Normal Adult Thymus Research Articles

Mouse thymic mast cells in normal state and after stress-induced atrophy

Background. Studying of thymic mast cells population in normal state and after stressinduced atrophy. Methods. The study was performed on 80 thymus of white outbred mice with using of histochemistry and immunohistochemistry methods. Sections of embryonic thymus were stained with toluidine blue. Adult mice were given a single injection of 2,5 mg of hydrocortisone for induction of thymic accidental transformation; sections were stained with toluidine blue and alcian blue-safranin. Within of immunohistochemical research paraffin sections of adult thymus were stained with polyclonal antibodies to synaptophysin and tyrosine hydroxylase with alcian blue stain. Results. Mast cells (MCs) appeared in thymus on 19th day of embryonic life and demonstrated mainly a medullar location. In adult animals MCs were observed only in the connective tissue of the capsule, interlobular septa, subcortex and perivascular space. Mast cells matured in thymus after accidental transformation. The localization of developing mast cells was changing from medullar and cortical to capsular. A morphological proximity between nerve terminals and mast cells has been observed in normal adult thymus. Some of these nerves were catecholaminergic. Conclusion. Possible important role of thymic mast cells and mast cellsnerves interaction in normal state and after accidental transformation is discussed.

Morphological and immunohistochemical changes to thymic epithelial cells in the irradiated and recovering rat thymus.

The present study analyzed morphological and immunohistochemical changes of thymic epithelial cells in the irradiated and recovering rat thymus. Observations showed the number of thymocytes was initially severely reduced after irradiation but abruptly increased on days 3 to 5 after 6 Gy and on days 7 to 11 after 8 Gy irradiation. To analyse the mechanisms for this abrupt recovery of the thymocytes after irradiation, the expression of p63 in the normal and irradiated thymus was immunohistochemically studied as the expression of this antigen may be related to the proliferation of epithelial cells. In the fetal thymus tissue, thymic epithelial cells were the principal cell type that stained strongly positive for p63. The sporadic expression of p63 was also observed in the normal adult thymus tissue, especially in the subcapsular region. An increased number of p63- positive cells in the thymus after irradiation indicates that repair or renewal of the thymic epithelial cells may be taking place because p63 is more specific to transient amplifying thymic epithelial cells. A RT-PCR analysis of p63 expression in irradiated and regenerating thymus tissue also showed an increased expression of p63 after irradiation compared with that of the normal thymus. These results suggest that changes in the thymic microenvironment-especially in relation to the repair and renewal of thymic epithelial cells- may have an important influence on thymocyte proliferation in the normal thymus as well as in the irradiated and recovering thymus.

Direct evidence for thymic function in adult humans.

The understanding of human thymic function and evaluation of its contribution to T cell homeostasis are matters of great importance. Here we report the development of a novel assay to quantitate the frequency and diversity of recent thymic emigrants (RTEs) in the peripheral blood of humans. Such cells were defined by the presence of T cell receptor (TCR) rearrangement deletion circles (DCs), episomal byproducts of TCR-beta V(D)J rearrangement. DCs were detected in T cells in the thymus, cord blood, and adult peripheral blood. In the peripheral blood of adults aged 22 to 76 years, their frequency was highest in the CD4(+)CD45RA(+) CD62L(+) subpopulation of naive T cells. TCR DCs were also observed in other subpopulations of peripheral blood T cells, including those with the CD4(+)CD45RO(-)CD62L(+) and CD4(+)CD45RO(+)CD62L(+) phenotypes. RTEs were observed to have more than one Vbeta rearrangement, suggesting that replenishment of the repertoire in the adult is at least oligoclonal. These results demonstrate that the normal adult thymus continues to contribute, even in older individuals, a diverse set of new T cells to the peripheral circulation.

