Ep-CAM transfection in thymic epithelial cell lines triggers the formation of dynamic actin-rich protrusions involved in the organization of epithelial cell layers.

Thymic epithelium is organized in a highly connected three-dimensional network through which thymocytes differentiate. The molecular mechanisms underlying this organization are still unknown. In thymic medulla, a major site of tolerance induction, the development of the epithelial cell net is tightly regulated by the needs of thymocyte selection. These reticulated epithelial cells express high levels of the Ep-CAM molecule. Using different thymic epithelial cell lines as a model system, we found that transfection of Ep-CAM enhances cell growth and leads to a rapid reorganization of the actin cytoskeleton by inducing the formation of numerous stress fibers and long cell protrusions. Finally, the crosslinking of the extracellular domain of a chimeric CD25ec/Ep-CAMic molecule is sufficient to trigger the formation of protrusions. These results suggest that expression of Ep-CAM might balance the organizing capacity of cadherin molecules and may be participating in the formation of a dynamic stromal cell network in the thymus.

Development, organization and function of the thymic medulla in normal, immunodeficient or autoimmune mice.

Thymopoiesis is initiated by the colonisation of the epithelial rudiment with blood-borne hemopoietic precursors. Their subsequent differentiation to the functionally mature T cell subsets is exquisitely linked to sequential interaction with a diverse array of thymic epithelial cells which form discrete microenvironments. The development and organisation of the epithelium, however, is in turn controlled by thymocyte subsets. In particular the medulla organization depends upon activating signals provided by mature thymocytes to epithelial and dendritic cells. These signals are lacking in RelB-deficient mice leading to the disorganization of the corticomedullary junction and abnormal negative selection despite normal thymocyte maturation. This thymic stromal cell architecture phenotype is found in autoimmune diseases suggesting that abnormalities in the establishment of medullary microenvironments might be linked to the development of autoimmunity.

Disorganization of thymic medulla precedes evolution towards diabetes in female NOD mice.

The thymic medulla is a complex microenvironment which plays a crucial role in central tolerance induction. Using a quantitative histological analysis of non-obese diabetic (NOD) mice, we show that the medulla undergoes several structural modifications during the course of the disease in NOD mice. Indeed, the majority of 70-day-old NOD mice show a scattering of medullary epithelial cells in the cortex which is associated with a reduction in the size of the medulla in heavily disorganized thymuses. The severity of this phenotype is shown to correlate with the subsequent appearance of diabetes in older female NOD mice. This trait is mainly controlled by non-major histocompatibility complex NOD genes since C57BL/6 H-2g(7) congenic mice have a normal medulla. It persists in conditions where effector lymphocytes that lead to diabetes are inhibited in periphery. These results suggest that primary alterations of the thymic stroma might play a role in the progression towards diabetes in NOD mice.

Thymocytes and RelB-dependent medullary epithelial cells provide growth-promoting and organization signals, respectively, to thymic medullary stromal cells.

The thymic medulla is composed of distinct epithelial cell subsets, defined in this report by the reactivity of two novel antibodies, 95 and 29, raised against mouse thymic epithelial cell lines. These antibodies were used to probe the development of medulla in wild-type or mutant thymuses. In CD3 epsilon-deficient mice where thymocyte maturation is arrested at the CD4- CD8- stage, few scattered 95+ and 29+ epithelial cells are found. When few mature thymocytes develop as in CD3- zeta/eta mice, expansion and organization of 95+ but not 29+ cells, becomes detectable. In RelB-deficient mice, T cell maturation proceeds normally but negative selection is inefficient due to the lack of thymic medulla and dendritic cells. Strikingly, 29+ epithelial cells are absent and 95+ medullary epithelial cells are scattered throughout the thymus, intermingling with CDR1+ cortical epithelium. In chimeric mice lacking only dendritic cells, the corticomedullary junction persists and both 95+ and 29+ epithelial cells are localized in the medulla. These results suggest that two types of signals are required for development of thymic medulla. A growth signal depends upon the presence of maturing thymocytes, but organization of the thymic medulla requires the presence of activated 29+ medullary epithelial cells.

Modulation of mRNA levels in the presence of thymocytes and genome mapping for a set of genes expressed in mouse thymic epithelial cells.

Modulation of gene expression in mouse thymic epithelium upon culture in the presence of thymocytes (coculture) was studied by comparison of hybridization signatures on a set of nearly 5000 mouse thymus cDNA clones. Forty-nine differentially expressed clones (usually down-regulated in coculture) were characterized by tag sequencing. Many of them corresponded to entities that had not been described previously in the mouse, and were further characterized by genome mapping. This set of genes appears to be involved in growth regulation and differentiation within the thymus.

Deletion of the mouse T-cell receptor beta gene enhancer blocks alphabeta T-cell development.

Intrathymic T-cell development requires temporally regulated rearrangement and expression of T-cell receptor (TCR) genes. To assess the role of the TCR beta gene transcriptional enhancer (Ebeta) in this process, mouse strains in which Ebeta is deleted were generated using homologous recombination techniques. We report that mice homozygous for the Ebeta deletion, whether a selectable marker gene is present or not, show a block in alphabeta T-cell development at the CD4-CD8- double-negative cell stage, whereas the number of gammadelta+ T cells is normal, few CD4+CD8+ double-positive thymocytes and no alphabeta+ T cells are produced. DNA-PCR and RNA-PCR analyses of thymic cells from homozygous mutants showed no evidence of TCR beta gene rearrangement although germ-line Vbeta transcripts were detected at a low level, in heterozygous T cells, the targeted allele is not rearranged. Thus, deletion of Ebeta totally prevents rearrangement, but not transcription, of the targeted beta locus. These data formally establish the critical role played by Ebeta in cis-activation of the TCR beta locus for V(D)J recombination during alphabeta T-cell development.