(A and B) H2-A^b–restricted HY-specific WT (black bars), CD28^Y170F (white bars), and p110δ^D910A (light gray bars) CD4⁺ T cells were injected i.v. into male mice (10⁷/mouse) that had received an i.p. injection of IFN-γ (600 U) 48 hours earlier. The following day, mice were sacrificed, and the presence of fluorescently labeled cells in the peritoneal cavity (A) and membrane (B) was assessed by flow cytometry and wide-field fluorescence microscopy, respectively. To facilitate visualization by flow cytometry, cells were double stained with an APC-conjugated anti-CD4 antibody following harvesting. The mean T cell numbers ± SEM observed in samples from at least 3 animals are shown. A: *P < 0.04, CD^28Y170F versus WT T cells; **P < 0.001, p110δ^D910A versus WT T cells; P < 0.005, p110δ^D910A versus CD^28Y170F T cells. B: *P < 0.03, CD^28Y170F versus WT T cells; **P < 0.002, p110δ^D910A versus WT T cells; P < 0.007, p110δ^D910A versus CD^28Y170F T cells. (C and D) HY-specific WT or p110δ^D910A CD4⁺ T cells that had either undergone antibody-mediated CD28 ligation (30 minutes at 37°C, PKH26-labeled) or had been pretreated with an antibody isotype control (IsC) (CFSE-labeled) were injected i.v. (10⁷/mouse) into male mice that had received an i.p. injection of IFN-γ 48 hours earlier. The presence of fluorescently labeled cells in the peritoneal cavity (C) and membrane (D) was assessed 24 hours later as described for A and B. The mean T cell number ± SEM observed in samples from at least 3 animals is shown. C: *P < 0.02 versus WT + IsC; D: *P < 0.02 versus WT + IsC.