APA1/1 epitope discerns synapses of CD8⁺T cells stimulated ex vivo with agonist but not with partial agonist peptides. (A, left) T-cell/DC synapses. Total spleen OT-I cells were incubated for 2 hours with 2 μM of the peptides indicated. T-cell/APC conjugates were identified in DIC images (left column) superimposed with the green marker. T-cell/DC synapses were visualized by antibody staining for CD11c (DC marker, green) and CD3ζ (T cell marker, blue). Exposure of the APA1/1 epitope is shown in red and indicated with arrows. (Right) Quantitation of T-cell/DC synapses. The percentage of T-cell/DC conjugates forming synapses positive for CD3ζ (□) and for APA1/1 (▪) is shown as the mean ± SD of 3 independent fields with a total of more than 30 cells for each data point. (B) Total T-cell/APC synapses. Samples of the experiment shown in panel A were labeled with the 25.D1.16 antibody, specific for OVA peptide-loaded H-2K^b, to detect all APCs bearing the OT-I TCR ligands. Quantitation of total T-cell/APC synapses was performed as in panel A. Images were acquired with a Zeiss Axiovert 200 microscope with a CCD camera.