T cell selection in the thymus

Expression of T cell antigen receptors (TCR) and their interaction with stromal ligands determine the fate of developing T cells in the thymus. To dissect molecular mechanisms involved in T cell selection, in vitro differentiation and selection system was developed using the thymocytes from TCR (F5) transgenic mice, thymic stromal cell lines derived from oncogene transgenic mice, and synthetic peptides presented by class I major histocompatibility complex (MHC). Chapter 3 describes mice carrying a temperature sensitive SV40 large T antigen under the control of class I H-2Kb promoter (H2ts mice). These mice develop normally except for the enlargement of the thymus in the adults. Conditionally immortalised thymic stromal cell lines were established from such hyperplastic thymic tissues. To compare antigen presentation capacities, thymic cortical epithelial cell lines and freshly isolated thymic dendritic cells were co-cultured with immature thymocytes or mature T cells from F5 TCR transgenic mice in the presence or absence of cognate peptide. The results show that cortical epithelial cells are as efficient as dendritic cells in negative selection of F5 thymocytes, but not in activating mature F5 T cells. In an attempt to establish cell lines which support positive selection of T cells, adherent cells in a thymic tumour of an H2ts mouse were purified using magnetic beads coated with antibodies against CD45, class II MHC, and a medullary epithelial marker. Several epithelial cell lines expressing class II H-2Ab' and a cortical marker ER-TR4 were established, and their function was assessed by reaggregate culture (chapter 5). Immature T cell lines were derived from mice which express c-myc proto-oncogene under the control of Thyl gene promoter (TM mice). These mice develop thymic tumours consisting predominantly of CD4+CD8+ (DP) cells which are mono- or oligo-clonal. Overexpression of c-myc is associated with increased apoptosis of thymocytes in vivo, and DP cell lines derived from the tumours retained their abilities to undergo apoptosis upon TCR stimulation. However, it was not possible to induce differentiation of these DP cells to CD4 or CDS single positive (SP) cells. Chapter 4 describes development of T cells in mice transgenic for an TCR which was isolated from a cytotoxic clone F5 specific for a peptide from influenza nucleoprotein and class I H-2Db. Ontogeny of T cells in F5 mice is largely similar to that in normal mice for expression of CD4, CDS, and TCR, except for slightly earlier appearance of immature CDS SP cells and DP cells. The data suggest that expression of functional ?? TCR enhances transition from CD4-CD8- (DN) to DP cells. Addition of cognate peptide in F5 fetal thymic organ culture causes severe block at the stage between immature CD8 SP and DP cells. A significant number of DP cells which develop in the presence of cognate peptide do not express F5 TCR and are likely to escape from cell death by expression of endogenous TCR since these do not appear in F5/RAG1-7- thymic lobes. Chapter 5 describes effects of thymic stromal cell lines and peptide analogues on F5 T cell development in vitro. F5 TCR transgenic mice were bred to non-selecting MHC backgrounds such as H-2q or ?-2 microgrobulin (?2m)-deficient mice, in which T cell development was arrested at DP stage. Total thymocytes, including T cell progenitors and stromal cells expressing nonselecting MHC, were then reaggregated with thymic cortical epithelial cell lines which express class I H-2b. The data show that development of F5q/q or F5/?2m-/- T cells can be restored by such epithelial cell lines. Further in an attempt to identify peptide/MHC ligands which mediate signals for positive selection of F5 T cells, peptide analogues were designed by introducing single amino acid substitutions in nonameric cognate peptide. The present study on F5 T cell development in fetal thymic organ culture confirms importance of side chains of residues at positions 4 and 7 of the peptide for interaction with TCR, as predicted from crystallographic data by others. Two of the peptides exhibit antagonistic activity and one of them appears to augment positive selection of F5 T cells if provided with suboptimal dose of cognate peptide. These data may imply significance of the presence of heterogeneous peptides in vivo for positive selection of T cells