Self-renewal and differentiation of human tk⁺ mH antigen-specific CTLs based on IL-7Rα expression. (A) HA1-specific CTLs generated after 2 rounds of antigenic stimulation were sorted for IL-7Rα expression by flow cytometry (left panels). The percentage of CD8⁺ T cells (x-axis) positive for the IL-7Rα (y-axis) is shown before and after sorting (arrows). Cytotoxicity (middle panels) is expressed as the percentage of lysis (y-axis) at different effector-to-target ratios (x-axis) against an HLA-A2^pos/HA1^neg lymphoblastoid cell line (HA1^neg LCL, ◊) or an HLA-A2^pos/HA1^pos LCL (HA1^pos LCL, ♦). The cytotoxicity of a HA1-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). Cytokine production (right panels) was evaluated by flow cytometry after antigenic stimulation (see “Generation, expansion and functional characterization of human mH antigen-specific CTLs”) and is expressed as the percentage of cells with the following profile of intracytoplasmic staining: IFN-γ⁻/IL-2⁺ (□), IFN-γ⁺/IL-2⁺ (▩), IFN-γ⁺/IL-2⁻ (■). Inset fold numbers indicate the expansion rate of sorted populations after PHA stimulation. (B) IL-7Rα expression (left panels), cytotoxicity (middle panels), and cytokine production (right panels) of the progeny of IL-7Rα⁺ HA-1–specific CTLs cells before and after re-sorting (arrows). After re-sorting, cells were re-stimulated with PHA. Fold growth of IL-7Rα⁺ cells from IL-7Rα⁺ cells was 10.4, fold growth of IL-7Rα⁻ cells from IL-7Rα⁺ cells was 4.7, and fold growth of IL-7Rα⁻ from IL-7Rα⁻ cells was 2.3. Results from the representative donor HA1-tk #1 are shown. (C) Relative telomere length (y-axis) of tk⁺ T cells generated with CD3/CD28-beads and IL-7/IL-15 (tk, □) and of tk⁺ mH antigen-specific CTLs (mH-tk) sorted as IL-7Rα⁺ (▩) or IL-7Rα⁻ (■) was measured by flow FISH. Means ± SD of relative telomere length from n = 2 HLA-A2^pos/HA-1^neg and n = 1 HLA-A2^pos/H-Y^neg donors and results from a paired t test statistical analysis are shown (*P < .05).