Calcium signaling and cellular interactions during T cell development in the three-dimensional thymic environment: A two-photon fluorescence microscopy study

Developmental events within the thymus guide immature thymocytes to become mature T cells capable of responding to foreign peptides bound to self major histocompatibility complex (MHC) molecules. Positive selection is the process ensuring affinity between recombined T cell receptors (TCR) on T cells and self MHC, and requires the delivery of TCR/MHC-dependent signals to thymocytes via three-dimensional interactions with thymic stromal cells. One such signal, elevation in intracellular free calcium ([Ca2+]i), is essential for positive selection; however, the shapes, amplitudes and durations of Ca2+ signals in thymocytes undergoing positive selection are unknown. The thymocyte-stromal cell interactions associated with Ca 2+ signals and positive selection are equally undefined. We performed real-time studies of cell-cell interactions and Ca2+ signals within 3-D thymic environments using two-photon microscopy. Chapter I provides a brief background on the signals and cellular interactions associated with positive selection. Chapter 2 examines the types of cellular interactions present during positive selection in reaggregate organ cultures. The results show that thymocytes are highly motile, and that MHC recognition on thymic stromal cells is necessary for the formation of sustained contacts. Chapter 3 describes a new thymic slice preparation providing measurements of thymocyte [Ca2+]i and motility within native tissue. We show that oscillations in [Ca2+]i during early stages of positive selection have characteristics consistent with activation of Ca 2+-dependent genetic pathways. These oscillations are necessary and sufficient to stop cells, a result that identifies a mechanism by which MHC recognition increases thymocyte-stromal cell contact time. We propose that this calcium stop signal allows thymocytes to reconcile the need to maintain a high degree of motility in the search for potentially rare antigens, and the need to stop at sites of recognition long enough to turn on genes supporting positive selection. Chapter 4 presents a quantitative model of cell-cell interactions yielding information on events operating on time scales longer than the length of data acquisition. Our application of time-lapse microscopy has provided the first recordings of Ca2+ signals and motility in the thymus, a deeper understanding of T cell development as it occurs in tissue, and powerful new tools for continued investigation.*.;*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Adobe Acrobat; QuickTime.