APA1/1 epitope distinguishes between synapses of CD4⁺T cells stimulated with agonist and with antagonist peptides. (A) T-cell/APC synapses. CH7C17 cells were incubated for 15 minutes with DAP-DR1-ICAM APCs loaded with 10 μM of the indicated peptides. DIC image of a representative T-cell/APC conjugate is shown for each condition. T cells were stained after fixation and permeabilization with anti-CD3ζ (green) and APA1/1 (red). Images were acquired with a Zeiss Axiovert 200 microscope with a CCD camera using a 100×/1.3 oil iris Plan-Neoplanar lens. (B) Quantitation of T-cell/APC synapses. Fifty to 60 T-cell/APC conjugates were counted per stimulation condition, and the percentage of cells with CD3ζ or APA1/1 concentrated in the IS is indicated as the mean ± SD of an experiment in duplicate. (C) T-cell stimulation. CH7C17 cells were stimulated for 24 hours with DAP-DR1-ICAM cells preloaded with the indicated concentrations of the agonist and antagonist peptides; induction of CD69 expression was evaluated by flow cytometry. Expression of CD69 is shown as the mean ± SD of an experiment in triplicate.