(A) Formation of the immunological synapse in IBP^trap/trap CD4⁺ T cells. CD4⁺ T cells were conjugated with A20 mouse B lymphoma cells loaded with or without 2 μg/ml SEE. After the indicated times of T and B cell contact, cells were stained with antibodies against either phosphorylated ZAP-70 or phosphorylated LAT and scored under a fluorescence microscope. Approximately 50 conjugates were counted per time point in at least 3 independent experiments. T and B cell conjugates without SEE (upper panels) and with SEE (lower panels) are shown. (B) Representative images for conjugates (phase), phosphorylated ZAP-70 (red) and CD4 (green) at 5 minutes in IBP^+/+ and IBP^trap/trap T cells, respectively. (C) Actin polymerization in IBP^trap/trap CD4⁺ T cells reconstituted with WT or mutant IBP retroviral vectors. Naive CD4⁺ T cells were infected with control YFP-RV (empty vector) and WT IBP–expressing (IBP-RV), IBP-Δ313-631–expressing (IBP-Δ313-631-RV), or constitutively active Rac2–expressing (CA Rac2-RV) retroviruses. Cells were harvested after 5 days and restimulated with anti-CD3ε antibodies followed by cross-linking with goat anti-hamster Ig at 4°C (control) or 37°C. Cells were then stained with biotin-labeled phalloidin followed by streptavidin-PE (PE-phalloidin) and actin polymerization measured by FACS. Data shown represent unstimulated cells (filled histogram) with overlay of stimulated cells (open histogram) after gating on YFP-positive cells.