Lymphocytes are the central players in the human adaptive immune response. In the body, individual T helper lymphocytes need to be activated first by physical contact with antigen- presenting cells (APC). T-cell contact with APCs initiated an activation cascade, which includes an increase in T-cell intracellular calcium, leading to T-cell proliferation, differentiation and lymphokine production. Calcium imaging are combined with optical manipulation to investigate the physical properties of T-cell activation. We study cell-cell contact requirements for T-cell activation using optical tweezers to control the orientation of T-cell/APC pairs and fluorescence microscopy to measure the subsequent T-cell intracellular calcium level [(Ca2+)i] response. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling. T cells which are presented with antigen at the leading edge have a higher probability of responding and a shorter latency of response than those contacting APCs or beads with their trailing end. Alterations in antibody density and bead size are used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal.
T-cell contact with antigen-presenting cells (APC) initiates an activation cascade which includes an increase in T-cell intracellular calcium [(Ca2+)i] and leads to T-cell proliferation and differentiation. Although T-cell/APC physical contact is required for an immune response, little is known about the patterns of cellular interaction and their relation to activation. We have combined fluorescence spectroscopy and imaging with optical manipulation to investigate the contact requirements for T-cell activation, using optical tweezers to control the orientation of T- cell/APC pairs and fluorescence microscopy to measure the subsequent (Ca2+)i response, detected as an emission shift from the combination of fura-red and oregon- green, two cytoplasmic (Ca2+) indicators. APCs or beads coated with antibodies to the T-cell receptor (TCR) are trapped with a near-infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling (2-5 micrometers /min). T cells contacted with antigen- presenting cells or antibody-coated beads entered a dynamic and reproducible program in the first 10 - 20 mins, including (Ca2+)i increase, changes in shape and motility, engulfment, and stable contact. T cells presented with antigen at the leading edge had a higher probability of responding (85%) and a shorter latency of response (50 secs) than those contacting APCs or beads with their trailing end (APCs: 30%, 150 secs; beads: 6%, 300 secs). Alterations in antibody density, quantified by FACS analysis, and bead size were used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal. Preliminary data show that T cell responses [response percentage, latency and (Ca2+)i pattern] depend on both antibody density and bead size.
In the body, individual T helper lymphocytes need to be activated first by physical contact with antigen-presenting cells (APC). T-cell contact with APCs initiates an activation cascade which includes an increase in T-cell intracellular calcium and leads to T-cell proliferation and differentiation. We have combined fluroescence spectroscopy and imaging with optical manipulation to investigate the physical properties of T-cell activation. We study cell-cell contact requirements for T-cell activation using optical tweezers to control the orientation of T-cell/APC pairs and fluorescence microscopy to measure the subsequent T-cell intracellular calcium level ([Ca2+]i) response. APCs or beads coated with antibodies to the T-cell receptor are trapped with a near- infrared titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling. T cells which are presented with antigen at the leading edge have a higher probability of responding and a shorter latency of response than those contacting APCs or beads with their trailing end. Alterations in antibody density and bead size are used to determine the spatial requirements for T cell activation and the minimum number of receptors which must be engaged in order to transmit a positive signal.
T-cell contact with antigen-presenting B cells initiates an activation cascade which includes an increase in T-cell intracellular calcium and leads to T-cell proliferation and differentiation. We studied cell-cell contact requirements for T-cell activation using an optical trap to control the orientation of T-cell/B-cell pairs and fluorescence microscopy to measure subsequent T-cell(Ca2+)i response. B cells or beads coated with antibodies to the T- cell receptor are trapped with a titanium-sapphire laser and placed at different locations along the T-cell, which has a polarized appearance defined by the shape and direction of crawling. T-cell (Ca2+)i is detected as an emission shift from the combination of fura-red and oregon- green, two cytoplasmic (Ca2+)i indicators. T- cells which are presented antigen at the leading edge have a higher probability of responding and a shorter latency of response than those contacting B-cells or beads with their trailing end.
Optical laser trapping microscopy has emerged as a powerful tool not only for the optical manipulation of cells and macromolecules, but also for the study of cellular physiological responses via force transduction and fluorescence imaging. We describe here the most recent results from our laboratory in the use and application of laser trapping microscopy to a variety of studies at the cellular and molecular levels. Fluorescence spectroscopy and imaging have been successfully combined with optical micromanipulation. A single near-infrared laser beam is used for two-photon fluorescence excitation and micromanipulation of trapped biological specimens. Cell viability is observed and monitored with a Nd:YAG laser ((lambda) equals 1064 nm) and an Al:GaAs diode laser ((lambda) equals 809 nm). Traps and conventional fluorescence imaging are also used simultaneously to examine T-cell activation dynamics.
T-cell contact with antigen-presenting B cells initiates an activation cascade which includes an increase in T-cell [Ca2+] and leads to T-cell differentiation and proliferation. We evaluated cell-cell contact requirements for T-cell activation by using an optical trap to control the orientation of T-cell/B-cell pairs and fluorescence microscopy to measure subsequent T-cell [Ca2+] responses. B cells were trapped with a titanium-sapphire laser tuned to 760 nm and placed at various locations along the T cells, which had a polarized appearance defined by shape and the direction of crawling. T-cell intracellular [Ca2+] was detected as an emission shift from the combination of fura-red and fluo-3, two cytoplasmic [Ca2+] indicators. T cells which were presented antigen at the leading edge had a higher probability of responding (84% vs. 31%) and a shorter latency of response (42 s vs. 143 s) than those contacting B cells with their trailing end. Similar results were obtained using beads coated with antibodies to the T-cell receptor. These findings demonstrate a role for initial T-cell/B-cell contact geometry in T-cell activation by showing that the T cell is a polarized antigen sensor.
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