Vav1^−/− T cells display defective T-cell/antigen-presenting DC interactions in vivo. (A) PKH26-labeled T cells (10⁷) were injected intravenously in syngeneic female mice that simultaneously received an intraperitoneal injection of Dby peptide–pulsed female-derived matured DCs labeled with CFSE, or DCs alone as a control. HY-specific CD4⁺ WT and Vav1^−/− T cells and DCs were injected alone as a control. In this model, T cells travel into the bloodstream and reach the peritoneal membrane and the spleen, whereas DCs travel out of the peritoneal cavity, through the peritoneal membrane, enter the bloodstream, and reach the spleen. The presence of T-cell/DC conjugates in the peritoneal membrane and spleen was quantified 24 hours later by wide-field fluorescence microscopy as described in “Methods.” The occurrence of cell-cell interactions was apparent as yellow fluorescence. Representative 40× images from the peritoneal membrane (A) and the spleen (D) are shown. The number of conjugates in the peritoneal membrane (A) and spleen (D) were averaged and quantified with the algorithm described in “Wide-field fluorescence microscopy and flow cytometry” in 10 × 10 images obtained from samples from at least 6 animals. In addition, the mean number of labeled T cells (not engaged by DCs) in the peritoneal membrane and cavity was measured as described in the legend to Figure 3, and is shown in panels B and C, respectively. No T-cell/DC conjugates were detected in the peritoneal lavage (data not shown). Error bars indicate standard error (**P < .01, ***P < .001).