Activation of HC/2G-1 T cells by DR4 loaded with soluble TRAIL. (A) CHO/CD58 cells were transduced with retroviruses that encode various truncations of the carboxyl terminus of DR4, and their recognition by HC/2G-1 T cells was measured by IFN-γ ELISA. The gray box in DR4 denotes the death domain. (B) A 96-well plate was coated with anti-CD2 Ab, control Ab, recombinant DR4-Fc, DR5-Fc, and Fc, individually or in various combinations as indicated. After blocking with PBS/FBS for 1 hour, recombinant soluble TRAIL (10 μg/mL) solubilized in the culture medium was added where indicated. After 3 hours, wells were rinsed to remove unbound soluble TRAIL and HC/2G-1 T cells were added. After an overnight culture, IFN-γ in the supernatant was measured by ELISA. Specificity controls (HEK-293 as the negative control and RCC#6 as the positive control) are shown in the right panel. (C) Titration of soluble TRAIL was done using the same method as in panel B. After coating with anti-CD2 Ab and recombinant DR4-Fc, DR5-Fc, DcR1-Fc, or DcR2-Fc, wells were blocked with PBS with 5% FBS for 1 hour, and serially diluted soluble TRAIL in the culture medium was added as indicated. After incubating for 3 hours, unbound TRAIL was removed by rinsing and HC/2G-1 T cells were added. IFN-γ in the supernatant was measured by ELISA after an overnight culture. Error bars indicate SEM.