Negative selection of T cells occurs throughout thymic development.

Thymic positive and negative selections govern the development of a self-MHC-reactive, yet self-tolerant, T cell repertoire. Whether these processes occur independently or sequentially remains controversial. To investigate these issues, we have employed tetrameric peptide-MHC complexes to fluorescently label and monitor polyclonal populations of thymocytes that are specific for moth cytochrome c (MCC)/I-Ek. In TCR beta mice tetramer-positive thymocytes are detectable even in the most immature TCR-expressing cells. In the presence of MCC peptide, thymocytes that bind strongly to MCC/I-Ek tetramers are deleted earlier in development and more extensively than cells that bind weakly. This negative selection of the MCC/I-Ek-specific cells occurs continuously throughout development and before any evidence of positive selection. Thus, positive and negative selections are independent processes that need not occur sequentially.

A T cell receptor-specific blockade of positive selection.

To investigate the influence of endogenous peptides on the developmental processes that occur during thymocyte selection, we have used monoclonal antibodies that preferentially recognize the major histocompatibility complex (MHC) molecule I-Ek when it is bound to the moth cytochrome c peptide (88-103). One of these antibodies (G35) specifically blocks the positive selection of transgenic thymocytes expressing a T cell receptor that is reactive to this peptide- MHC complex. Furthermore, G35 does not block the differentiation of transgenic T cells bearing receptors for a different I-Ek-peptide complex. This antibody recognizes a subset of endogenous I-Ek-peptide complexes found on a significant fraction of thymic antigen-presenting cells, including cortical and medullary epithelial cells. The sensitivity of G35 to minor alterations in peptide sequence suggests that the thymic peptide-MHC complexes that mediate the positive selection of a particular class II MHC-restricted thymocyte are structurally related to the complexes that can activate it in the periphery.

Lethality induced by a single site-specific double-strand break in a dispensable yeast plasmid.

Cells of the yeast Saccharomyces cerevisiae are delayed in the G2 phase of the cell cycle following chromosomal DNA damage. This arrest is RAD9-dependent and suggests a signaling mechanism(s) between chromosomal lesions and cell cycling. We examined the global nature of growth inhibition caused by an HO endonuclease-induced double-strand break (DSB) at a 45-bp YZ sequence (from MAT YZ) in a non-yeast region of a dispensable single-copy plasmid. The presence of an unrepaired DSB results in cellular death even though the plasmid is dispensable. Loss of cell viability is partially dependent on the RAD9 gene product. Following induction of the DSB, 40% of RAD+ and 49% of rad9 delta cells [including both unbudded (G1) and budded (S plus G2) cells] did not progress further in the cell cycle. The remaining RAD+ cells progressed to form microcolonies (< 30 cells) containing aberrantly shaped inviable cells. For the rad9 delta mutant, the majority of the remaining cells produced viable colonies accounting for the greater survival of the rad9 delta strain. Based on the profound effects of a single nonchromosomal DNA lesion, this system provides a convenient means for studying the signaling effects of a DNA lesion, as well as for designing strategies for modulating cell proliferation.