AP1903 sensitivity of LV'VFas+ T cells correlates with the level of transgene expression. (A) LV'VFas+ T cells (5 × 109/m2) were infused to macaque no. 3 and AP1903 (0.1 mg/kg) was given on day 1 and then every other day for 10 doses. PBMCs collected before and at the indicated days after infusion were analyzed by a quantitative real-time PCR assay for the presence of an LV'VFas sequence. Percentages (%) LV'VFas+ cells within PBMCs are as indicated. (B) Residual LV'VFas+ T cells express reduced levels of ΔLNGFR that are up-regulated by activation. PBMCs from macaque no. 3 before (upper left panel) and 1 day (upper right panel) or 23 days (lower left panel) after the T-cell infusion were stained with both anti-LNGFR and anti-CD3 mAbs, and evaluated by flow cytometry. The cells were gated on CD3+ T cells and the percentage of cells positive for both ΔLNGFR and CD3 are indicated in the upper right of each panel. The residual ΔLNGFR+ T cells present in PBMCs on day 23 (lower left panel) were sorted based on expression of ΔLNGFR and activated in vitro using anti-CD3 mAb alone or both anti-CD3 and anti-CD28 mAbs, respectively, in the presence of autologous γ-irradiated PBMCs. After 5 days of culture, cells were stained with both anti-LNGFR and anti-CD3 mAb (lower right panel). (C) Aliquots of LV'VFas+ (▪) cells were exposed on day 5 to 8 of the culture for 2 hours to 10 nM AP1903 or medium alone. Cells were cultured for 24 hours, and cell death as compared to the untreated cells was assessed by trypan blue exclusion or staining with 7-AAD. Aliquots of parental T cells (□) derived from pretreatment PBMCs were also stimulated and served as negative controls. Data are shown as the mean (± SD) of 3 experiments.
