Expansion and cytotoxicity of human tk⁺ mH antigen-specific CTLs. (A) The transduction efficiency (left panels) of peripheral blood lymphocytes (PBLs) from HLA-A2^pos/HA-1^neg donors either after activation with an anti-CD3 mAb (OKT3) and culture with IL-2 or after activation with CD3/CD28-beads and culture with IL-2 (B + IL-2), IL-7 (B + IL-7) or a combination of IL-7 and IL-15 (B + IL-7/IL-15) was evaluated by flow cytometry and is shown as the percentage of T cells positive for the NGFR marker gene (y-axis) over the FSC (x-axis). Flow cytometry was also used to measure (middle panels) and to isolate (right panels) CD8⁺ T cells (x-axis) binding the HLA-A2^HA-1 tetramer (y-axis). In the case of RV-mediated transduction, HLA-A2^HA-1 tetramer-binding CD8⁺ T cells were gated for NGFR expression. Inset fold numbers indicate the expansion rate after PHA stimulation. Cytotoxicity is expressed as the percentage of lysis (y-axis) at different effector-to-target ratios (x-axis) against an HLA-A2^pos/HA-1^neg lymphoblastoid cell line (HA1^neg LCL, ◊) or an HLA-A2^pos/HA1^pos LCL (HA1^pos LCL, ♦). The cytotoxicity of an HA-1–specific cytotoxic T lymphocyte clone (HA1 clone) against the HA1^pos LCL is shown as reference (□). Maximal background cytotoxicity of the HA1 clone against the HA1^neg LCL was always < 5% (not shown). Results from 1 donor representative of n = 3 are shown. (B) The same experimental procedure was applied to HLA-A2^neg/H-Y^neg (female) donors using appropriate reagents and control cell lines. Results from 1 donor representative of n = 4 are shown. (C) The expansion of HA-1–specific CTLs (HA1 CTLs, left panel) generated after either 2 rounds (HA1^×2, ▿) or 4 rounds of antigenic stimulation (HA1^×4, ▾) is expressed as fold growth. The cytotoxicity of HA1 CTLs (right panel) generated after either HA1^×2 (◊) or HA1^×4 (♦) is expressed as a relative percentage over that of the HA1 clone (see “Functional characterization of human mH antigen-specific CTLs”). Each symbol represents the result from a single donor. (D) The expansion (left panel) and cytotoxicity (right panel) of HA-1–specific CTLs generated from B + IL-7/IL-15 tk⁺ T cells (HA1-tk CTLs) after either HA1^×2 or HA1^×4 are shown. Each symbol represents the result from a single donor. (E-F) Experiments were repeated with H-Y–specific CTLs generated from PBLs (HY CTLs) or from B + IL-7/IL-15 tk⁺ cells (HY-tk CTLs) of HLA-A2^neg/H-Y^neg donors after either 2 (HY^x2) or 4 (HY^x4) rounds of antigenic stimulation, using appropriate reagents and control cell lines. Expansion (left panels) and cytotoxicity (right panels) are shown. Each symbol represents the result from a single donor. Results from a paired t test statistical analysis are shown (*P < .05, **P < .01).