(A–C) HY-specific H2-A^b–restricted WT and p110δ^D910A CD4⁺ T cells or HY-specific H2-K^k–restricted CD8⁺ C6 T cells were seeded onto IFN-γ–treated female- and male-derived syngeneic EC monolayers grown on 3-μm-pore Transwells. T cell migration was assessed as described in Figure 1. Error bars for 3 averaged experiments are shown. *P < 0.05 versus migration through female ECs at all time points except 2 hours. (D and E) Male and female C57BL/6 mice were injected i.p. with 600 U IFN-γ or PBS. Two days later, mice received an i.v. injection of PKH26-labeled HY-specific CD4⁺ T cells (10⁷/mouse). The presence of labeled cells in the peritoneal membrane (D) and cavity (E) was assessed 24 hours later by wide-field fluorescence microscopy and flow cytometry, respectively. A cluster of 3 PKH26-labeled cells is visible in the membrane of male recipient of WT HY-specific T cells. Due to the presence of an autofluorescent population of non-T cells often detected in FL-2 (also in control mice that received IFN-γ but no T cells; far-left dot plot, “saline”), cells were double stained with an APC-conjugated anti-CD4 antibody following harvesting, and the percentage of PKH26 (FL-2)–labeled T cells gated in the CD4⁺ T cell population is shown in the dot plots and the graph representing cumulative data from at least 3 animals. The mean ± SEM observed in samples from at least 3 animals is shown on the right. *P < 0.04 versus female mice.