LFA-1 activation requires a polarized TCR stimulus and immobilized ICAM-1. (A) T cells were either left untreated or incubated on ice with biotinylated anti-CD3, and then stimulated at 37°C with either soluble streptavidin (soluble αCD3) or coverslip-adsorbed streptavidin (immobile αCD3). Some cells were concomitantly stimulated with either soluble ICAM-1 (sol. ICAM), coverslip-adsorbed TS1/22 (im. TS1/22), or coverslip-adsorbed ICAM-1 (im. ICAM). Images show single confocal planes near the contact site. (B) Contact area of cells from A. (C) Proportion of LFA-1 in the open conformation on different stimulatory surfaces. Data represent at least 70 cells per condition from a single representative donor; n = 3 donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.