Age-related deregulation of Aire and peripheral tissue antigen genes in the thymic stroma of non-obese diabetic (NOD) mice is associated with autoimmune type 1 diabetes mellitus (DM-1)

Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed ""promiscuous gene expression"" (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80(+) mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Inducible gene expression in fetal thymic epithelium: A new BAC transgenic model.

The thymus is the site of T cell development. Several stromal and hematopoietic cell types are necessary for the proper function of thymic selection and eventually peripheral immunity. Thymic epithelial cells (TECs) are essential for T cell lineage commitment, expansion, and maturation in the thymus. We were interested in developing an in vivo model in which exogenous gene expression could be transiently induced in embryonic TEC (Tet-On system). To this end, we have generated a bacterial artificial chromosome (BAC) transgenic mouse line in which the reverse tetracycline-dependent transactivator (rtTA) is expressed under the control of the Foxn1 promoter, a transcriptional factor indispensable for TEC development. To analyze the expression pattern and efficiency of this novel mouse model, we crossed the Foxn1-rtTA founder with a Tet-Responsive Element (TRE)-LacZ GFP mouse reporter to obtain a double transgenic mouse. In the presence of doxycycline, rtTA can interact with TRE and induce the expression of GFP and LacZ. In this double transgenic mouse, we observed that GFP expression was high, inducible and limited to TEC in fetal thymus. In contrast, in adult thymus, when TEC development and maturation is completed, GFP was barely detectable. Therefore, Foxn1-rtTA represents a new and efficient transgenic mouse model to induce genes of interest specifically in fetal thymic epithelium. genesis 51:717-724. © 2013 Wiley Periodicals, Inc.

Thymus involution in alloxan diabetes: analysis of mast cells

We previously reported that alloxan-induced diabetes results in reduction in the number and reactivity of mast cells at different body sites. In this study, the influence of diabetes on thymic mast cells was investigated. Thymuses from diabetic rats showed marked alterations including shrinkage, thymocyte depletion, and increase in the extracellular matrix network, as compared to those profiles seen in normal animals. Nevertheless, we noted that the number and reactivity of mast cells remained unchanged. These findings indicate that although diabetes leads to critical alterations in the thymus, the local mast cell population is refractory to its effect. This suggests that thymic mast cells are under a different regulation as compared to those located in other tissues.

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.

DL4-mediated Notch signaling is required for the development of fetal αβ and γδ T cells.

T-cell development depends upon interactions between thymocytes and thymic epithelial cells (TECs). The engagement of delta-like 4 (DL4) on TECs by Notch1 expressed by blood-borne BM-derived precursors is essential for T-cell commitment in the adult thymus. In contrast to the adult, the earliest T-cell progenitors in the embryo originate in the fetal liver and migrate to the nonvascularized fetal thymus via chemokine signals. Within the fetal thymus, some T-cell precursors undergo programmed TCRγ and TCRδ rearrangement and selection, giving rise to unique γδ T cells. Despite these fundamental differences between fetal and adult T-cell lymphopoiesis, we show here that DL4-mediated Notch signaling is essential for the development of both αβ and γδ T-cell lineages in the embryo. Deletion of the DL4 gene in fetal TECs results in an early block in αβ T-cell development and a dramatic reduction of all γδ T-cell subsets in the fetal thymus. In contrast to the adult, no dramatic deviation of T-cell precursors to alternative fates was observed in the fetal thymus in the absence of Notch signaling. Taken together, our data reveal a common requirement for DL4-mediated Notch signaling in fetal and adult thymopoiesis.

Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment.

Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor-mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.

An adult thymic stromal-cell suspension model for in vitro positive selection.

Presented here is a cell-suspension model for positive selection using thymocytes from alphabeta-TCR (H-2Db-restricted) transgenic mice specific to the lymphocytic choriomeningitis virus (LCMV) on a nonselecting MHC background (H-2d or TAP-1 -/-), cocultured with freshly isolated adult thymus stromal cells of the selecting MHC type. The thymic stromal cells alone induced positive selection of functional CD4- CD8+ cells whose kinetics and efficiency were enhanced by nominal peptide. Fibroblasts expressing the selecting MHC alone did not induce positive selection; however, together with nonselecting stroma and nominal peptide, there was inefficient positive. These results suggest multiple signaling in positive selection with selection events able to occur on multiple-cell types. The ease with which this model can be manipulated should greatly facilitate the resolution of the mechanisms of positive selection in normal and pathological states.

