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.
Highlights
Each conventional mature T cell expresses one variety of T cell receptor (TCR) on its surface and is capable of precisely recognizing and responding to an extremely low dose of an appropriate agonist peptide presented on major histocompatibility complex by an antigen presenting cell
We propose a new ex vivo model for analysis of negative selection based on stimulation of TCR transgenic thymocytes with pMHC tetramers with subsequent analysis of TCR signalinginduced apoptosis by activated caspase 3 staining
Intracellular staining for activated caspase 3 (ActCasp3) with subsequent analysis by flow cytometry has been extensively used for quantitative analysis of apoptosis in a variety of model systems
Summary
Each conventional mature T cell expresses one variety of T cell receptor (TCR) on its surface and is capable of precisely recognizing and responding to an extremely low dose of an appropriate agonist peptide presented on major histocompatibility complex (peptide-MHC, pMHC) by an antigen presenting cell. Thymocytes expressing functional TCR on their surface successfully pass the positive selection test in the thymic cortex whereas cells failing to achieve survival signal via TCR-pMHC interaction are eliminated by ‘‘death by neglect’’ [1,2,3]. Ex vivo simulation of negative selection has been extensively used to study signaling events downstream of TCR stimulation in immature thymocytes.
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