THYMIC ENVIRONMENT AND T-CELL DIFFERENTIATION

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The stromal cells comprising the thymic environment influence a number of important aspects of T-lymphopoiesis. These include the expression of non- clonotypic, lineage restricted cell surface molecules and antigen receptors, and the positive and negative selection of immature T cells based on their antigen receptor specificity. Although the importance of the thymic environment to these processes is well established, the role(s) played by the non-lymphoid cells comprising that environment remains poorly understood. Progress in understanding the role of thymic stromal cells in T cell differention has been hampered by a lack of knowledge regarding the thymic epithelium (TE). The proposed work will generate murine TE cell lines and mAbs detecting TE cell surface molecules and employ these reagents to characterize the ability of TE to support several aspects of T cell differentiation in vitro. First, the nature of the stromal cell molecules that influence an early stage of thymocyte development, the progression from a CD4-CD8(lo) phenotype to a CD4+CD8+ population will be identified. This will be accomplished by screening anti-stromal cell mAbs for their ability to affect this phenotypic change in short term co cultures with thymocytes and a stromal cell line shown to modulate the process. Antibodies with inhibitory activity will be used to biochemically characterize the molecules they recognize and to isolate the cDNA encoding these stromal cell surface molecules using a direct expression cloning-approach. Second, cell lines representative of cortical TE will be generated. Recent technical advances made in this laboratory (flow cytometric identification of viable cortical TE) will facilitate a rapid and systematic analysis of optimal culture conditions for the propagation of this cell type. Isolated cortical TE cells will be used to generate mAbs specific for cortical TE cell surface molecules, to identify the cytokines produced by cortical TE,and to examine the functional activity of cortical TE in vitro. Third, the Z210R.1 thymic stromal cell line isolated by this laboratory and previously shown to support a novel pattern of B-lymphopoiesis in vitro will be assessed in terms of it's ability to support T-lymphopoiesis. Stromal cell surface molecules involved in these processes will be identified with the use of anti- stromal cell mAbs raised against this cell line. In addition, a novel cytokine cloned from this stromal cell line will be assessed in terms of it's effect on thymocyte maturation and function. The proposed work is significant in that it will help define the thymic environment in terms of stromal cell heterogeneity, the cell surface molecules expressed by TE cells,the cytokines they elaborate, and how these molecules and cytokines influence T cell differentiation. This information should result in a better understanding of thymic stromal cell contributions to T cell differentiation and may lead to the identification of cytokines and/or cell interaction molecules with therapeutic value.