The remaining blood was used to isolate PBMCs using previously described methods [25] (link). PBMCs were thawed, washed and stained with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen), and then stained with the following fluorescently-conjugated monoclonal antibodies: CD8-QDOT 605 and CD4-PE-Texas Red (Invitrogen, Grand Island, NY, USA); CD3-Pacific Blue, CCR5-PE-Cy5, CD38-PE, HLA-DR-FITC, PD-1 Alexa Fluor 647, CD45RA-PE-Cy7 (BD Biosciences, San Jose, CA, USA); and CCR7-APCeFluor 780 (eBioscience, San Diego, CA, USA). Naïve and memory T cell subsets were defined by quadrant gating with FMO controls on a CD45RA versus CCR7 plot.
For cytokine staining, PBMCs were stimulated for 18–22 h at 37°C with overlapping peptide pools corresponding to HIV-1 Con B Gag peptides (NIH 8117) in the presence of 0.5 µg/mL Brefeldin A and 05 µg/mL Monensin (Sigma-Aldrich, St. Louis, MO, USA). A control well with no stimulation was run in parallel for each sample. Cells were washed and stained with AARD, fixed, and permeabilized for intracellular staining with antibodies against CD3-Pacific Blue, IFNγ -FITC, IL-2-PE (BD BioSciences), CD4-PE Texas Red, and CD8-QDOT 605 (Invitrogen). Data were compensated and analyzed using FlowJo V9 (TreeStar, Ashland, OR, USA).
For cytokine staining, PBMCs were stimulated for 18–22 h at 37°C with overlapping peptide pools corresponding to HIV-1 Con B Gag peptides (NIH 8117) in the presence of 0.5 µg/mL Brefeldin A and 05 µg/mL Monensin (Sigma-Aldrich, St. Louis, MO, USA). A control well with no stimulation was run in parallel for each sample. Cells were washed and stained with AARD, fixed, and permeabilized for intracellular staining with antibodies against CD3-Pacific Blue, IFN