Dominant-negative hTERT-transduced T cells disappear early from bulk cultures. (A) Human polyclonal CD4⁺ T cells transduced with either DN-hTERT (Asp868Ala, Asp869Ala)–GFP (•), hTERT-GFP (○), or control GFP (▵) were mixed with untransduced cells in a 50%:50% ratio and cultured in parallel for 53 days to determine the lifespan compared with the untransduced T cells. The graph shows the percentage of GFP-positive cells during the culture. Results are representative of 3 experiments. (B) CD8⁺ T-cell clone transduced and cultured as described in panel A. Results are representative of 2 experiments. (C) CD4⁺ T-cell clone MoT-72 was transduced with either DN-hTERT (Asp712Ala, Val713Ile)–GFP (•), hTERT-GFP (○), or control GFP (▵), and the percentage of GFP-positive cells was measured weekly during the culture. The graph shows the percentage GFP-positive cells during the culture. Transduction efficiencies at the onset of the cultures were as follows: hTERT-GFP, 20%; GFP control, 35%; and DN-hTERT–GFP, 15%. Results are representative of experiments with 6 different CD4⁺ T-cell clones. Panels A-C show that in both CD4⁺ and CD8⁺ T-cell cultures, ectopic expression of DN-hTERT decreased the lifespan compared with untransduced T cells. (D) Cumulative number of population doublings of the untransduced GFP-negative cells in the mixed culture containing DN-hTERT (•) or GFP-transduced T cells (▵). The graph shows that the untransduced T cells were growing equally well in both mixed cultures, indicating that the decrease in percentage DN-hTERT–GFP–expressing T cells in DN-hTERT-GFP–transduced cultures was not due to an enhanced proliferation of the untransduced T cells compared with untransduced T cells in the control GFP-transduced cultures.