PD-L1 promotes attachment and synapse formation of virus-specific CD8⁺ T cells on planar bilayers. Real-time TIRFM imaging was used to assess the ability of GFP⁺ CD8⁺ P14 cells to form stable synapses on planar bilayers and to examine the distribution of ICAM-1/PD-L1 after synapse formation. (A) Purified GFP⁺ CD8⁺ P14 cells from day 7 spleens of Arm or CL13-infected mice were loaded onto planar lipid bilayers containing ICAM-1^AF405 (cyan, 200 molecules/µm²) and H-2D^bGP_33-41 (unlabeled, 185 molecules/µm²), in the presence or absence PD-L1^AF568 (red, 275 molecules/µm²). Representative images show P14 cell synapses ∼15 min after they were placed on the bilayers. Images represent data from at least three independent experiments per condition. See corresponding Video 5. (B and C) Histograms show the frequency of day 7 Arm (B) or CL13 (C) GFP⁺ P14 cells that formed synapses, unstructured synapses, or were moving after placement on bilayers with or without PD-L1. P14 cells were incubated on the bilayers for 10 min before imaging, and thus the denoted time points reflect the number of min after this initial incubation period. Histograms reflect the number of GFP⁺ P14 cells per condition: Arm no PD-L1 (n = 78 cells), Arm PD-L1 (n = 71 cells), CL13 no PD-L1 (n = 130 cells), CL13 PD-L1 (n = 80 cells). Asterisks denote statistically significant differences between the two conditions (no PD-L1 vs. PD-L1) at each time point (P < 0.05). Data are representative of two independent experiments.