TCR affinity controls duration of stable CD8⁺ T cell–DC interactions in vivo. (A) Experimental layout. T cell–DC interactions in popliteal LNs were analyzed by 2PM in the presence of s.c. peptide-pulsed DCs at the indicated time points after i.v. T cell transfer. (B) Representative 2PM images of OT-I T cell tracks (white lines) or OT-3 T cell tracks (blue lines) in the presence of peptide-pulsed DCs at early (2–10 h) or late (24–32 h) time points after T cell transfer. Arrowheads point to stable (>30 min) OT-I T cell–DC (closed) or OT-3 T cell–DC (open) interactions. Bar, 20 µm. (C and E) Single–T cell speeds at each time point. Polyclonal T cell speeds are included for comparison. Percentages refer to tracks in dashed-line boxes showing single-cell speeds of >7 µm/min. (D and F) T cell–DC interaction times. Each dot represents a single track (C and E) or interaction (D and F). Bars indicate mean (C and E) or median (D and F). (C–F) Data are pooled from at least two independent experiments with four or more mice/eight image sequences per condition and time point. Statistical significance was analyzed for entire datasets by Kruskal-Wallis tests with Dunn’s posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001.