(A) HY-specific H2-A^b–restricted WT and P110δ^D910A T cells (5 × 10⁵/well) were seeded onto untreated syngeneic female-derived EC monolayers grown on 3-μm-pore Transwells, and T cell migration was monitored as described in Methods. Results are expressed as a percentage of migrated T cells at the given time points and reported as the average of 3 experiments of identical design. Error bars are shown. (B) HY-specific H2-A^b–restricted WT and P110δ^D910A T cells (5 × 10⁵/well) were seeded onto ICAM-1–coated (2 μg/ml) 35-mm dishes, and their migration was observed for 25–30 minutes by time-lapse microscopy. A representative example of a series of 3 independent experiments with similar design is shown. Mean migration speed ± SEM is shown. (C and D) PKH26-labeled HY-specific H2-A^b–restricted CD4⁺ WT and P110δ^D910A T cells were injected i.v. into syngeneic female mice. T cell localization in the indicated tissues was assessed 24 hours later by wide-field fluorescence microscopy. To minimize the effect of arbitrary choice of field, tissue infiltration was quantified by randomly selecting ten ×10-magnified fields from tissue samples from at least 3 animals and assessing the number of fluorescent cells in each field. Each panel shows a representative tissue image. The mean T cell infiltration ± SD observed in samples from at least 3 animals is shown in D.