Early Intrathymic Precursor Cells Acquire a CD4low Phenotype

AbstractCD4low cells are a population of lymphoid lineage-restricted progenitor cells representing the earliest precursors present in the adult thymus. Paradoxically, thymic progenitors with a similar phenotype in fetal mice and adult RAG-2-deficient (RAG-2−/−) mice lack this characteristic low-level expression of CD4. We now show that radiation-induced differentiation of CD4+CD8+ double positive thymocytes in RAG-2−/− mice results in the appearance of low levels of CD4 on thymocytes that are phenotypically identical to CD4low progenitor cells present in the normal adult thymus. This suggests that CD4 surface expression can be passively transferred from double positive cells to early progenitor thymocytes. Analysis of mixed bone marrow chimeras, reconstituted with hematopoietic stem cells from both CD4−/− (CD45.2) and CD4wt (CD45.1) congenic mice, revealed a CD4low phenotype on cells derived from CD4−/− bone marrow cells. Furthermore, these CD4−/−-derived “CD4low” progenitors were capable of reconstituting lymphocyte-depleted fetal thymi, with all thymocytes displaying a CD4−/− phenotype. This directly demonstrates that genetically CD4-deficient thymic progenitor cells can passively acquire a CD4low phenotype. Moreover, CD4 expression on CD4low progenitor thymocytes is sensitive to mild acid treatment, indicating that CD4 may not exist as an integral cell surface molecule on this thymocyte population. Our findings demonstrate that low-level CD4 surface expression can be passively acquired by intrathymic progenitor cells from the surrounding thymic microenvironment, suggesting that other cell surface molecules expressed at low levels may also result from an acquired phenotype.

Altered major histocompatibility complex restriction in the NK1.1+Ly-6Chi autoreactive CD4+ T cell subset from class II-deficient mice.

We previously demonstrated selective enrichment of major histocompatibility complex (MHC) class II-specific autoreactive T cells in a subset of mouse CD4+ thymocytes. Here we show that a significant fraction of these autoreactive cells in the normal adult thymus expresses NK1.1 and high levels of Ly-6C and also exhibits flexibility in MHC restriction. In normal mice, this NK1.1+Ly-6Chi subfraction accounts for 10-50% of the CD4+ autoreactive subset and is enriched for MHC class II-restricted autoreactive cells as determined by mixed leukocyte reaction frequency analysis, similar to NK1.1-Ly-6C-CD4+ autoreactive cells. In contrast, in the thymus of class II-deficient littermate mice, NK1.1+Ly-6Chi cells account for most of the mature heat stable antigen (HSA)-CD4+ fraction and exhibit MHC-restricted non-class II autoreactivity. Thus, NK1.1+Ly-6ChiCD4+ T cells show flexibility in MHC class restriction, but their autoreactivity remains MHC dependent.

Developmental and Anatomical Patterns Of IL‐2 Gene Expression in Vivo in The Murine Thymus

Interleukin-2 (IL-2) is a potent growth factor that mature T lymphocytes synthesize and use as a proliferation signal. Much controversy has arisen concerning whether it is used to drive the extensive proliferation of immature pre-T cells in the thymus. Immature thymocytes acquire the competence to express IL-2 at an early stage, but it has remained uncertain whether they are activated to exercise this competence in vivo. Therefore, we have used in situ hybridization and immunohistochemistry on serial sections obtained from fetal and adult thymuses of normal C57BL/6 mice and of mice bearing the scid defect to determine where, when, and whether IL-2 is expressed in vivo. Our results show a striking spatial and temporal pattern of IL-2 expression in the normal fetal thymus. We detected a burst of IL-2 mRNA accumulation at day 14.5 of gestation, which rapidly decreased by day 15. At day 15, we observed maximal IL-2 protein production that subsequently decreased by day 16 of gestation. Both in situ hybridization and immunohistochemical staining revealed an unexpectedly strict localization of IL-2 expressing cells to patches around the periphery of the fetal thymus, creating a previously unrecognized compartment of high IL-2 protein content. IL-2 production in the day-15 fetal thymus appeared to be unaffected by the scid mutation, indicating that this response is likely to be T-cell receptor (TcR)-independent. Several features distinguish the IL-2 induction pattern in the adult thymus from that in the fetal thymus. In the normal adult thymus, IL-2-expressing cells are extremely rare (found at a frequency of 10-7), but they are reproducibly detectable as isolated cells in the outer cortex and subcapsular region of the thymus. Unlike the fetal thymic IL-2 producers, the IL-2 producers in the adult thymus are completely eliminated in mice homozygous for the scid mutation. This suggests that the IL-2-expressing cells in the normal adult thymus are of a more mature phenotype than the immature, TcR-negative cells that accumulate in the scid adult thymus. Thus, our work demonstrates that two developmentally distinct types of cell interactions induce IL-2 expression in vivo: one, a broadly localized interaction in day 14‑15 fetal thymus that is unaffected by the scid mutation; the other, a rare event that occurs asynchronously from late fetal through adult life, but which is completely eliminated by the scid defect. These results imply that significant differences exist between the physiological processing of thymocytes in the fetal and postnatal thymic microenvironments.

