C57BL/6 female mice received 3 skin grafts: one from a syngeneic male mouse (A), one from a syngeneic female mouse (B), and the third from a CBA/Ca male mouse (C). Four days following grafting, HY-specific A^b-restricted WT (PKH26-labeled) CD4⁺ T cells (10⁷/mouse), were injected i.v. Graft infiltration by labeled T cells was assessed 24 hours later by wide-field fluorescence microscopy, as described in the legend to Figure 1, C and D. Original magnification, ×10. The mean values ± SD observed in samples from at least 3 animals are summarized in D. *P < 0.03 versus female and CBA/Ca graft infiltration. Parallel experiments were run in which C57BL/6 female mice engrafted with C57BL/6 male-derived skin 4 days earlier were coinjected with either WT (PKH26-labeled, red) or p110δ^D910A (CFSE-labeled, green) HY-specific CD4⁺ T cells (E and F) or with untreated (PKH26-labeled, red) or IC87114-treated (CFSE-labeled, green) HY-specific CD4⁺ T cells (G and H). Grafts were removed 24 hours later, and 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. The few CFSE-labeled cells are indicated by an arrowhead. Nuclei are stained by DAPI (blue). A–C show representative ×10-magnified tissue images, while the images shown in E and G were taken at a ×20 magnification. The mean T cell infiltration ± SD observed in samples from at least 3 animals is shown; *P < 0.03 (F); *P < 0.01 (H).