CD8+ T cells stably interact with VV-infected cells in DC-ablated mice. (A) MPM images of untreated or DTx-treated CD11c-DTR-eGFP animals that were given 1.0 × 107 Cell Tracker Red (red) labeled OT-I cells before DC depletion. Images acquired 6 hpi with VV-SIINFEKL (nonfluorescent). The macrophage rich-region (MRR) was identified by in vivo uptake of FITC-dextran (green). (B) Percentage of OT-I cells per 63X field (238 × 238 × 85 µm) located in the MRR in untreated or DTx-treated mice. Results were analyzed with an unpaired t test. (C) Time-lapse MPM images of OT-I cells (red) in the LN of untreated (top panels) or DC-ablated (bottom panels) mice 6–10 hpi with VV-NP-S-eGFP (green). White circles indicate stable contacts. For untreated mice, 106 OT-I cells were transferred; for DTx-treated, 107 (it was necessary to transfer more OT-I cells into DTx-treated mice than untreated mice due to decreased homing to the LN following DTx-mediated ablation). (D) Calculation of contact times between OT-I cells and VV-infected cells over the course of a 30 min imaging session. (E) Plot of OT-I cells’ movement (step size) between individual frames of a 30 min movie in untreated vs. DC-ablated mice. We classified movement under 0.5 µm between frames as a pause step. (F) Calculation of the percentage of T cells arresting in each condition. (G) Color-mapped plot of T cell tracks during a 30-min movie. Each box on grid is 20 × 20 µm. Increasing track speed is colored from purple to red (bottom bar). (H) Calculation of average OT-I cell speed in untreated or DTX-treated mice. Results are shown from one experiment of three to six with similar results. No differences between untreated and DTx-treated mice were statistically significant according to unpaired Student’s t tests. Time is shown in minutes. Scale bars are shown in micrometers.
