TETARs functionally expresses both introduced TCRs simultaneously and retains the ability to kill target cells with the second TCR after antigen-specific down-modulation of the first TCR. (A) CD8⁺ T cells were transfected with the gagTCR or with a combination of the gagTCR and the murinized (mur) nefTCR (quantities as indicated). RNA-transfected CD8⁺ T cells were stained with the gag/HLA-A2 dextramer and sorted for dextramer-positive cells 2-3 hours after electroporation. These positive cells were used as effector cells in a cytokine production assay 6 hours after electroporation. Irradiated EBV-transformed B cells loaded with gag peptide, nef peptide, a combination of gag and nef peptide, or, as a negative control, an influenza nucleoprotein (INP) peptide (CTELKLSDY) were used as stimulator cells at a ratio of 1:1 to the effector cells. IL-2 release was measured by a cytometric bead array after overnight stimulation. Cytokine production by CD8⁺ T cells transfected with gagTCR and stimulated with gag peptide (mean value IL-2 secretion 3.5 ng/mL) was set to 100%, and other values were normalized to that. Average values of 4 standardized independent experiments ± SEM are shown. (B-C) CD8⁺ T cells were transfected with the human gagTCR, the murinized nefTCR, or a combination of the gagTCR and the murinized nefTCR (as indicated). Twelve to 16 hours after electroporation, these reprogrammed T cells were cocultured at a ratio of 1:1 with irradiated EBV-transformed B cells loaded with gag (open bars) or nef (solid bars) peptide. Two hours after stimulation, these cells were used in a standard 4-6–hour cytotoxicity assay in which additionally Cr⁵¹-labeled EBV-transformed B cells were added. These labeled B cells were loaded with the gag peptide (B) or the nef peptide (C) and served as target cells at a ratio of 1:60 (target-to-effector ratio). Data of 1 representative experiment of 3 are shown. Error bars indicate the SD of triplicate values. Negative values were set to zero.