A combination of shear flow and chemokines sustains Vav1^−/− T-cell migration. (A,B) PKH26-labeled HY-specific CD4⁺ WT or Vav1 T cells (3 × 10^b) were injected intraperitoneally in syngeneic male mice that had previously received an intraperitoneal injection of IFNγ to induce MHC class II expression and antigen presentation. The percentage of T cells remaining in the peritoneal cavity and T-cell infiltration of the peritoneal membrane were evaluated 24 hours later by flow cytometry and wide-field fluorescence microscopy, respectively, as described in the legend to Figure 2. Representative examples of peritoneal lavage dot plots and peritoneal membrane sections (×10 magnification) are shown. The mean number of cells in the peritoneal membrane and lavage from at least 4 mice are shown. Error bars indicate standard error (*P < .05, **P < .01). (C) HY-specific WT and Vav1^−/− CD4⁺ T cells were perfused at 37°C over male-derived EC-coated slides at a fixed shear stress of 2.5 dyn/cm² for 10 minutes. Some ECs were pretreated with IFNγ for 48 hours to induce antigen presentation (indicated as IFNγ, C). In some experiments, ECs were overlaid with CXCL10 (300 ng/mL) for 2 hours prior to use in the flow assay. Slides were then removed, gently washed with warm PBS, and exposed to 0.05% trypsin-EDTA solution to obtain a cell suspension. The number of labeled T cells in this suspension was evaluated by flow cytometry (by gating on the small lymphocyte population and comparing the number of green and red fluorescent cells). The graphs summarize data obtained from at least 3 experiments. Error bars indicate standard error (*P < .05, **P < .01).