Modulation of cytokeratin expression in the hamster thymus: evidence for a plasticity of the thymic epithelium.

Cytokeratin (CK) expression was investigated, by means of immunocytochemistry, in the hamster thymic epithelium during ontogeny, as well as in primary cultures and upon glucocorticoid hormone treatment in vivo. As compared to the distribution pattern of distinct monoclonal antibody-defined cytokeratins in the normal adult thymus, CK modulation was evidenced in the three situations studied. During thymus ontogeny, both cytokeratins of simple lining epithelia, as CK8 and CK18, as well as the CK1/CK10 pair (typical marker of terminal stage of keratinization), were expressed since early stages of thymus development. They were located in the central region of thymic lobules preceding the cortical-medullary distinctions. This differed from what had been previously shown for mouse thymus ontogeny, revealing that the interspecific diversity in the distribution pattern of thymic cytokeratins occurred early in fetal life. A modulation of CK expression was also detected when hamster thymic epithelial cells (TEC) were led to grow in culture, with a down-regulation of CK19 contrasting with an enhancement of CK18 expression. This diverged from the maintenance of the in situ pattern when human TEC were cultured. Last, in vivo hydrocortisone treatment, known to increase the numbers of KL1+ cells in the mouse thymus medulla, promoted a cortical expression of the CK1/CK10 pair in the hamster thymus. Taken together, our findings demonstrate a continuous plasticity of the thymic epithelium, at least regarding cytokeratin expression, and enlarge the concept of interspecific diversity of intrathymic CK distribution in conditions as morphogenesis, in vitro system, and responsiveness to glucocorticoid hormone treatment.

Sequential appearance of thymocyte subpopulations and T cell antigen receptor gene messages in the mouse thymus after sublethal irradiation.

The sequential differentiation patterns of thymocyte were observed with cell surface phenotypes and the expression of T cell antigen receptor in 800 rad irradiated adult mice. Thymus was severely reduced in size and cell number by day 5 after whole body irradiation and rapidly recovered from day 7 to day 14. Surface marker analysis on day 5 after irradiation showed thymocytes with Thy-1low L3T4+/Lyt-2- dominantly existed and suggested that these cells were radioresistant-survived cells. On the other hand, thymocytes on day 7 were composed of a large number of Thy-1high L3T4+/Lyt-2+ blast-like cells and a relatively high proportion of Thy-1high L3T4-/Lyt-2- cells which expressed a large amount of gamma-chain gene messages but scarcely any alpha- and beta-chain gene messages similar to the fetal thymocytes. On day 14, thymocytes were composed mostly of Thy-1high H-2low L3T4+/Lyt-2+ subpopulation which expressed a remarkably low level of gamma-chain gene messages, and high levels of alpha- and beta-chain transcripts analogous to those of normal adult thymus. Taken together, intrathymic radioresistent stem cells for T thymocytes seem to proliferate and differentiate after irradiation with the same pattern as was seen in a fetal thymus development.

Dynamics of early T cells: prothymocyte migration and proliferation in the adult mouse thymus.

The object of this review has been to consider precursor cell migration into the normal adult thymus, using the mouse model. We have presented a series of experiments and discussed them in the context of other relevant experiments in the literature. The conclusions, qualified in the text, can be summarized as follows: There is a continual input of precursor cells into the normal undepleted adult thymus. The daily input of precursors under normal circumstances is very low (e.g. several per day). Once a precursor enters the pool of proliferating cells inside the thymus, its proliferation is limited to only several weeks. There is no permanent endogenous stem cell. There are a number of different precursor microenvironments in the thymus with different controls, since the kinetics of early (bone marrow-derived) and late (thymus-derived) precursors is quite different. All of these points require further analysis, and we have presented a minimal model as a basis for further experiment.

Tumor-specific antigen of murine T-lymphoma defined with monoclonal antibody.

