CD2–CD58 interaction at cell–bilayer interface mediates signaling in the absence of direct TCR stimulation. (A) A schematic of the experimental system is shown. Jurkat T cells interacted with planar lipid bilayers containing GPI-linked CD58 molecules (the ligand for CD2) supported on a glass substrate. (B) Signaling events were examined for T cells interacting with bilayers containing GPI-linked CD58 (40 molecules/µm²) or ICAM-1 (an adhesion molecule used as a control; 10 molecules/µm²). Similar results were obtained over a wide range of ligand densities in the bilayer (Fig. S1 A). Actin-GFP cells, imaged by spinning-disk confocal microscopy, spread on CD58 but not ICAM-1 bilayers. Intracellular calcium (measured by Fura-2 emission ratio [340 nm/380 nm] at 2 min of cell contact; see Materials and methods), cell surface expression of CD69 (fixed cell staining with an FITC-labeled anti-CD69 antibody at 3 h), and tyrosine phosphorylation (immunofluorescence with an antiphosphotyrosine antibody at 2 min) were elevated to a greater extent on CD58 compared with ICAM-1 bilayers. Asterisks and dashed lines indicate single cells. More than 50 cells were measured in all assays, and the data are representative for more than three independent experiments. Data are quantified as relative intensity to control experiment (ICAM-1 bilayer). pY, phosphotyrosine. Error bars indicate SEM (n > 50). Bars, 10 µm.