B7-H1 signaling to tumor cells is required for molecular shield. (A) Schematic of the wild-type B7-H1 (B7-H1) and cytoplasmic domain-truncated B7-H1 (ΔB7-H1) genes. IgV indicates IgV domain; IgC, IgC domain; TM, transmembrane domain; CY, cytoplasmicdomain; GFP, gene encoding GFP. (B) The expression of wild-type B7-H1 (B7-H1; top left) or truncated B7-H1 (ΔB7-H1) on P815 cells (top right) after transfection was determined by flow cytometry using anti-mouse B7-H1 mAb (10B5) or murine PD-1Ig fusion protein. These cell lines were also stained by anti–H-2L^d mAb. (C) Activated 2C CTLs were incubated at indicated E/T ratios with ⁵¹Cr-labeled mock/P815, B7-H1/P815, or ΔB7-H1/P815 cells in the presence of control (Ctl) IgG or anti-mouse B7-H1 mAb (10B5) for 4 hours. CTL activity was determined in a ⁵¹Cr release assay. Each point is the mean of triplicates with SD. The data are representative of at least 3 experiments. (D) Equal mix of mock/P815 and B7-H1/P815 cells (top), ΔB7-H1/P815, and B7-H1/P815 cells (middle), or mock/P815 and ΔB7-H1/P815 (bottom) were coincubated with 2C CTLs for 4 hours. The E/T ratio is 2.5:1. Cells were harvested and analyzed by flow cytometry. The numbers in the graph indicate the percentage of cells in H-2D^d+ population. The data are representative of at least 3 experiments.