Figure 4.

B7-H1 mAb promotes programmed cell death of activated CD4⁺ T cells. (a) Human CD4⁺ T cells were cultured with 10 μg/ml of immobilized control (ctl) Ab, B7-H1 mAb, PD-1Ig in the presence of precoated optimal dose (500 ng/ml) of CD3 mAb. The cells were analyzed by FACS to determine apoptotic cells (positive in AV and negative in PI staining). (b) Blocking of B7-H1 mAb-induced apoptosis by soluble B7-H1Ig. Control Ig or B7-H1Ig at 10 μg/ml was preincubated with immobilized control Ab or B7-H1 mAb for 30 minutes before the addition of T cells. Percentages of apoptotic CD4⁺ T cells were shown at 72 hours of culture. (c) Expression of apoptotic genes by B7-H1 mAb costimulation. Purified human CD4⁺ T cells (5 × 10⁶) were cultured with immobilized B7-H1 mAb or control mAb at 10 μg/ml in the presence of optimal dose of CD3 mAb. The mRNA levels of each gene from B7-H1 mAb-stimulated T cells were presented as the fold induction, relevant to that from control mAb-treated T cells. (d) FACS analysis of expression of TRAIL protein on CD4⁺ T cells 3 days after anti-CD3/B7-H1 mAb costimulation. (e) Purified human CD4⁺ T cells were stimulated in the presence of indicated mAb or fusion protein in immobilized form as indicated in the legend of a and the activated form of caspase-3 at the indicated time point was stained by FAM-DEVD-FMK and subjected to FACS analysis. (f) Blocking of B7-H1 mAb-induced apoptosis of activated CD4⁺ T cells by anti–IL-10 neutralizing mAb. Purified human CD4⁺ T cells were stimulated with immobilized CD3 mAb and B7-H1 mAb for 72 hours, and the apoptotic cells were analyzed by AV and PI staining. Control Ab and mAbs to Fas ligand, IL-2, or IL-10 at 10 μg/ml was included at the beginning of the culture.