Measuring T-cell Responses by ICS

T-cell responses in PBMC were measured by flow cytometric intracellular cytokine staining (ICS) as previously described [36 (link)]. One million PBMC were incubated with overlapping 15-mer peptide pools spanning HIV-M (subtype B) Gag or Nef, and co-stimulated with CD28 and CD49d antibodies (BD Biosciences) for 1 hour, followed by incubation with Brefeldin A (Sigma-Aldrich) for 8 hours. Similarly, stimulation using human cytomegalovirus (HCMV) 15-mer peptide pools spanning IE1 and pp65 served as positive controls, while no antigen incubation served as background controls. Cells were surface stained with antibodies against CD3, CD4, CD8, CD95, CD28, and amine-reactive dye, and then fixed with 2% paraformaldehyde (PFA). BD FACS Permeabilizing Solution (BD Biosciences) was used to permeabilize the membranes before staining intracellularly for IFN-γ, TNF-α, and CD69. Samples were collected on a BDLSR-II instrument with FACsDIVA version 6.1, and analyzed with FlowJo v10 (Tree Star) by gating on singlet, live, CD3+, CD4+ or CD8+ cells. Positive responding CD4+ and CD8+ memory (CD95+) T-cells were measured by Boolean gating on CD69+/TFN-α+ and/or CD69+/IFN-γ+ cells. Presented as HIV positive responses were sums of individual HIV Gag and Nef responses, while positive HCMV responses were sums of individual HCMV IE1 and pp65 responses.