Regional specification of thymocyte signaling and migration in the thymus [electronic resource] : a two-photon microscopic study

T cell development involves stereotyped movements of developing thymocytes through specific stromal microenvironments in thymus, with immature double-negative (DN, CD4-CD8-) and double-positive (DP, CD4+CD8+) thymocytes found in cortex, and mature single-positive (SP, CD4+CD8- or CD4-CD8+) thymocytes in medulla. Critical events occur in each of these locations, notably positive selection, which leads to survival and maturation of DPs and occurs in cortex, and negative selection, which leads to elimination of autoreactive thymocytes and can occur in cortex or medulla depending on the type of antigen. Several key questions remain about the mechanisms underlying these developmental transitions. How are thymocytes properly localized to cortex and medulla? Is there further specialization of stroma within these regions for supporting thymocyte migration or signaling? How do Ca2+ signals, which are critical for both positive and negative selection, differ between these two developmental pathways? In this thesis, we utilize thymic slices, which preserve native stromal architecture and support thymocyte development, to address these questions. Two-photon microscopy of fluorescently labeled thymocytes within slices allows us to non-invasively image the migration, localization, and signaling of single thymocytes in real time, providing a better understanding of how these behaviors are regulated during development and also as a function of location in tissue. In Chapter 2, we address the question of how developing thymocytes are localized to their proper tissue locations in thymus. When layered on the cut surface of slices, purified stages of thymocytes localize to their proper thymic environments, with immature DNs and DPs restricted to cortex, and SPs concentrating in medulla. Remarkably, we find evidence for two distinct mechanisms controlling proper localization of immature versus mature thymocytes. Pre-selection DPs are strictly localized to cortex by their inability to migrate on medullary substrates. SPs localize to medulla via CCR7-dependent chemotaxis, which is directly demonstrated, as well as a novel GPCR-mediated signaling module permitting SP migration on medullary substrates. In Chapter 3, we examine differences in Ca2+ signaling between positive versus negative selection, as well as between distinct locations in thymus, and clarify the role of Ca2+ in controlling motility during negative selection. We find larger Ca2+ signals during early stages of negative selection relative to positive selection, and stronger signaling in response to selecting antigens in the inner cortex near medulla. Sustained Ca2+ signals during negative selection are largely not required for stopping cells, but do contribute to prolonging motile arrest of a proportion of the thymocyte population. Overall these results point to several key events during the transition from cortical DPs to medullary SPs that may enhance the efficiency of selection. As DPs pass through a specialized inner cortical environment, they may become more responsive to peptide ligands leading to positive and negative selection. Stronger Ca2+ signals during negative compared to positive selection in DPs and SPs may help to commit thymocytes to death over maturation. Finally, the ability to migrate in medulla, which thymocytes acquire post-positive selection, may represent an important developmental switch confining pre-selection cells to cortex for further testing against selecting antigens, while permitting post-positive selection thymocytes to undergo an additional layer of testing against tissue-specific antigens, followed by maturation and exit.

Keywords: Molecular and Cellular Physiology.
Advisor: Lewis Richard (Richard Sheridan)
Davis Mark M.
Dolmetsch Ricardo E.
Smith Stephen J.