PD-L1 diminishes pZAP-70 clusters in synapse-forming virus-specific CD8⁺ T cells on planar bilayers. Fixed sample TIRFM imaging was used to assess proximal TCR signaling by measuring fluorescence intensity and distribution of phosphorylated ZAP-70 and TCR microclusters on synapse-forming GFP⁺ CD8⁺ P14 cells. (A) Purified GFP⁺ CD8⁺ P14 cells from day 7 spleens of CL13-infected mice were incubated with TCRβ (H57) Fab^AF568 (red) and loaded onto planar lipid bilayers containing ICAM-1^AF405 (blue, 200 molecules/µm²) and H-2D^bGP_33-41 (unlabeled, 185 molecules/µm²), in the absence or presence of PD-L1 (unlabeled, 275 molecules/µm²). P14 cells were fixed 5 min after placement on the bilayers and stained with anti-pZAP-70^AF647 (green). (B) Dot plots show quantification of mean intensity fluorescence (background corrected raw mean intensities) for p-ZAP-70 and TCRβ. The asterisk denotes a statistically significant difference (P < 0.05). Each dot represents an individual P14 T cell for the two conditions (no PD-L1, n = 255 cells; PD-L1, n = 305 cells). Horizontal black bars denote the mean of each group. Data are representative of two independent experiments. (C) Radial distributions of after raw intensities (fluorescence) for ICAM-1, TCRβ, and p-ZAP-70 were calculated and plotted for day 7 CL13 GFP⁺ CD8⁺ P14 cells interacting with bilayers. Intensity distributions are shown from cell center to periphery (µm) in all graphs. For illustration purposes, P14 cells with a spreading area >93 µm² are shown. Note that the presence of PD-L1 in the bilayers decreases p-ZAP-70 and increases ICAM-1, but has no impact on TCRβ expression. Graphs are representative of two independent experiments.