AIM assay

PBMCs were thawed and washed, and CD8⁺ T cells were depleted using human CD8 MicroBeads (Miltenyi Biotec, 130-045-201). CD8⁺ T cell–depleted PBMCs were cultured in 24-well plates at a concentration of 10 × 10⁶ cells/ml in RPMI 1640 supplemented with Hepes, penicillin/streptomycin, and 10% human serum (Sigma-Aldrich). Cells were rested for 3 h and then stimulated with 0.5 µg/ml of HIV-1 consensus B Gag pool, CMV peptide pool, and CEFT peptide pool (PM-HIV-CONB, PM-PP65-2, PM-CEFT-MHC-II, all from JPT Peptide Technologies) for 18 h at 37°C, 5% CO₂. A MOG peptide pool (0.5 µg/ml, PM-MOG, JPT Peptide Technologies) served as negative control. SEB-stimulated cells (0.5 µg/ml, Toxin Technology, BT202) served as positive control. Cells were stained for viability with Aquavivid (Life Technologies) at 1/200 in PBS for 20 min, 4°C; incubated with human FcR Block (Miltenyi Biotec, 130-059-901) at 1/70 in PBS-FBS 1% for 10 min, 4°C; and stained with antibodies to surface markers for 30 min, 4°C with Brilliant Violet Buffer (BD Biosciences, 566349) at 1/4 in PBS-FBS 1% (see Table S4 for antibody staining panel).