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Clonal deletion of T cells specific for self-antigens in the thymus has been widely studied, primarily by approaches that focus on a single receptor (using TCR transgenes) or a single specificity (using peptide-MHC tetramers). However, less is known about clonal deletion at the population level. In this article, we report an assay that measures cleaved caspase 3 to define clonal deletion at the population level. This assay distinguishes clonal deletion from apoptotic events caused by neglect and approximates the anatomic site of deletion using CCR7. This approach showed that 78% of clonal deletion events occur in the cortex in mice. Medullary deletion events were detected at both the semimature and mature stages, although mature events were associated with failed regulatory T cell induction. Using this assay, we showed that bone marrow-derived APC drive approximately half of deletion events at both stages. We also found that both cortical and medullary deletion rely heavily on CD28 costimulation. These findings demonstrate a useful strategy for studying clonal deletion within the polyclonal repertoire. [ABSTRACT FROM AUTHOR]

We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic &brg;2M(-/-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified. These pep-tide-selected thymocytes have the same phenotype as mature CD8+ T cells and can respond to antigen. Those peptides with the ability to induce positive se-lection were all variants of the antigenic peptide and were identified as TCR antagonist peptides for this re-ceptor. One peptide tested, E1, induced positive selec-tion on the &brg;2M[-l-) background but negative selec-tion on the &brg;2M(+l-) background. These results show that the process of positive selection is exquisitely peptide specific and sensitive to extremely low ligand density and support the notion that low efficacy li-gands mediate positive selection. [ABSTRACT FROM AUTHOR]