Notch1 deficiency dissociates the intrathymic development of dendritic cells and T cells.

Thymic dendritic cells (DCs) form a discrete subset of bone marrow (BM)-derived cells, the function of which is to mediate negative selection of autoreactive thymocytes. The developmental origin of thymic DCs remains controversial. Although cell transfer studies support a model in which T cells and thymic DCs develop from the same intrathymic pluripotential precursor, it remains possible that these two types of cells develop from independent intrathymic precursors. Notch proteins are cell surface receptors involved in the regulation of cell fate specification. We have recently reported that T cell development in inducible Notch1-deficient mice is severely impaired at an early stage, before the expression of T cell lineage markers. To investigate whether development of thymic DCs also depends on Notch1, we have constructed mixed BM chimeric mice. We report here that thymic DC development from Notch1(-/)- BM precursors is absolutely normal (in terms of absolute number and phenotype) in this competitive situation, despite the absence of Notch1(-/)- T cells. Furthermore, we find that peripheral DCs and Langerhans cells are also not affected by Notch1 deficiency. Our results demonstrate that the development of DCs is totally independent of Notch1 function, and strongly suggest a dissociation between intrathymic T cell and DC precursors.

Quantitative impact of thymic clonal deletion on the T cell repertoire.

Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus. Some thymocytes with appropriate receptors are stimulated to undergo differentiation to the fully mature state (positive selection), whereas others with strongly autoreactive receptors are triggered to undergo programmed cell death before completing this differentiation process (negative selection). The quantitative impact of negative selection on the potentially available repertoire is currently unknown. To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection. In such chimeras, the number of mature thymocytes was increased by twofold as compared with appropriate control chimeras This increase in steady-state numbers of mature thymocytes was not related to proliferation, increased retention, or recirculation and was accompanied by a similar two- to threefold increase in the de novo rate of generation of mature cells. Taken together, our data indicate that half to two-thirds of the thymocytes able to undergo positive selection die before full maturation due to negative selection.

Promoter IV of the class II transactivator gene is essential for positive selection of CD4+ T cells.

Major histocompatibility complex class II (MHCII) expression is regulated by the transcriptional coactivator CIITA. Positive selection of CD4(+) T cells is abrogated in mice lacking one of the promoters (pIV) of the Mhc2ta gene. This is entirely due to the absence of MHCII expression in thymic epithelia, as demonstrated by bone marrow transfer experiments between wild-type and pIV(-/-) mice. Medullary thymic epithelial cells (mTECs) are also MHCII(-) in pIV(-/-) mice. Bone marrow-derived, professional antigen-presenting cells (APCs) retain normal MHCII expression in pIV(-/-) mice, including those believed to mediate negative selection in the thymic medulla. Endogenous retroviruses thus retain their ability to sustain negative selection of the residual CD4(+) thymocytes in pIV(-/-) mice. Interestingly, the passive acquisition of MHCII molecules by thymocytes is abrogated in pIV(-/-) mice. This identifies thymic epithelial cells as the source of this passive transfer. In peripheral lymphoid organs, the CD4(+) T-cell population of pIV(-/-) mice is quantitatively and qualitatively comparable to that of MHCII-deficient mice. It comprises a high proportion of CD1-restricted natural killer T cells, which results in a bias of the V beta repertoire of the residual CD4(+) T-cell population. We have also addressed the identity of the signal that sustains pIV expression in cortical epithelia. We found that the Jak/STAT pathways activated by the common gamma chain (CD132) or common beta chain (CDw131) cytokine receptors are not required for MHCII expression in thymic cortical epithelia.

