LFA-1–ICAM-1 interactions are maintained by ongoing F-actin dynamics. Ex vivo CD4⁺ T cells were treated with inhibitors as in Fig. 5 and allowed to spread on planar bilayers functionalized with anti-CD3 and Alexa Fluor 488–labeled ICAM-1. Cells were then fixed and stained with TS2/4, Kim127, and TS1/22. Bar, 5 µm. Representative cells (A) and population average projections (B). (C) Quantification of ICAM-1 enrichment under the cell after subtracting background levels of ICAM-1. Values are normalized to the untreated control. (D) Ratio of unligated to total LFA-1 defined by staining with TS1/22 and TS2/4, respectively. Data represent 100–150 cells per condition from a single representative donor; n = 3 donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001.