Thymic nurse cells (TNCs) are cortical epithelial cells that contain up to 50 CD4+CD8+alphabetaTCR/low/CD69- thymocytes within specialized vacuoles in their cytoplasm. A subset of the internalized population proliferates and differentiates to the CD4+CD8+alphabetaTCR/high/CD69+ stage, while another subset undergoes apoptosis and is degraded within the TNC. Antibodies, which block the thymocyte/TNC interaction in vitro, lead to an 80% reduction in the number of thymocytes that develop in fetal organ cultures. Together, these data lead to the hypothesis that all thymocytes, which make functional alpha/betaTCR rearrangements, migrate through the TNC complex. Subsequent to antigen driven selection, a subset of those cells is released from the complex and continues to progress through the thymus to become part of the mature T cell repertoire. Recent work has verified that macrophages also enter the TNC complex and form intimate associations with the internalized thymocytes. CFDA SE labeled peritoneal macrophages were shown to home to the TNC complex when injected IP into C57BL/6 mice. These findings suggest that macrophages may play a role in the selection events that occur within the TNC complex. The experiments proposed for this study are designed to show that peripheral macrophages circulate freely through the thymus and interact dynamically with the TNC microenvironment. The central role of macrophages in removing apoptotic cells, provides for a mobile source of peripheral self-antigens, not expressed in the thymus, which may be used to ensure self-tolerance. Using adoptive transfer of TNC complex and peritoneal macrophages, isolated from green fluorescence protein (GFP)-expressing mice, into either C57BL/6 or HY TCR/trans Rag-/- mice, we propose to; 1) determine the developmental fate of intra- TNC thymocytes subsequent to their release; 2) determine if intra-thymic and intra-TNC macrophages represent a unique population of macrophages, or one that is continuous with the circulating peritoneal macrophage population; 3) develop a model system to determine if macrophages present peripheral antigens to developing T cells in the TNC microenvironment. These studies will determine the role of the TNC microenvironment in shaping the mature T cell repertoire, and will lead to future studies of the complex cell/cell interactions and molecular mechanisms involved. Demonstrating a role for circulating peripheral macrophages in presenting antigens to developing thymocytes will lead to a new way of thinking about self-tolerance induction and the development of autoimmune disease.
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