IL-7 and IL-15 preserve high proliferative potential, self-renewal ability, and low sensitivity to activation-induced cell death in gene-modified T_CM CD8 and CD4 lymphocytes. Immune-selected TK⁺ lymphocytes harvested at day 10 and unmanipulated CD3⁺ lymphocytes (PBLs) from the same donor were labeled by CFSE, plated at the concentration of 10⁶/mL, and cocultured with irradiated allogeneic PBMCs at a 1:1 ratio for 7 days. Viable cells were then harvested and restimulated by the same conditions for additional 7 days. No cytokine was added. 1st indicates first allostimulation; 2nd, second allostimulation. (A) Number of viable CD8⁺ and CD4⁺ lymphocytes that had proliferated, as measured by CFSE dilution (CFSE^dim), 6 days after each stimulation, were calculated according to the formula: (no. of viable cells × % of CFSE^dim cells). baCD3/CD28⁺ IL-7/IL-15 (■), baCD3/CD28⁺ IL-7 (), baCD3/CD28⁺ IL-2 (), aCD3⁺ IL-2 (□), or PBLs (). N = 7 or more per group from 4 independent experiments with 9 independent donors. Allo Ag indicates cells stimulated with allogeneic irradiated PBMCs; no Ag, mock-stimulated cells. Nonparametric analysis was performed. *P < .05; **P < .01. (B) CCR7 expression was measured on CFSE-stained TK⁺ lymphocytes and PBLs 6 days after first and second allogeneic stimulation. Contour plots show CFSE content (x-axis) versus CCR7 expression (y-axis) on CD3⁺ lymphocytes. Results from 1 representative of 5 independent experiments with 10 independent donors are shown. Histograms show averages plus or minus SD of the percentage of CCR7-expressing cells gated in CFSE^dim CD3⁺ lymphocytes. baCD3/CD28⁺ IL-7/IL-15 (■), baCD3/CD28⁺ IL-7 (), baCD3/CD28⁺ IL-2 (), aCD3⁺ IL-2 (□), or PBLs (). Results of 5 independent experiments with 10 independent donors are shown. *P < .05; **P < .01. Asterisks only on a bar indicate significant differences versus aCD3⁺ IL-2. (C) CD127 expression was measured on CFSE-stained TK⁺ lymphocytes and PBLs 6 days after first and second allogeneic stimulation. Contour plots show CFSE staining (x-axis) versus staining with CD127 (y-axis) in gated CD3⁺ lymphocytes after 1 and 2 stimulation cycles. Results from 1 representative of 6 independent experiments with 11 independent donors are shown. Histograms show averages plus or minus SD of the percentage of CD127-expressing cells gated in CFSE^dim CD3⁺ lymphocytes. baCD3/CD28⁺ IL-7/IL-15 (■), baCD3/CD28⁺ IL-7 (), baCD3/CD28⁺ IL-2 (), aCD3⁺ IL-2 (□), or PBLs (). Results of 6 independent experiments with 11 independent donors are shown. *P < .05; **P < .01, versus aCD3⁺ IL-2. (D) Activation-induced cell death was measured on CFSE-stained TK⁺ lymphocytes and PBLs after stimulation with escalating OKT3 concentrations. The percentage of To-Pro-3⁺ cells in CFSE^dim T lymphocytes was measured by flow cytometry. baCD3/CD28⁺ IL-7/IL-15 (■), baCD3/CD28⁺ IL-7 (), baCD3/CD28⁺ IL-2 (), aCD3⁺ IL-2 (□), or PBLs (). N = 3 per group from 2 independent experiments with 3 independent donors. *P < .05; **P < .01, versus aCD3⁺ IL-2.