Cd4 precp

To test the effect of BAGN on HIV in vitro infection, PHA-blasts from HIV sero-negative donors were generated. One-half of the target cells were pretreated by adding 2 mM BAGN (Sigma) to the PHA-blast at day 2 after PHA stimulation, i.e., BAGN was added overnight from day 2 to 3. At day 3, both BAGN pretreated and non-pretreated PHA-blast were pelleted by centrifugation at 400 g for 5 min and re-suspended in ~200 μl residual volume. Cells were then infected with NL4-3 at a multiplicity of infection (MOI) of 0.01 and incubated at 37°C in a 5% CO₂ atmosphere for 4 h. The cells were then washed twice with 10 ml of R20 and 0.5 × 10⁶ cells were plated at a density of 1 M PHA-blast/ml in 48 well plates. Cultures were maintained at 37°C and 5% CO₂ for 5 days in R20/50 containing different concentrations of BAGN (range: 0, 0.02, 0.2, and 2 mM) and intracellular p24 production was measured by flow cytometry. Briefly, Live/Dead violet fluorescence fixable dead cell stain reactive kit (Invitrogen) was added to cultured cells, followed by extracellular staining with antibodies: CD3-PE-Cy7, CD4-PreCp, and CD8-V500 (BD Biosciences). The cells were fixed and permeabilized (Fix and Perm, Invitrogen) for intracellular staining with the p24 reactive KC57-PE antibody (Coulter). Cells were run on an LSR II instrument (Becton Dickinson) and analysis performed using FlowJo software.

PHA-blast target cells from 10 HIV-uninfected donors, either BAGN-treated or -untreated, and stained for surface activation markers and HIV co-receptors expression, were further used for the VRAs. At day 3, both BAGN-treated and -untreated target cells were infected (MOI = 0.001) with the viral stocks produced with or without BAGN in the culture medium. Cells were cultured for 7 days in R20/50, with or without 0.2 mM BAGN, at 37°C in a 5% CO₂ containing atmosphere. Secretion of p24 into the culture supernatant was measured by ELISA (Fujirebio) and intracellular p24 production was measured by flow cytometry. Briefly, cells were stained with Live/Dead violet fluorescence fixable dead cell stain reactive kit (Invitrogen) followed by extracellular staining with antibodies: CD3-APC-H7, CD4-PreCp, and CD8-APC (BD Biosciences). The cells were fixed and permeabilized (Fix and Perm, Invitrogen) while staining for HIV Gag p24 using the KC57-FITC antibody (Coulter). As before, cells were collected on an LSR II instrument (Becton Dickinson) and analysis performed using the FlowJo software.