Flow cytometric analysis of HIV-1–specific immune responses by intracellular cytokine staining

After thawing, PBMCs were incubated at 37°C overnight at a concentration of 2 × 10⁶ cells/mL in RPMI medium containing 10% FBS and penicillin/streptomycin. The next day, 1 × 10⁶ cells were stimulated with Gag pooled peptides (final concentration 1 μg/mL in DMSO provided by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 Con B Gag Peptide Pool cat #12425), for 6 hours in the presence of brefeldin A and monensin (BD). The percentage of CD8+ or CD4+ T cells producing interferon gamma (PE-dazzle, clone 4S.B3, Biolegend), tumor necrosis factor alpha (Alexa fluor 700, clone mAb11, eBioscience) or interleukin-2 (BV785, clone MQ1-17H12, Biolegend), or degranulation as detected by CD107a expression (APC, clone H4A3, Biolegend), was measured by flow cytometry, and the frequency of positive cells was determined after subtraction of the frequency measured in wells incubated with DMSO alone. Examples of gating strategies are shown in S2 Fig.