Antigen-driven Vav1^−/− T-cell migration. HY-specific CD4⁺ WT and Vav1^−/− T cells (3 × 10⁵) were seeded onto IFNγ-treated antigenic (male) or nonantigenic (female) syngeneic EC monolayers grown on transwells. The mean percentage migration at the indicated time points from 3 experiments of similar design is shown. (B) T cells (1 × 10⁶) were plated on 35-mm dishes coated with IFNγ-treated antigenic (male) or nonantigenic (female) ECs and allowed to migrate for 50 minutes. Pictures were taken every 30 seconds and analyzed as described in “Methods.” Transmigrating T cells were defined as changing phase (ie, turning from bright to dark once under the endothelial monolayer). Mean percentage of cells transmigrating was calculated from 3 independent experiments from a sample of 100 cells per movie. (C,D) HY-specific CD4⁺ WT and Vav1^−/− T cells labeled with PKH26 (red) and CFSE (green), respectively, were coinjected intravenously into syngeneic male or female recipients that had previously received an intraperitoneal injection of IFNγ. Labeled T-cell enrichment of the peritoneal lavage was analyzed by flow cytometry 24 hours later (C). The mean percentage of CD4⁺-labeled (PKH26 or CFSE) T cells in the peritoneal lavage from at least 3 animals is shown (C right panel). Nuclei are stained by DAPI (blue). Retention of T cells into the peritoneal membrane (D) was analyzed by wide-field fluorescence microscopy as described in the legend to Figure 2. The mean number of cells in 6 tissue samples from at least 3 mice is shown (D right panel). Error bars indicate standard error (*P < .05, **P < .01).