WASp in DCs is required for pSMAC formation. (A) Confocal analysis of ICAM-1/GFP-transduced DCs that formed an IS with OT-II cells. From left to right panels show DIC, GFP signal, pseudocolor of GFP and 3D reconstruction of the contact interface area between the cells, projected en face, scale bars represent 5 μm. Right panel shows schematic representation of classic bull's-eye immune synapse and SMAC distribution (B) Quantification of data shown in panel A, data are shown as the ratio of the mean fluorescence intensity of the interface area divided by the fluorescence of the peripheral plasma membrane, data are shown as average ± SEM and are from 4 independent experiments, C57BL/6 n = 34 cells, WAS KO n = 31 cells, P values are indicated (Student t test). (C) 3D reconstruction of the interface between DCs and T cells from z-sections obtained by confocal imaging, in top panels LFA-1 is in green, CD45 in red, in bottom panels TCR is in green, asterisks indicate cSMAC foci. (D) Quantitative evaluation of IS formation based on appearance (focused cSMAC or multifocal), data are percentage of cells with classic IS appearance from at least 10 experiments with C57BL/6 n = 98 cells, WAS KO n = 87 cells and Y293F WASp n = 35 cells, P values are indicated (Fisher exact).