Blocking IL-27 signaling reverses inhibition of HIV infection. (A) Inhibition of IL-27 signal transduction by blocking JAK activation interrupts STAT1 and STAT3 phosphorylation. Macrophages were treated with JAK inhibitor at 1 or 10 nM for 1 hour before addition of IL-27, and after 30 minutes, processed for Western blots using antibodies to STAT1, STAT3, p-STAT1, and p-STAT3 (representative data, n = 3). (B) Inhibition of JAK activation blocks IL-27 downstream transcription of IFN-α. Macrophages pretreated for 1 hour with JAK inhibitor (0-10 nM) before addition of IL-27 exhibited reduced induction of IFN-α after 30 minutes as determined by RT-PCR (mean ± SEM, *P = .01; n = 3). (C) Monocyte-derived macrophages were treated or not with IFN receptor antibody (CD118 at 10 μg/mL) 20 to 30 minutes before IL-27 (100 ng/mL) and APOBEC3A determined by RT-PCR after 24 hours (mean ± SEM, *P = .01; representative triplicate data, n = 8). (D) Monocyte-derived macrophages (n = 4) were infected with HIV, washed, and then treated or not with IFN receptor antibody (CD118 at 10 μg/mL), which was added 20 to 30 minutes before IL-27 (100 ng/mL). ELISA for p24 was used to monitor HIV replication in the treated and untreated cultures (mean ± SEM, *P = .01, **P = .001).