Investigating T Cell Receptor Signals In Situ by Two-Photon Microscopy of Thymocytes Expressing Genetic Reporters in Low-Density Chimeras.

Abstract

T cell development is a dynamic process accompanied by extensive thymocyte migration, cellular interactions, and T cell receptor (TCR) signaling. In particular, thymic selection processes that ensure a functional, self-tolerant repertoire require TCR interactions with self-peptide presented by major histocompatibility complex molecules expressed by specialized thymic antigen-presenting cells. The quantity and quality of these TCR signals influence T cell fate. Two-photon microscopy, which enables live imaging of cells in intact tissue, has emerged as a powerful tool to gain insights into thymocyte migration and TCR signaling during T cell development in situ. Here we describe the generation of non-irradiated, low-density chimeric mice by neonatal injection of adult bone marrow engineered to express fluorescent TCR signaling reporters for imaging by two-photon microscopy. We also describe how the thymic lobes from chimeric mice are prepared for live imaging of thymocyte behavior and TCR signaling events. While we focus on imaging TCR signals associated with T cell development in the thymus, these techniques can also be adapted to study TCR signaling in mature T cells in peripheral lymphoid organs.