Morphological evidences indicate that the interference of cimetidine on the peritubular components is responsible for detachment and apoptosis of Sertoli cells

Cimetidine, referred as antiandrogenic agent, has caused alterations in the seminiferous tubules, including alterations in the peritubular tissue and death of myoid cells by apoptosis. Regarding the structural and functional importance of the peritubular tissue for the maintenance of Sertoli cells (SC), we purpose to investigate the SC-basement membrane interface, focusing the morphological features of SC and their interaction with the basement membrane in the affected tubules by cimetidine. Ten animals were distributed into two groups, control (CG) and cimetidine (CmG) which received saline solution and 50 mg of cimetidine per kg of body weight, respectively, for 52 days. The testes were fixed, dehydrated and embedded for analyses under light and transmission electron microscopy. Paraffin sections were submitted to the TUNEL method; sections of testes embedded in glycol methacrylate were submitted to PAS method and stained by H&E for morphological and quantitative analyses of Sertoli Cells. In the CmG, the SC nuclei were positive to the TUNEL method and showed typical morphological alterations of cell death by apoptosis (from early to advanced stages). A significant reduction in the number of Sertoli Cells was probably due to death of these cells by apoptosis. A close relationship between SC nuclear alterations (including a high frequency of dislocated nuclei from the basal portion) and damage in the peritubular tissue was observed. The ultrastructural analysis showed a parallelism between the gradual advancement of apoptotic process in SC and detachment of the anchoring sites (hemidesmosomes) of SC plasma membrane from the lamina densa. The presence of portions of lamina densa underlying the detached hemidesmosomes indicates a continuous deposition of lamina densa, resulting in the thickening of the basal lamina. The results indicate a possible disarrangement of the SC cytoskeleton, including the focal adhesion structure. These alterations are related to SC apoptosis and probably result from disturbs induced by cimetidine on the peritubular tissue.

Testicular interstitial cells, and steroidogenic detection in the protogynous fish, Synbranchus marmoratus (Teleostei, Synbranchidae)

The swamp eel, Synbranchus marmoratus, is a freshwater protogynic diandric species. Primary males develop directly as males while secondary males arise from the sex reversal of females. Fishes from Argentine and Brazil inland waters were collected, examined and compared for this study. In order to characterize the interstitial testicular compartment, light and electron microscopy techniques and an enzyme histochemical examination for steroidogenic cells detection were used. The interstitial compartment of S. marmoratus is composed of Leydig and myoid cells, collagen fibers, blood cells, macrophages,and amyelinic nerves. At the ultrastructural level, no differences were observed in the interstitial tissue, either between specimens from the different sampling sites or between primary and secondary males. Leydig cells are present in all testes examined throughout the year. A cytoplasmatic reaction of 3beta-HSD was detected only in Leydig cells during sex reversal and in both type of males, mainly during the regressed and early maturation classes (autumn and winter). Leydig cells possess the typical fine structural characteristics associated with steroidogenesis. Furthermore, in both type of males, during sex reversal and after the spawning period, the number of granulocytes and macrophages present in the testes increased, suggesting that they could be involved in phagocytosis and resorption of damaged cells. (C) 2004 Elsevier Ltd. All rights reserved.

Rhabdomyomatous carcinoma of the thymus

Myoid cells have been described in the thymus in association with a variety of benign and malignant conditions. The presence of a benign rhabdomyomatous component in a malignant primary thymic epithelial neoplasm, however, is extremely rare. A case of poorly differentiated carcinoma of the thymus arising in the posterior mediastinum containing a prominent rhabdomyomatous component is described. The patient, a 70-year-old woman, was seen for a large posterior mediastinal mass. An open chest biopsy revealed an extensively necrotic tumor composed of islands of atypical cells with vesicular nuclei and prominent nucleoli with numerous mitotic figures admixed with abortive glandular structures. Interspersed with the malignant epithelial components were islands of large cells containing brightly eosinophilic cytoplasm and small, round, excentrically placed nuclei. Immunohistochemical studies showed strong positivity of the epithelial cells for cytokeratin and strong positivity of the myoid cells for pan-actin, desmin, and myogenin. The possible relationship of the rhabdomyomatous component of this tumor with the myoid cells of the thymus is discussed.