Synapse Assay.
LB27.4 B cells (H-2d/b–restricted B cell hybrid; American Type Culture Collection) were incubated with OVA peptide (OVA329–339) for 4 h at 37°C followed by centrifugation at 200 g for 5 min with an equal number of lymph node T cells from transgenic mice or from WAS−/−/OT-II lymphocytes transfected with pEGFP-C3 or pDSRED expression constructs. Samples were incubated at room temperature for 10 min and the cells were resuspended and plated onto poly-l-lysine–coated coverslips (Biocoat; Becton Dickinson) before fixation in 3% paraformaldehyde. Synapse formation was scored as the percent conjugates (T cell in physical contact with an APC) showing actin accumulation at the T cell–APC interface.