Most dividing T cells during priming are not in contact with APCs. (A–E) Visualization of T cell division dynamics in the lymph node. GFP⁺ OT-I CD8⁺ T cells were adoptively transferred and stimulated by an i.v. injection of OVAp. At various time points, recipient mice were subjected to intravital imaging of the popliteal lymph node. (A) The graph shows the frequency of dividing T cells observed per hour of imaging. Two to four videos were analyzed for each time point. Data are shown as mean ± SD. (B) Time-lapse images showing the typical phases of T cell division (motility, arrest, mitosis, and recovery of motility). (C and D) Tracks (C) and velocity (D) for the dividing cell shown in B (black) and the two daughter cells (red and blue). (E) Timing for the indicated phases of T cell division measured after T cell arrest. 16 dividing T cells were tracked over time. Each dot represents the value for the different phases for each of these 16 division events. Mean values are shown in red. Results are representative of eight independent experiments. (F and G) Analysis of T cell division upon in vivo stimulation with DCs. Recipient mice were adoptively transferred with GFP-expressing OT-I CD8⁺ T cells and injected in the footpad with OVAp-pulsed CFP-expressing DCs. Intravital imaging of the draining popliteal lymph node was performed at 48 h. (F) The vast majority of dividing T cells (green) are not in contact with an antigen-bearing DC (cyan) at the time of mitosis. White arrows show the cell before division. Blue and red arrows show the daughter cells. (G) The graph shows the distance between dividing T cells and the closest DCs. T cells forming stable interaction (but not dividing) with DCs were scored similarly for comparison. Mean values are shown in red. Results are representative of three independent experiments. Significance testing was performed with the nonparametric Mann-Whitney test. ***, P < 0.001. Bars, 15 µm.