LFA-1 activation intermediates are organized into a concentric array by a T cell–intrinsic mechanism. (A) LFA-1 conformational states and antibody binding sites. Inactive LFA-1 is present in a bent conformation on the surface of T cells and exhibits low affinity for ligand. Talin binding to the β chain leads to unbending, yielding the extended intermediate affinity conformation. F-actin flow and ligand engagement cause the hybrid domain of the β chain to swing out, resulting in the high affinity open conformation that efficiently mediates adhesion and signaling. Putative binding sites for monoclonal antibodies are marked with asterisks. (B) Human primary CD4⁺ T cells were conjugated to SEE-pulsed Raji B cells (asterisk) for 25 min, labeled for 5 min with m24 (β2 open), and then fixed and labeled with CF405M-phalloidin to detect F-actin and monoclonal antibodies TS2/4 (αL total) and Kim127 (β2 extended). Z stacks of whole conjugates were collected and rendered in 3D in the IS plane (arrowhead). (right) Radial intensity profiles of synapses from multiple conjugates were analyzed as described in Materials and methods and normalized with the maximum intensity for each antibody set equal to 1. Data represent mean ± SEM. (C and D) Cells were allowed to spread on planar bilayers coated with anti-CD3 and 0.1 µg/ml ICAM-1 (C) or coverglasses adsorbed with anti-CD3 and ICAM-1 (D) and analyzed as in B. Bars, 5 µm.