IL-4 is essential for anti-PD-1–mediated enhancement of vaccine-specific CD8 ⁺ T cell responses. (A and B) C57BL/6 mice were injected into the footpad with 2 × 10⁶ p.f.u. of MVA-HIV-B. After 3 days, mice were treated i.v. with anti-PD-1 or a control isotype. The draining lymph nodes were analyzed on day 6. (A) Production of TNF-α and IFN-γ by CD8⁺ T cells after ex vivo stimulation by A3L, A8R, A19L, K3L, or B8R peptides, five known H-2^b–restricted MVA epitopes. Compiled from two independent experiments with seven to eight mice per group. Uns., unstimulated. (B) IL-4 production measured in the lysate of the lymph node draining the site of MVA injection. Compiled from two independent experiments with seven to eight mice per group. (C–E) C57BL/6 mice were injected into the footpad with 2 × 10⁶ p.f.u. of MVA-HIV-B. After 3 days, mice were either untreated or treated i.v. with anti-PD-1 Ab combined with two injections of anti-IL-4 mAb or with anti-PD-1 with two injections of an isotype control. Representative FACS contour plots (C) and quantification (D) of H2-K^b-B8R tetramers⁺ CD8⁺ T cells. Compiled from three independent experiments with 11–12 mice per group. (E) Quantification of TNFα⁺ and IFNγ⁺ double-producing CD8⁺ T cells after ex vivo stimulation by K3L or B8R peptides (n = 4–9 mice per group). Statistical analyses were performed using two-way ANOVA (A, D, and E) and t test (B). *, P < 0.05; **, P < 0.01; ***, P < 0.001.