A panel of hybridomas was constructed by fusion of P3X63Ag8 myeloma cells with spleen cells from a BALB/c mouse that had been immunized with a C57BL/Ka x-ray-induced lymphoma, C6XL. One of forty-three hybridomas secreting antibodies reactive with the tumor cells was found to be unreactive with normal spleen cells in a radioimmunometric assay. This antibody, designated 124-40, was unreactive with normal adult thymus, spleen, lymph node, or bone marrow cells, or with fetal spleen or thymus cells in radioimmunometric or radioimmunoprecipitation assays. Flow microfluorometric analysis of these nonmalignant lymphoid cells failed to reveal subpopulations reactive with MAb 124-40. The antibody was highly specific for the lymphoma cells used for immunization and did not react with a panel of other spontaneous or x-ray-induced or chemically induced lymphomas. The antigen reactive with MAb 124-40 was isolated by radioimmunoprecipitation and found to be a glycoprotein composed of disulfide-bonded subunits of 39,000 m.w. and 41,000 m.w. A cell surface component of similar structure, but not reactive with MAb 124-40 could be detected by two-dimensional electrophoresis in extracts of purified T cells, but not B cells. These results suggest that the apparently individually specific lymphoma antigen reactive with MAb 124-40 might be a clonally expressed epitope carried by a T cell surface component.

Development of cytolytic T lymphocyte precursors in organ-cultured mouse embryonic thymus rudiments.

The appearance of immunologically competent cells in the organ-cultured mouse fetal thymic rudiment has been investigated. Fetal thymuses removed at 14 d of gestation and cultured for 7-21 d were assayed for their content of cytolytic T lymphocyte precursors (CTL-P) directed against H-2d alloantigens. Whereas CTL-P were undetectable within fetal thymus until 18-19 d of gestation, their frequency in the organ-cultured fetal thymus was similar to, or greater than that found in the normal adult thymus. This direct demonstration of the appearance of alloreactive CTL-P in a closed in vitro system should provide an accessible model for the investigation of interactions between developing T cells and the thymic microenvironment.

Development of thymus, parathyroids, and ultimo-branchial bodies in NMRI and nude mice.

AbstractIn the NMRI mouse embryo, the thymus develops from the third endodermal pouch and the third ectodermal cleft. The cervical vesicle, formed not by the closure of the sinus cervicalis but by an invagination of the dorsal segment of the ectoderm between the third and fourth branchial clefts, contributes to the formation of the thymus. The intense proliferation of the ectoderm of the third cleft on the eleventh day covers the endodermal part.The thymus is thus composed of a central endodermal region and a peripheral ectodermal region. The normal adult thymus, then, has a mixed origin, the cortical cells being ectodermal, and the medullary cells endodermal in origin.The fourth endodermal pouch gives rise to the ultimo‐branchial body, which becomes entirely incorporated within the thyroid on the fourteenth day. There is no formation of a thymus IV nor of a second pair of parathyroids from the fourth endodermal pouch.In the Nude mouse embryo, the third branchial pouch and cleft, as well as the cervical vesicle, develop normally for the first 11½ days. From this point on, the ectoderm of the third cleft ceases to develop further. The endoderm is, now, no longer covered by ectoderm and, deprived of its normal inducing agent, ceases to develop further. Thymic dysgenesis is thus ectodermal in origin. The fourth endodermal pouch develops normally. No development of an accessory “thymus‐like” structure from the fourth pouch was observed. The dysgenetic thymus originates entirely from the third branchial pouches and clefts.In both Nude and NMRI embryos, the parathyroids develop at about 11½ days from a very limited area in the dorsal region of the cranial wall of the third endodermal pouch between the pharyngo‐branchial and ecto‐branchial ducts. Morphometric analysis shows that the volume of the parathyroids is the same in both strains of mice at each stage of development; nor does their microscopic appearance differ. Thus, mutation in the Nude mouse does not affect the development of the parathyroids from the third pouch, even though the first anomalies in the development of the thymus are observed at the precise moment at which the parathyroid primordium appears.

An ultrastructural study of nine thymomas

Nine thymomas, a normal adult thymus, and tissue culture of one thymoma were studied ultrastructurally. The histologic types of thymoma included lymphocytic, epithelial, mixed and spindle cell varieties. Two of the tumors were invasive and one was associated with myasthenia gravis. Despite the histologic dissimilarities, the complex anatomic interrelationships involving lymphocytes, epithelial cells, and blood vessels as seen in the thymus tended to be preserved in all but one of the thymomas. The exception was the spindle-cell thymoma which contained only rare lymphocytes and simpler vascular structures. The findings include variations in the frequency of demosomes and cytoplasmic fibrils of the epithelial cells, the occasional presence of lymphocytes within the laminated venules, and the unusual finding of a gland-like structure in one of the tumors. Correlations between ultrastructural changes and clinical behavior could not be made.