negative selection assay based on stimulation of t cell receptor transgenic thymocytes with peptide-mhc tetramers负选择分析基于刺激t细胞受体转基因胸腺细胞peptide-mhc四聚体.pdfVIP

Negative Selection Assay Based on Stimulation of T Cell Receptor Transgenic Thymocytes with Peptide-MHC Tetramers Vasily Rybakin*, Nicholas R. J. Gascoigne* Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, California, United States of America Abstract 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. Citation: Rybakin V, Gascoigne NRJ (2012) Negative Selection Assay Based on Stimulation of T Cell Receptor Transgenic Thymocytes with Peptide-MHC Tetramers. PLoS ONE 7(8): e43191. doi:10.1371/journal.pone.0043191 Editor: Jose Alberola-Ila, Oklahoma Medical Research Foundation, United States of America Received June 7, 2012; Accepted July 20, 2012; Published August 10, 2012 Copyright: 2012 Rybakin, Gascoigne. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes