(A) Proteasome activity, assessed by in-gel assay, of naive CD8⁺ T cells following 4 hours of culture with vehicle, proteasome inhibitor, or proteasome activator. Immunoblotting was performed with anti–β-actin antibodies to confirm equal loading of samples. (B) Proteasome activity, assessed by flow cytometry, of cells cultured with vehicle (black line), proteasome inhibitor (red line), or proteasome activator (blue line). (C) Proteasome activity, assessed by bioluminescent chymotrypsin-like proteolytic assay, of cells cultured with proteasome inhibitor (red bar) or proteasome activator (blue bar) and normalized to vehicle control (black bar). (D) Flow cytometry analysis (left) and MFI (right) of T-bet and IRF4 at 72 hours after activation in CD8⁺ T cells transiently treated for 4 hours with vehicle, proteasome inhibitor, or proteasome activator prior to activation with anti-CD3 and anti-CD28 antibodies. (E) Percent specific cytotoxicity of OT-I CD8⁺ T cells treated transiently with vehicle (black line), proteasome inhibitor (red line), or proteasome activator (blue line) prior to culture with T cell–depleted splenocytes and OVA peptide. CD8⁺ T cells were incubated with an equal ratio of peptide-pulsed and unpulsed splenocytes in varying effector cell to target cell ratios. Cytotoxicity was calculated as the difference in the percentage of live events between pulsed and unpulsed target cells, normalized to the live percentage of unpulsed target cells. Data are representative of at least 2 independent experiments (A–C) or least 3 biologic replicates from 3 independent experiments (D and E). Error bars represent SEM of 3 replicates. *P < 0.05, **P < 0.01, ***P < 0.001 (C and D, 1-way ANOVA with Dunnett’s post-test; E, repeated measures 1-way ANOVA).