Pe conjugated antibody

For characterization of Tregs, PBMCs were incubated first with EMA and Gamunex™, followed by indirect staining for CD3 with a primary CD3 antibody (OKT3 supernatant) and a Pacific Orange-conjugated secondary antibody (Invitrogen). After blocking the non-specific binding of the secondary antibody with mouse serum (Merck Millipore, Darmstadt, Germany), cells were directly stained with CD4-Pacific Blue, CD45RA-Alexa Fluor-700, CD8-peridinin-chlorophyll protein (PerCP), CD279-PerCP-Cy5.5, CD127-Alexa Fluor-647 (BioLegend) and CD25-APC-Cy7 (BD Biosciences). The cells were then fixed and permeabilized using the human FoxP3 kit (BioLegend) and the cells were stained for intracellular FoxP3 using a PE-conjugated antibody (BioLegend) according to the manufacturer’s instructions.
For characterization of MDSCs, PBMCs were stained with a cocktail of lineage (Lin) markers (CD3, CD19, CD56)-Brilliant Violet 605 (BioLegend, BD Biosciences), CD14-Brilliant Violet 711 (BioLegend), CD45-V500, CD15-FITC, HLA-DR PerCP-Cy5.5, CD11b APC-Cy7, CD33 Alexa Fluor-700 (BD Biosciences), and CD124-APC (R&D Systems, Minneapolis, MN, USA). All samples were measured using a BD LSRII (BD Biosciences) immediately after staining.

Direct-infection latency model target cells were prepared as above. Immediately prior to the initiation of assays, cells were stained with pooled PE conjugated antibodies directed against CD69, CD25, and HLA-DR (all from Biolegend, stained at 5 μl antibody in 100 μl 1% FBS PBS buffer). Cells were then washed, labeled with anti-PE microbeads (Miltenyi Bioetc) following the manufacturer’s instructions, and depleted of labeled cells using an AutoMACS system.

CD4⁺ TSCM produced cytokines, IFN-γ, IL-2, IL-17 and TNFα were detected by intracellular staining the cells with PE-conjugated antibodies (Biolegend) following restimulation of cells with PMA, after 4 days in vitro culture of cells in the presence 10 µg/ml of HIVgp140 antigen (NIBSC, Potters Bar, UK).

Cells were harvested with Accutase and fixed in 4% paraformaldehyde. Cells were centrifuged at 1,500 ×g for 5 min and resuspended at 5 × 10⁶ cells/mL in PBS containing 0.5% bovine serum albumin (BSA). Aliquots containing 10⁵ cells were incubated with individual phycoerythrin- (PE-) conjugated antibodies or isotype control PE-conjugated IgGκ for 30 min at room temperature and then washed in PBS supplemented with 3% FBS. Samples were analyzed using a FACS Aria flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and the data were analyzed using CELLQUEST software (Becton Dickinson). The following monoclonal antibodies were used: PE-conjugated antibodies against CD73 (mouse IgG1κ; Biolegend, San Diego, CA, USA), CD90 (mouse IgG1κ; Biolegend), CD105 (mouse IgG1κ; Biolegend), and CD166 (mouse IgG1κ; Santa Cruz Biotechnology, Texas, USA). PE-conjugated isotype control mouse IgG1κ (Biolegend) was used as the control.

CAR-expressing cells were labeled with the anti-c-myc mAb (clone 9E10; Sigma-Aldrich) or the isotype control (mouse IgG1, Southern Biotech, Milan, Italy), followed by a secondary antibody (PE-conjugated goat anti-mouse IgG; Southern Biotech). Cell surface markers were labeled using APC-, FITC- or PE-conjugated antibodies to CD4, CD8, CD57, CD27, CD28, CD62L (BioLegend, San Diego, CA, USA), CCR7 (eBioscience, San Diego, CA, USA), and the relative isotype controls purchased from the same companies.