PD-1 blockade restores HIV-specific CD4 T-cell effector functions. (A-C) Kinetic analysis of cytokine production by CD8-depleted PBMCs stimulated with HIV Gag peptide pool in the presence of PD-L1 blocking antibody or isotype control antibody. (A) mRNA levels of IL-2, IFN-γ, and IL-13 were measured by quantitative RT-PCR and normalized to the housekeeping gene GAPDH. (B) Representation of the transcription kinetics of IL-2 and IFN-γ mRNA. (C) Cytokine secretion as measured in the supernatants by Luminex bead arrays. (D) Representative example of the mRNA and the secreted levels of IL-2 and IFN-γ at 48 hours after stimulation. (E) Statistical analysis of data on a cohort of 16 untreated subjects indicated a significant increase in IL-2 (median fold increase = 2.26, P = .0006) and IFN-γ (median fold increase = 2.83, P = .0005) in the presence of PD-L1 blocking antibody. For IL-13 analysis, we used 12 untreated subjects (median fold increase = 1.74, P = .021; Wilcoxon matched-pairs test).