Figure 7.

Dominant-negative c-Myc inhibits the generation of memory CD8⁺ T cells. Naive CD8⁺ TCRVβ5⁺ T cells from OT-I TCR Tg mice were stimulated with peptide and APCs. On day 2/3, T cells were transduced with retroviral vectors expressing GFP alone (Mig) or GFP with dominant-negative (dn) c-Myc (Mig-dnMyc). On day 5 of primary culture, 5 × 10⁴ GFP⁺ CD8 cells were sorted and adoptively transferred into Thy1.1 congenic mice that were infected i.p. with VV-OVA on the following day. After various days, memory progenitors or memory T cells from the spleen and LNs were analysed. Five mice were used for each time point. (a) DnMyc transduction. On day 5 of primary culture, GFP⁺ T cells were sorted and analysed for c-Myc and β-actin by immunoblot. Similar data were obtained in three experiments. In Western blots, β-actin was used as internal control. For Western blots belonging to the same experiment, bands pertaining to different proteins were cropped either from the same blot or multiple gels were run under similar experimental conditions. (b) The frequencies of Thy1.2⁺ at day 35 post-infection of VV-OVA, gating on CD8⁺ cells. Data are representative of three independent experiments (p < 0.001 between Mig and Mig-dnMyc; one-way ANOVA). (c) Functional analysis using IFN-γ. At day 35 post-infection of VV-OVA, splenocytes were stimulated with OVA peptide for intracellular IFN-γ staining, gating on Thy1.2⁺ cells. Data are representative of three independent experiments (p < 0.01 between Mig and Mig-dnMyc; one-way ANOVA). (d) The absolute numbers of CD8⁺ Thy1.2⁺ at indicated day post-infection of VV-OVA. Data are represented as the mean ± s.e.m. (p < 0.001 between Mig and Mig-dnMyc on days 7, 14, 21, 28 and 35; two-way ANOVA).