The heterogeneous epithelial compartment of the thymic environment plays a central but ill defined role in T cell differentiation. In particular, interactions between immature thymocytes and cortical thymic epithelium are thought to mediate the process of positive selection, where thymocytes bearing antigen receptor molecules with low affinity for self peptides are thought to be rescued from programmed cell death by interactions with cortical TE. An understanding of this important aspect of thymocyte differentiation has been hampered by a lack of information regarding cortical epithelial cells; the cytokines produced , potential cell interaction molecules expressed by these cells, and lack of a simple experimental approach to define the nature of the cellular interactions between thymocytes and cortical thymic epithelium in vitro. The proposed studies will utilize cortical epithelial cell lines isolated in this lab that can support the positive selection of thymocytes from TCR transgenic mice that bear class I restricted antigen receptors. Cytokine production by cortical TE will be defined by RT-PCR analyses and bioassay. The activity of cortical TE cell lines will be compared with that of purified populations of normal thymic epithelial cells and the mechanisms that regulate quantitative and qualitative aspects of cytokine production by these cells will be examined. A co-culture system developed in this laboratory to examine positive selection in vitro will be exploited to define a number of biological and methodological parameters of positive selection and expanded to test the hypothesis that this TE cell line can also support earlier stages of thymocyte differentiation as well. Finally, cortical TE cell lines will be used to generate mAbs defining cell surface molecules . These mAbs will be screened for their ability to perturb positive selection in vitro and other proximal stages of thymocyte differentiation thought to be supported by cortical TE. Using these mAbs in conjunction with direct expression cloning methodologies, cDNA molecules encoding cortical TE cell surface molecules involved in the positive selection process or other aspects of thymocyte development will be isolated and sequenced. This genetic information will be used to generate soluble forms of these stromal cell surface molecules which can be employed to identify potential ligands for cortical TE cell surface molecules that may be expressed by thymocytes. Characterizing the cellular interactions between thymocytes and cortical TE cells that may be involved in thymocyte differentiation and positive selection should provide new insight regarding the contribution of thymic epithelium to normal T cell development. Furthermore, defining the functional activity of thymic epithelium at a mechanistic level should greatly facilitate our long range effort to identify the age-related alterations of the senescent thymic environment that results in thymic involution and to design strategies to impede or reverse this process.
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