Intracellular Cytokine Staining of PBMC

PBMC were plated in a 96-well plate (10⁶ cells per well). PBMC stimulation was performed in 10% FBS/RPMI media in the presence of 1 μg/ml anti-CD28 and anti-CD49d and Brefeldin A (BD Biosciences, San Diego, CA) and stimulated with HIV peptides (New England Peptide, Gardner, MA) of 15-mer overlapping by 11 amino acids representing HIV subtype E-Env (TH023; 162 peptides) and HIV subtype B-Gag (LAI; 120 peptides). PBMC supplemented with DMSO was used as a negative control. After 6 h of stimulation at 37 °C, 5% CO₂ EDTA (20 mM, Sigma) was added and incubated for 15 min. Subsequently, PBMC were fixed and permeabilized using FACS lysing solution and FACS permeabilizing solution 2 (BD) according to the manufacturer’s instructions. The following antibodies were added for 60 min at room temperature in the dark: CD4-fluorescein isothiocyanate (FITC), CD3-allophycocyanin (APC), IFNγ-phycoerythrin (PE), Il-2-phycoerythrin (PE), and CD8-PerCP-Cy5.5 (all BD Biosciences). PBMC were washed and fixed with 1% paraformaldehyde. The analysis was performed using a FACSCalibur flow cytometer (BD Immunocytometry Systems). ICS data were provided to us by the trial investigators in either an ICS-positivity score (call) format and aggregate value format^{2 (link)}. ICS analytes included CD154, IFNγ, IL-4, IL-2, IL-17α, and TNFα.