Figure 2.

Retroviral transduction of Bcl-xL and Survivin promotes passive proliferation and survival of CD8⁺ T cells in vitro. Naive CD8⁺ T cells from OT-I TCR transgenic mice were stimulated with peptide/APCs, and transduced on days 2/3 with retroviral vectors expressing GFP, GFP with Bcl-xL, GFP with Survivin, or GFP with Bcl-xL and Survivin, and then recultured without any further stimulation. (a) Primary passive proliferation on day 4 and day 6 were measured in unseparated cultures by pulsing with tritiated thymidine for 20 hr. Data are representative with mean of three independent experiments (* P<0.05, Student’s unpaired t-test). (b) GFP⁺Vβ5⁺ T cell recovery normalized to take into account differences in initial transduction efficiency between cultures. Numbers of GFP⁺ cells present on day 4 were assigned a value of 100%, and numbers surviving on day 6 and day 8 were used to calculate the percentage recovery relative to day 4. Data represent the mean ± S.D. percentage change from three separate experiments (* P<0.05, ** P<0.01, Student’s unpaired t-test). GFP⁺ CD8⁺ T cells on day 6 were sorted, and protein expression of Survivin and β-actin was also determined by western blotting, Data are representative of three independent experiments. (c) CD25 expression on day 4 was analyzed by flow cytometry, after gating on live CD8⁺ GFP⁺ T cells. Data are representative of three independent experiments. (d) Apoptosis of GFP⁺ CD8⁺ T cells on day 6 based on staining of Annexin V and 7-AAD and analyzed by flow cytometry. Data are representative of three independent experiments.