Schematic design of the artificial Fas receptor and persistence of autologous LV'VFas T cells. (A) The Fas signaling domain is incorporated in a fusion protein (LV'VFas) of human origin that consists of the extracellular and transmembrane portions of the human low-affinity nerve growth factor receptor (ΔLNGFR), 2 copies of the FKBP12 containing a single amino acid substitution at position 36 (F36V) as drug-binding domains, and the cytoplasmic domain of Fas. The LV'VFas protein remains inert unless clustering and Fas-mediated apoptosis are induced by the F36V-binding drug AP1903. The fusion gene is introduced into T cells using a retroviral vector and expressed from the MoMLV-LTR. (B-E) In vitro selection of LV'VFas-modified T cells and sensitivity to AP1903. (B-C) Analysis of ΔLNGFR expression in T cells either unmodified (B) or transduced with the LV'VFas retroviral vector and enriched using ΔLNGFR-coated microbeads (C). T cells were stained with anti-LNGFR and anti-CD3 mAbs and analyzed by flow cytometry. The percentage of cells positive for both ΔLNGFR and CD3 are indicated in the upper right quadrant. Data are shown for transduced T cells selected for infusion into macaque no. 2. (D) Macaque LV'VFas⁺ T cells are sensitive to AP1903-induced cell death in vitro. Aliquots of LV'VFas⁺ T cells were exposed for 2 hours to 10 nM AP1903 (solid line) or media alone (dashed line), stained after 24 hours with 7-AAD, and examined by flow cytometry. Cells were considered to be positive for 7-AAD if they stained outside the gate indicated by the bisected line. The number of LV'VFas T cells that stained positive for 7-AAD after exposure to AP1903 is indicated (74.0%). Data are shown for a representative experiment. (E) Aliquots of unmodified (○) or LV'VFas⁺ T cells (•) were exposed 4 times (48 hours apart) for 2 hours to medium alone or to medium containing 10 nM AP1903. Cells were washed, and the survival of LV'VFas⁺ T cells as compared to the untreated cells was evaluated 24 hours later by trypan blue exclusion.