Simultaneous transfection of two human TCRs, but not of one human TCR combined with one murinized TCR, leads to a competitive effect that impairs their function. (A) CD8⁺ T cells were transfected with the α and β chains of the human gagTCR, the human nefTCR, or a combination of the human gagTCR and the human nefTCR by RNA electroporation (RNA quantities as indicated), or (B) T cells were electroporated with 10 μg of TCR RNA encoding the gagTCR, the human (hum) nefTCR, or the murinized (mur) nefTCR, or 2 μg of the human or the murinized nefTCR combined with 12 μg of the gagTCR. These T cells were used as effector cells in a cytokine-production assay 2-4 hours after electroporation. The transfection volume was equalized in all transfections with water. EBV-transformed B cells, either loaded with the gag peptide (solid bars) or with the nef peptide (open bars), were used as stimulator cells at a ratio of 1:1 to the effector cells. TNF secretion into the supernatant was measured with a cytometric bead array after overnight incubation. Cytokine production by CD8⁺ T cells transfected with 10 μg of the human gagTCR and stimulated with the gag peptide (mean value TNF secretion 15.6 ng/mL) was set to 100%, and other values were normalized to that. Average values of 3 independent standardized experiments ± SEM are shown.