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Negative selection assay based on stimulation of T cell receptor transgenic thymocytes with peptide-MHC tetramers

Vasily Rybakin1, Nicholas R J Gascoigne

  • 1Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, United States of America. vrybakin@scripps.edu

Plos One
|August 18, 2012
PubMed

Thymocyte negative selection is a requirement for the development of self tolerance. Although it is possible to assay the induction of cell death in thymocytes in vitro using antibody cross-linking, this stimulus is much stronger than the normal range of T cell receptor ligands that could be encountered during normal development. Signaling in thymocytes is finely balanced between positive and negative selection stimuli, where a negative selecting ligand can be only marginally higher affinity than a positive selecting ligand. We have therefore developed an assay for the induction of negative selection that can distinguish such cases, and that is amenable to high-throughput analysis. The assay is based on the induction of activated caspase 3 in thymocytes expressing a defined T cell receptor, after stimulation with MHC-peptide tetramers in vitro for 24 hours or less.

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