In vitro characterization of HMMR-specific T-cell clones and TCR-transgenic lymphocytes. (A) Clones induced by in vitro priming using DCs prepared from an HLA-A2⁻ donor pulsed with HLA-A2 and HMMR ivt-RNA. Lytic capacity (percentage specific lysis) was assessed in a standard 4-hour chromium-release assay using K562-A2 (HLA-A2⁺, HMMR⁺) cells as a positive target and HLA-A2⁺ T2 cells pulsed with an irrelevant peptide (flu) as a negative control. (B) Lytic capacity, shown as the percentage specific lysis by HMMR-specific CTL67 and CTL150 of THP-1 and K562-A2 and T2 cells pulsed with the flu or ILS peptide of HMMR, respectively at an effector-to-target ratio of 1:5. (C) Cytokine secretion by CTL67 and CTL150 given in nanograms per milliliter for 2 × 10³ cells 24 hours after stimulation with the 4 target cells described in panel B. (D) Flow cytometry staining of TCR150-transgenic lymphocytes showing expression in CD3, CD4, and CD8 T cells (from left to right). (E) IFN-γ ELISA of 4 × 10⁴ lymphocytes stimulated with 2 × 10³ tumor cells for 24 hours. THP-1 and K562-A2 cells were used as positive stimulating cells, whereas T2 cells pulsed with flu peptide served as an HMMR⁻ control. PBLs were transduced with TCR150 or GFP control vector. Mock PBLs served as a background control. Data are given in nanograms per milliliter. (F) Specific lysis of target cells THP-1 (A2⁺; HMMR⁺), K562-A2 (A2⁺; HMMR⁺), mel624.38 (A2⁺; HMMR⁺), and K562 (A2⁻; HMMR⁺) mediated by untransduced PBLs (mock; ○) or TCR150-transgenic PBLs (●).