (ST) and retinoic acid (RA) APA showed an increase in activity in the thymic cortex. In addition, ST and RA induced AMPP, ATPP and TPPP activity in cortical reticular cells up to the same pattern as in medullary reticular cells. After ethanol (ET) administration severe damages occurred. The thymic cortex was free of DPP IV-positive lymphocytes; the medullary reticular cells showed reduced or no GGT and occasionally an increased APM activity. Dexamethasone (DEXA) given to normal or zinc-deficient rats produced the most severe lesions; thymocytes with DPP IV activity were completely absent in the cortex and medulla. In Zn-deficient pregnant rats similar alterations were observed as after ET.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2'-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20-40% and 60-80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF. The majority of thymocytes present at Day 9 and at Day 14 in inhibitor-treated rats had the characteristics of subcapsular cortical thymocytes which are probably the most ancestral of the thymocytes. Thus, an induced ADA deficiency blocked the proliferation and differentiation of subcapsular cortical thymocytes which are the precursors of cortical and medullary thymocytes.     

Lymphocytes in thymomas are tolerant to self-MHC.

Neoplastic epithelial cells of thymoma apparently retain the function of the cortical epithelial cells of normal thymus because a large number of nonneoplastic T cells in thymomas are often CD4+8+. However, the lack of medullary structure suggests that thymomas may lack some of the function of the normal thymus, especially the function of the medullary interdigitating cells to induce tolerance to self-antigens on T cells. Thymoma is often associated with autoimmune diseases, most frequently, myasthenia gravis. This suggests that the microenvironment of a thymoma may not be able to induce T cell tolerance to self-antigens. We addressed this question by testing the lymphocytes in thymomas for proliferative responses to mitogens and allogeneic or autologous stimulator cells. The lymphocytes in thymomas proliferated consistently in response to PHA and to allogeneic cells even when the response to OKT3 was undetectable. However, neither the thymoma lymphocytes nor the peripheral blood lymphocytes in these patients proliferated in response to autologous cells.