Anti–PD-1 treatment restores antiviral T cell signaling and cytokine production, resulting in death. (A and B) Upstream (p-ZAP-70) and downstream (p-JNK; p-ERK) T cell signaling was quantified immediately ex vivo 30 min (A) or 16 h (B) after administration of anti–PD-1 or isotype control antibodies (n = 10 mice). Signaling was quantified simultaneously in P14 and SMARTA cells. Asterisks denote a statistically significant (P < 0.05) increase in signaling when compared with the corresponding isotype control group. (C and D) Serum concentrations of TNF (C) and IFN-γ (D) were measured by ELISA 24 h after administration of anti–PD-1 or isotype control antibodies (n = 5 mice). The asterisk denotes a statistically significant (P < 0.05) difference from the control group. (E) Q-PCR was used to quantify IFN-γ expression in flow cytometrically sorted CD8⁺ P14 and CD4⁺ SMARTA cells 16 h after injection of anti–PD-1 or isotype control antibodies. The asterisk denotes a statistically significant (P < 0.05) difference from the control group. (F) Survival was monitored in B6 (black), IFN-γ^−/− (green), and TNF^−/− (red) mice after injection of anti–PD-1 antibody on day 7 (n = 5 mice). B6 receiving isotype control antibody (blue) served as a control for this experiment. All mice receiving anti–PD-1 expired except for IFN-γ^−/− mice. The data presented in this figure are representative of 2–4 independent experiments.