In the absence of WASp contacts between DCs and T cells are less functional. (A-B) Recording of calcium oscillations resulting from DC-T-cell interactions, shown are stills from a 30-minute recording (6 frames/min), calcium was measured by Fluo4 intensity and is depicted as pseudocolor, arrows indicate when cells are in contact. In the last panels, analysis of Fluo4 fluorescence intensity is plotted for representative T cells together with the velocity of the cells and manual tracking of contacts with a DC, gray boxed area indicates when T cell is in contact with a DC. (C) Quantification of data from panels A and B, shown is the SD of fluorescence intensity expressed as a percentage of the average fluorescence within the indicated recording time. Data are the mean of 3 independent experiments and shown as average ± SEM, C57BL/6 n = 72-86 cells, WAS KO n = 64-94 cells, P values are indicated (1-way ANOVA). (D) Analysis of ZAP70 phosphorylation resulting from TCR stimulation. Top panels show immunoblot with anti phospho-tyrosine after immunoprecipitation with anti–ZAP70, bottom panels show immunoblot with anti-ZAP70 as loading control. Representative blots of 4 independent experiments are shown. (E) Densitometry analysis of Western blot analysis shown in panel D. Data are shown as box-and-whisker plots (minutes to max) and P values are indicated (Student t test).