The function of thymic nurse cells and intra-TNC macrophages during T-cell development

Pleuripotent precursor thymocytes migrate from the bone marrow or fetal liver and enter the thymus where a series of developmental changes occur. One such interaction involves thymocytes and thymic nurse cells (TNCs). TNCs are unique cortical epithelial cells and have been implicated in MHC restriction. To further elucidate the role of TNCs in MHC restriction, we developed TNC cell lines by transforming freshly isolated TNCs with a temperature sensitive SV40 virus, tsA58. In culture, tsTNC-1 was shown to bind and internalize immature thymocytes bearing the αβTCRloCD4+CD8 + phenotype. A subpopulation of the internalized thymocytes became apoptosis within the TNC cytoplasm, whereas TNC-interactive non-apoptotic thymocytes egress into the medium and display the mature CD4+CD8 +CD69+ phenotype. To further decipher the function of TNCs with respect to thymocyte development, three defined investigations are presented: (1) the mechanism employed by TNCs to eradicate apoptotic thymocytes within cytoplasmic vacuoles; (2) determining whether macrophages reside within TNC complexes and (3) determining the role of peripheral macrophages and TNCs in thymocyte development. In the first study, we examined the mechanism employed by TNCs to remove dead thymocytes that bide within its cytoplasm. Confocal microscopic studies confirmed that TNC-specific lysosomes degrade apoptotic thymocytes. We then investigated whether TNC-internalized cells represent a mixed or homogeneous cell population. Immunofluorescence data clearly identified the presence of internalized thymocytes as well as peripheral macrophages within the cytoplasm of TNCs. These results implied that non-resident thymic macrophages could transport peripheral self-antigens to the thymus. To test this hypothesis, we conducted experiments exploiting the H-Y and DO11.10 mouse transgenic systems. When male macrophages were intraperitoneal injected (i.p) into H-Y female transgenic mice, the CFDA-labeled macrophages migrated into the thymus and induced negative selection of immature thymocytes as indicated by TUNEL and annexin-V staining. Similarly, FRCS analysis data show that i.p injected cOVA peptide sensitized macrophages caused a rapid deletion of the immature αβTCRloCD4+CD8 + thymocytes. Taken together, these data suggest that TNCs and macrophages participate in the processes of negative and positive selection during T cell development.