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. 2016 Jan 20;6(1):150208. doi: 10.1098/rsob.150208

Figure 5.

Figure 5.

C-Myc regulates the expression of survivin to modulate the development of memory CD8+ T cells. Naive CD8+ TCRVβ5+ T cells from OT-I, OT-I/Cd28−/− or OT-I/Ox40−/− 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 survivin (Mig-survivin). 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, the transferred T cells from the spleens and LNs were analysed. Five mice were used for each time point. (a) C-Myc transduction. On day 5 of primary culture, GFP+ T cells were sorted and analysed for c-Myc, survivin, aurora B, bcl-xL 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+ T cells at day 35 post-infection of VV-OVA, gating on CD8+ cells. Data are representative of three independent experiments (p < 0.001 between Wt and Cd28−/− or Ox40−/− with Mig; p < 0.01 between Wt and Cd28−/− or Ox40−/− with Mig-survivin; two-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 < 0.001 between Wt and Cd28−/− or Ox40−/− with Mig; p < 0.05 between Wt and Cd28−/− or Ox40−/− with Mig-survivin; two-way ANOVA). (d) Numbers of CD8+ Thy1.2+ T cells at indicated day post-infection of VV-OVA. Data are represented as the mean ± s.e.m. (p < 0.01 between Mig and Mig-survivin in Ox40−/− on days 14, 21 and 28; two-way ANOVA). (e) Numbers of CD8+ Thy1.2+ T cells at day 35 post-infection of VV-OVA. Data are represented as the mean ± s.e.m. from three independent experiments (*p < 0.05; two-way ANOVA).