IL-27 suppresses HIV replication. (A) PHA-stimulated CD4⁺ T cells infected with X4 virus and PHA-stimulated PBMCs infected with R5 virus were cultured for 7 days in the absence or presence of 12.5% of VLP-Sup from PBMCs. To neutralize IFNs (IFN-α and IFN-β), MCP-2, IL-10, or CCR5 ligands, 10 μg/mL of each neutralizing antibody was added in the culture. To neutralize CCR5 ligands, an antibody mixture containing 10 μg/mL of each antibody to MIP-1α, MIP-1β, and RANTES was used. IL-27 in VLP-Sup from PBMCs was immunodepleted as described in “Materials and methods.” As a replication control, the infected cells were cultured in the absence of the VLP-Sup. On the 4th day after infection, 50% of culture supernatants were changed with fresh culture media alone. HIV-1 replication was measured by p24 antigen capture assay. The neutralization and the immunodepletion assay were performed at least 3 times, and results show the means ± SEs. (B) Relative expression of IL-27 mRNAs in PBMCs and MDMs were assessed by RT-PCR. PHA-stimulated PBMCs and MDMs were cultured in the absence or presence of 1 μg/mL VLPs for 36 and 24 hours, respectively. A total cellular RNA was isolated, and RT-PCR was performed as described in “Materials and methods.” As a control, RT-PCR was performed using RNA from SV40VLP-treated PHA-simulated PBMCs. (C-E) X4-infected CD4⁺ T cells (C) or PBMCs (D) were cultured in the presence of different concentrations of recombinant human IL-27. On the 4th day after infection, 50% of culture supernatants were changed with fresh culture media containing IL-27. HIV-1 replication was measured on 7th day by p24 antigen capture assay. MDMs were infected with R5 virus (E) and then cultured for 10 days in the presence of IL-27. Half of the culture supernatants were changed on the 4th and 7th day with fresh media with IL-27. HIV-1 replication was measured by p24 antigen capture assay. HIV-1 replication was measured by p24 antigen capture assay. Data show means ± SDs and are representative of 5 experiments.