F-actin dynamics regulate the organization of activated LFA-1. (A) Ex vivo CD4⁺ T cells spreading on bilayers coated with anti-CD3 in the presence or absence of ICAM-1 were subjected to the indicated experimental paradigm. After fixation, cells were labeled with TS2/4 and Kim127. (B) Representative synapses. Bar, 5 µm. (C) Mean fluorescence intensity distributions for cell populations. (D–F) Cells were treated as in B, and the relative labeling intensity of LFA-1 in the extended conformation (D), open conformation (E), or total LFA-1 (F) was determined. (G and H) Ratio of extended (G) or open (H) conformations of LFA-1 to total LFA-1 at the IS. Data represent 130–250 cells per condition from a single representative donor; n = 3 donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.