Antibody engagement of the TCR induces the exposure of the APA1/1 epitope in primary cells. (A) Pull-down assay. Spleen cells from C57BL/6 mice stimulated for 5 minutes with 10 μg/mL soluble anti-CD3 145-2C11 or left nonstimulated (NS) were lysed in Brij96, and the lysate was incubated with GST-Nck beads or with GST. Precipitated proteins were subjected to immunoblotting and were probed first with anti-CD3ζ and subsequently with anti-CD3ϵ M20. Total lysate (TL) was run as a control of loading. (B) Induced exposure of the APA1/1 epitope. Spleen cells from C57BL/6 mice were stimulated (STIM) with soluble anti-CD3 for 5 minutes at 37°C or were left nonstimulated (NS), fixed, permeabilized, and stained with APA1/1. Staining with an irrelevant IgG1 antibody was carried out in parallel on stimulated cells as a control (gray shaded curves). APA1/1 immunofluorescence on gated CD4⁺ and CD8⁺ T cells is shown. The diagram to the right shows the quantitative increase in mean fluorescence intensity (MFI) after stimulation (in reference to the nonstimulated cells, □) and SD from data collected in triplicate. To quantify total TCR levels, permeabilized spleen T cells were stained either with anti-CD3ϵ antibody 145-2C11 or with antibody M20, recognizing the C-terminal end of CD3ϵ (CD3ϵcit). (C) Protein tyrosine kinase (PTK) and temperature independence of APA1/1 epitope exposure. Spleen T cells were stimulated with anti-CD3 for 5 minutes at 37°C in the presence or absence of 20 μM PP2, for 1 minute at 37°C, or for 5 minutes on ice. For PP2 treatment, cells were preincubated with the drug for 30 minutes before stimulation. ▪ indicates stimulated samples; □, nonstimulated samples.