Effect of IFN type I antibodies on TRAIL production by monocytes from HIV-1–uninfected donors. (A) Monocytes were cultured 3 days in presence of AT-2 HIV-1_MN alone (•) or with blocking antibodies against IFN-α (2000 neutralizing U/mL; ▦), IFN-β (500 neutralizing U/mL; ▨), or both (□). The level of sTRAIL was quantified by ELISA. (B) Monocytes were cultured for 1 day in presence of AT-2 HIV-1_MN and with or without blocking antibodies against IFN-α and IFN-β. The level of TRAIL mRNA was quantified by real-time PCR analysis. (C) Monocytes were cultured for 3 days in presence of recombinant IFN-α or IFN-β (10 ng/mL). The level of sTRAIL was quantified by ELISA. (D) IFN-α (□) and IFN-β production (•) by monocytes cultured for 3 days in presence of microvesicles (control), AT-2 HIV-1_MN, or AT-2 HIV-1_Ada. Mean values with standard errors are shown for 4 independent experiments in panels A-C for each condition tested. (E) STAT1 and STAT2 expression. Monocytes were cultured 24 hours in presence of AT-2 HIV-1_MN, AT-2 HIV-1_Ada, recombinant IFN-α/β (10 ng/mL), or HIV-1_MN plus anti–IFN-α/β antibodies. Production of STAT1 and STAT2 was analyzed by Western blot; β-actin was used as a loading control. (F) Blocking assay. Monocytes were cultured for 3 days with AT-2 HIV-1_MN or HIV-1_Ada and in presence of isotype control antibody or the CD4 binding inhibitor soluble CD4 (2 μg/mL). IFN-α (□) and TRAIL levels (•) were quantified by ELISA. (G) Monocytes were cultured for 3 days with AT-2 HIV-1_MN and in presence of isotype control antibody; the CD4 binding inhibitor soluble CD4 (2 μg/mL); the CXCR4 inhibitor AMD-3100 (2 μg/mL), or the fusion inhibitor T20 (2 μg/mL). Monocytes cultured without AT-2 HIV-1_MN were used as control. TRAIL level was quantified by ELISA. Mean values with standard errors are shown for 3 independent experiments for each condition tested (panels F-G).