Isotype control mouse igg1κ

PBMCs were cultured in 96-well U-bottom plates in R10 medium [RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), HEPES] alone or with anti-CD3/CD28 beads (Life Technologies, 1 bead:2 cells), R848 (1 μg/ml), or LPS (1 μg/ml, Invivogen) (46 (link)). All cultures included anti-retroviral agents (5 μM tenofovir, 50 nM raltegravir; Selleck Chemicals) to prevent viral replication from HIV donors and to account for the immunomodulatory effects of anti-retroviral agents on the stimulation of PBMCs from healthy donors. Supernatants were collected and HIV-1 virus was undetectable by p24 ELISA (Perkin Elmer). For IFN-γR blockade experiments, PBMCs were stimulated with R848 or LPS as described earlier in the presence of either anti-human CD119/IFN-γR α-chain (clone GIR-208) or isotype control mouse IgG1 κ (Biolegend). Both antibodies were used at a final concentration of 50 μg/ml and culture medium was supplemented every day to maintain this concentration of antibody. Cells from each HIV+ donor were run as an independent experiment (batch), whereas CD4+ T cell–depleted PBMCs were analyzed in parallel with whole PBMCs from the same donor (paired batch).