Negative selection assay based on stimulation of T cell receptor transgenic thymocytes with peptide-MHC tetramers.
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.
Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, United States of America. vrybakin@scripps.edu
Vasily
V
Rybakin
Nicholas R J
NR
Gascoigne
D000818:Animals; D017209:Apoptosis / Q000276:immunology; D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation / Q000276:immunology; D017637:Clonal Deletion / Q000276:immunology*; D006649:Histocompatibility Antigens / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D008822:Mice, Transgenic; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D060168:Thymocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism
D006649:Histocompatibility Antigens; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; D053148:Caspase 3