Retroviral transduction after baCD3/CD28 activation and culture with γ-chain cytokines results in central memory TK⁺ lymphocytes. (A) PBMCs were stimulated with either baCD3/CD28⁺ IL-7/IL-15 (■), baCD3/CD28⁺ IL-7 (), baCD3/CD28⁺ IL-2 (), aCD3⁺ IL-2 (□), or baCD3/CD28 only (▦). At 48 and 72 hours after stimulation, cells were transduced with the cell-free supernatant carrying the retroviral vector SCFMM-3 Mut2. Transduction efficiency was measured by flow cytometry at day 7. Averages plus or minus SD of 9 independent experiments from different donors are shown. **P < .01 versus all other samples. (B) TK⁺ lymphocytes were cultured until day 21. Cytokines were added every 3 to 4 days. Immune-selected cells were counted with trypan blue exclusion at days 7, 9, 15, and 21. To evaluate and compare TK⁺ lymphocyte expansion, while avoiding biases related to variable transduction efficiency, cell expansion was calculated according to the following formula: (no. of cells at day X/no. of cells at day 7). baCD3/CD28⁺ IL-7/IL-15 (◆), baCD3/CD28⁺ IL-7 (), baCD3/CD28⁺ IL-2 (), aCD3⁺ IL-2 (□), or baCD3/CD28 only (black square with dotted line). Median of 3 independent experiments from different donors is shown. *P < .05; **P < .01 versus baCD3/CD28⁺ IL-7/IL-15. (C) CD4/CD8 ratio was measured on transduced lymphocytes by flow cytometry at day 10. Averages plus or minus SD of 9 independent experiments from 9 independent donors are shown. **P < .01 versus all other samples. (D) Expression of CD45RA and CD62L was assessed on TK⁺ lymphocytes at day 10 by flow cytometry. Relative distribution plus or minus SD of T_NA (CD45RA⁺CD62L⁺, ▥), T_CM (CD45RA⁻CD62L⁺, ▨), T_EM (CD45RA⁻CD62L⁻, ▩), and T_E (CD45RA⁺CD62L⁻ ▩) lymphocytes on CD3 TK⁺ lymphocytes are shown. N = 6 or more per group from 10 independent experiments with 10 independent donors. **P < .01, versus all other samples. (E) Expression of CD27 and CD28 was assessed on TK⁺ lymphocytes at day 10 by flow cytometry. Relative distribution plus or minus SD of CD27⁺CD28⁺ (▨), CD27⁺CD28⁻ (▩), CD27⁻CD28⁺ (▥), and CD27⁻CD28⁻ (▩) cells in CD3 TK⁺ lymphocytes are indicated. N = 5 or more per group from 9 independent experiments with 9 independent donors. *P < .05; **P < .01, versus aCD3⁺ IL-2. (F) At day 10, TK⁺ lymphocytes were stimulated by PMA/ionomycin and analyzed by flow cytometry for IFNγ and IL-2 production. Averages plus or minus SD of IFNγ⁻IL-2⁺ (▨), IFNγ⁺IL-2⁺ (▩), and IFNγ⁺IL-2⁻ (▩) cells in CD3 TK⁺ lymphocytes are indicated. N = 5 per group from 5 independent experiments with 5 independent donors. *P < .05; **P < .01 versus aCD3⁺ IL-2.