Primary T cells transduced with the high-affinity TCR show lower peptide sensitivity compared with Tax-TCR transduced T cells. (A) Freshly transduced primary T cells had the following percentage of CD8+Vβ13+ T cells: Tax TCR = 26%; M1-TCR = 24%; M2-TCR = 25%; and M3-TCR = 26%. Bulk transduced T cells (1 ×105) were cocultured with T2 cells at a 1:1 ratio of transduced T cells–to–T2 cells loaded with pTax peptide or pHuD peptide at the stated concentrations. After 18 hours of stimulation, the supernatant was removed to determine IFN-γ release by ELISA. Stimulation with T2 cells pulsed with the control pWT235 peptide or without peptide defined the background level of IFN-γ release. This represents 4 individual experiments that demonstrated similar results. (B) Primary T cells transduced with the Tax TCR and affinity-matured versions and having undergone 5 rounds of weekly peptide stimulation had the following percentage of CD8+Vβ13+ T cells: Tax TCR = 97%; M1-TCR = 99%; M2-TCR = 99%; and M3-TCR = 99%. T cells (2 × 105) were stimulated with 2 × 105 T2 cells loaded with decreasing concentration of pTax peptide. After an 18-hour coculturing period, cells were stained with tetramer. Shown is the relative loss of tetramer-positive T cells after peptide stimulation. The experiment represents 3 individual experiments that showed similar results. (C) Primary T cells that were transduced with the Tax TCR or the affinity-matured version and expanded using 5 rounds of weekly peptide stimulation had the following CD8+/Vβ13 expressing cells: Tax TCR = 89%; M1-TCR = 93%; M2-TCR = 91%; and M3-TCR = 90%. T cells (2 × 105) were stimulated with either 2 × 105 HeLa GFP-positive cells that had been transduced with either the GFP plasmid or the GFP-Tax gene fusion plasmid, or were stimulated with the same transduced HeLa cells that were loaded with 100μM exogenous pTax peptide. Cells were cocultured in the presence of anti-CD107a, brefeldin A, and GolgiStop for 18 hours. Shown are the percentages of CD8+ and CD107a+ T cells. The figure represents 5 independent experiments that demonstrated similar results.
