CCR4 is required for efficient interactions between post-positive selection thymocytes and thymic DCs. (A) The migration index of Ccr4^+/+ and Ccr4^−/− thymocytes toward supernatant from thymic DC cultures was assayed using in vitro Transwell chemotaxis assays, as in Fig. 8 C. Means + SEM were compiled from three independent experiments, with three technical replicates per experiment. A line at 1 indicates no chemotaxis. *, P < 0.05 (unpaired Student’s t test). (B) A maximum intensity projection of Ccr4^+/+ or Ccr4^−/− CD4SP cells (red) migrating among DCs (yellow) in a live CD11c-EYFP thymic slice. Color-encoded time tracks depict the location of individual CD4SP cells throughout the 15-min time course. Bar, 50 µm. Videos 4 and 5 show corresponding two-photon microscopy time-lapse imaging data. Mean cell velocities (C), migratory straightness (D), frequency of thymocyte/DC contacts (E), and percent of time in which CD4SP cells contacted DCs (F) were quantified from two-photon time-lapse imaging data as in B. Data are compiled from five independent experiments, with a total of five mice per group. Each dot represents a single tracked cell, and bar and whiskers indicate means ± SD. Mean values are listed above each plot. (C–F) *, P < 0.05 (Mann-Whitney test). n = 832, Ccr4^+/+ thymocytes; n = 1,119, Ccr4^−/− thymocytes.