| All adaptive immune responses are mediated by lymphocytes. Lymphocytes have cell- surface receptors for antigen that are encoded in rearranging gene segments. There are two main classes of lymphocytes, B lymphocytes (B cells) and T lymphocytes (T cells), which mediate humoral and cell-mediated immunity respectively. Typical T cell activation assays are based on populations of T cells. Although these types of population studies provide valuable information on T cell activation, they can not provide insights into dynamic events of early T cell activation upon T cell-APC interactions. Recent advances in optical techniques have enabled a single-cell approach to immunology. Video-microscope techniques, together with the development of new fluorescent probes, are helping to clarify events during antigen presentation, calcium signaling and effector functions including gene activation, cell proliferation and apoptosis.;In this dissertation, I study T lymphocytes at the single-cell level with the latest developed optical techniques. The first goal of this dissertation is to map the polarity and sensitivity of a murine hybridoma 1E5 T cell line. Calcium imaging combined with an optical trap enabled the T-cell contact requirements and polarity to be investigated. 6 mum-diameter anti-CD3 mAb-coated beads induced calcium signaling with ∼ten-fold higher frequency and ∼four-fold shorter latency upon contact with the leading edge of the T cell, compared with the trailing edge. T cell response percentage, latency, and calcium signaling pattern depended on antibody density on bead. Engagement of no more than 340 T cell receptors is sufficient to initiate Ca2+ signaling.;The second goal of this dissertation is to examine continuous-wave diode-laser induced two-photon fluorescence excitation of three calcium indicators, indo-1, oregon-green, and fluo-3. The near 2.0 log-log power-dependence of fluorescence intensity versus incident laser power indicates that the fluorescence from these three calcium indicators was excited by two-photon absorption process of 809-nm laser light. Calcium-dependent emission spectra of indo-1 and oregon-green for two-photon excitation under wavelength of 809-nm have also been measured.;The third goal of dissertation is to study cell viability and DNA denaturation at the single-cell level by two-photon fluorescence excitation in CW Al:GaAs diode laser optical traps. Two-photon fluorescence emission spectra are presented for CHO cells and T lymphocytes loaded with various fluorescent probes. This single-beam method is demonstrated to be a safe tool to monitor the viability of optically trapped cells, even under intense 809-nm diode laser illumination. The dynamics of cellular necrosis is monitored by adding ethanol to the cell suspension during trapping.;The fourth goal of this dissertation is to study the mechanisms by which protein isoprenylation inhibitor perillyl alcohol may mediate its immunosuppressive properties on T cells. POH alone is effective in preventing rejection in a rat cardiac transplant model. Here, I show that POH disrupts T cell morphology and inhibits T cell proliferation and calcium response to anti-CD3 mAb stimulation.;Furthermore, I show that POH induces apotosis in human T cells as well as antigen-specific murine 1E5 T cells, with activated human T cells being 10-fold more sensitive to POH-induced apoptosis as compared to resting T cells. (Abstract shortened by UMI.). |
