Transfer of antigen specificity by retroviral transfer of TCR-D115 and TCR-T58. (A) PBLs of a healthy donor were transduced with TCR-D115 or TCR-T58. Unsorted TCR-transduced PBLs were analyzed on day 10 for transgenic TCR expression using irrelevant B7-pp65 and A2-pp65 multimers and specific A2-tyr multimers. Untransduced PBLs showed no multimer binding (0.1%, data not shown). Percentages of multimer+CD8+ T cells are displayed in the top right quadrant. (B-C) The IFN-γ release of unsorted TCR-transduced PBLs after stimulation with T2 cells loaded with graded amounts of tyrosinase peptide (10−12 M to 10−5 M) at a ratio of 2:1. (B) The relative IFN-γ release is displayed in percentage. (C) The specific IFN-γ release is presented as picograms per milliliter. (D) Functionality of unsorted TCR-transduced PBLs was measured by IFN-γ release using autologous HLA-A2+ PBMCs loaded with tyrosinase peptide (10−11 M to 10−6 M) as stimulating cells at ratio of 2:1. Untransduced PBLs (▲) showed no peptide-specific IFN-γ release. (E) The HLA-A2+tyrosinase− tumor cell lines Mel-A375, RCC-26, and MaCa1/A2 were exogenously loaded with either 10−5 M irrelevant flu peptide (f) or 10−5 M tyrosinase peptide YMD (t) and IFN-γ secretion was measured by ELISA and given as picograms per milliliter. (F) Specificity of recognition was assessed by IFN-γ release after coculture with the tumor cell lines from left to right: MaCa1 (HLA-A2−tyrosinase−); SK-Mel-28 (HLA-A2−tyrosinase+); Mel-A375, RCC-26, PancTu 1, MaCa1/A2, and UTS CC 1588 (all HLA-A2+tyrosinase−); Mel-624.38, Mel-93.04A12, SK-Mel-23, SK-Mel-29, and WM-266-4 (all HLA−A2+tyrosinase+). T indicates CTL without stimulating cells.
