The finding that more than 152 tissue-restricted antigens are expressed by thymic medullary epithelial cells is redefining the importance of thymic central tolerance induction in the prevention of autoimmune diseases. One of the tissue-restricted antigens in the thymus is proinsulin, and in both mice and humans, reduced thymic proinsulin levels have been shown to predispose to Type 1 diabetes. Using transgenic mice expressing a functional beta-Galactosidase gene under the regulation of the Ins2 promoter we have determined that between 1-3% of all medullary thymic epithelial cells express proinsulin and that these cells are frequently part of the Hassall's Corpuscles like structures in mice. Using a cross between the beta-Galactosidase expressing mice and Immortomice (expressing SV40 large T Antigen under the regulation of the MHC I promoter), we have isolated and cultured two proinsulin and two non-proinsulin producing medullary epithelial cell lines. Microarray analysis and RT-PCR analysis of the cell lines revealed the over-expression of approximately 50 genes (>4 fold or more) in the proinsulin producing lineage, versus the non proinsulin producing lineage, and approximately half the over-expressed genes can be considered tissue-restricted antigens. We do not find any evidence for chromosomal clustering of the over-expressed genes nor do we report the expression of any other pancreatic n-cell antigens or specific pancreatic proinsulin regulatory proteins (Pdx-1, Glut-2 or GCK) within the proinsulin producing cell lines but we do detect their expression in whole thymus. Our results suggest that chromosomal clustering is not a phenomenon associated with thymic tissue-restricted antigen expression and that the mechanisms allowing for thymic tissue-restricted antigen expression are not related to the expression mechanisms of such antigens in peripheral tissues.

No description available.

The development of T cells in the thymus is a tightly regulated process. Any defect in thymic differentiation could result in autoimmune disorders, inability to ward off infections or neoplasm. Early thymocyte development requires signals mediated through the preTCR complex by the associated CD3 chains (gamma, delta, epsilon, and zeta). Research conducted towards this project has revealed that signaling modules within the intracytoplasmic domain of CD3epsilon is absolutely required for this process. Interestingly, our results emphasized the importance of the proline-rich sequence motif in preTCR mediated signaling events, such as the proliferation of double negative thymocytes and the regulation of TCR surface expression on double positive thymocytes in a stage-specific manner. The outcomes of this project may provide a better understanding of the mechanism of preTCR-mediated thymocyte differentiation and the role of CD3 chains in these processes.

No description available.

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.