hTERT-transduced CD4 or CD8⁺T cells grow equally to untransduced cells. (A) Cultures of untransduced or hTERT–puro-transduced cells of CD4⁺ T-cell clone MoT-72 or CD8⁺ T-cell clone AKR103 were labeled with CFSE and stimulated with feeder cells and PHA. The fraction of dividing cells and the number of cell divisions in each culture was calculated from the mean decrease in CFSE fluorescence intensity at day 8 (MoT-72) or at day 7 (AKR103). MoT-72: 64% proliferating cells with a mean decrease in CFSE fluorescence from 586 (unstimulated cells) to 50, which corresponds to an average of 3.5 cell divisions. MoT-72-hTERT: 63% proliferating cells with a mean decrease in CFSE from 569 to 77, corresponding to 2.9 cell divisions. AKR103: 98% proliferating cells with a mean decrease in CFSE from 610 to 23, which represents 4.7 cell divisions. AKR103-hTERT: 91% proliferating cells with a mean decrease in CFSE from 459 to 21 (= 4.5 cell divisions). The proliferation of the T cells upon stimulation was identical between hTERT-transduced and untransduced cells at 3 different time points measured at biweekly intervals during the culture. The graphs are representative of the 3 independent experiments. (B) Activation-induced cell death. MoT-72 cells and hTERT–puro-transduced MoT-72 cells were stimulated with anti-CD4– and anti-CD28–coated beads and IL-2 (bottom panels) or cultured in IL-2 alone (top panels). The presence of apoptotic cells detected by annexin V binding was analyzed by flow cytometry on day 2. Numbers in the graphs indicate the percentage of annexin V binding cells (7-AAD–positive cells were excluded).