Recently activated T cells do not effectively stop upon TCR engagement. (A–D) CD8⁺ T cell dynamics during the course of activation in vivo. (A) Experimental set-up. GFP⁺ OT-I CD8⁺ T cells were adoptively transferred, and recipients were stimulated by an i.v. injection of OVAp. At various time points, recipient mice were subjected to intravital imaging of the popliteal lymph node. (B–D) Graphs show the velocity (B), arrest coefficient (C), and straightness index (D) of individual cells at the indicated time point. Each dot represents an individual track, and the velocity represents the mean speed of the track. (E–G) CD8⁺ T cell arrest in response to the antigenic peptide is dependent on T cell activation status. (E) Experimental set-up. GFP⁺ OT-I CD8⁺ T cells were adoptively transferred, and recipients were stimulated by an i.v. injection of OVAp. At 30 h, a second cohort of SNARF-labeled OT-I CD8⁺ T cells was adoptively transferred. Intravital imaging of the popliteal lymph node was performed 18 h later. OVAp was reinjected during the imaging experiment. (F) T cell tracks (15 min long) of the first (green) and second cohort (red) of T cells obtained from a representative experiment are shown before and after OVAp reinjection. Bar, 15 µm. (G) Graphs show the velocity, arrest coefficient, and straightness index for individual T cells of the first and second cohorts before and after OVAp reinjection. Results are compiled from two videos obtained in one representative experiment out of three. Significance testing was performed with the nonparametric Mann-Whitney test. **, P < 0.01; and ***, P < 0.001. See also Fig. S1.