Motility of Vav1^−/− T cells in vitro. (A) Mean expression (from at least 3 independent experiments) of LFA-1 by HY-specific CD4⁺ WT and Vav1^−/− T cells 7 days following antigen stimulation. (B) Mean adhesion (from at least 3 independent experiments) by WT and Vav1^−/− T cells to ICAM1 (2 μg/mL)–coated 96-well plates at the indicated time points. (C) Mean migration (from 4 independent experiments) by WT and Vav1^−/− T cells 6 hours after plating onto ICAM1-coated transwells. (D) Mean migration by WT and Vav1^−/− T cells through syngeneic female EC monolayers. Migration was measured at 2, 4, 6, and 24 hours. The percentage of migrated cells was calculated by dividing the number of cells in the bottom chamber by the total input of T cells from the mean of 3 experiments. (E,F) Migration by WT and Vav1^−/− T cells plated on ICAM1-coated dishes was analyzed by time-lapse microscopy. The number of cells migrating was quantified by counting motile T cells (E). T cells were tracked using Kinetiq tracking software (Kinetiq Media, Chichester, United Kingdom) and their migratory speed (F, μm/min) was quantified using Mathematica spreadsheets. The mean percentage of motile cells and mean speed was calculated from data of 3 independent experiments. (G) WT and Vav1^−/− T-cell migration in response to CXCL10 through a transwell was assessed by counting the cells in the bottom chamber at 2, 4, and 6 hours. Percentage migration was calculated by dividing the number of cells in the bottom chamber by the original number of cells plated on the transwell. The mean percentage migration from 4 independent experiments is shown. Error bars indicate standard error (*P < .05, ** P < .01, ***P < .001).