CTL Response Measurement using IFN-γ ELISPOT

IFN-γ enzyme-linked immunospot (ELISPOT) kit and AEC substrate set (BD Pharmingen, San Diego, CA) were used to measure CTL responses in the clinical study, as reported previously [14 (link)]. Peripheral blood mononuclear cells (PBMCs) were obtained from patients and were frozen before vaccination and at the end of each course. Frozen PBMCs were thawed and used for in vitro sensitization. Briefly, PBMCs were cultured in 1 mL of complete media (prepared with a mix of AIM-V and RPMI, 50 % of each) containing 10 % fetal bovine serum in a 48-well plate with 10 μg/mL of HIG2-9-4 peptide and 20 IU/mL of IL-2 at 37 °C with 5 % CO₂, for 2 weeks. On day 7, half of the medium was removed from each well and 500 μL of fresh medium containing epitope peptide, as described above, was added to sensitize. After a 2-week incubation, CD4-positive cells were removed by using a Dynal CD4 positive isolation kit (Invitrogen, Carlsbad, CA, USA), and harvested cells were co-cultured with peptide-pulsed T2 cells (1 × 10⁵ cells per well) at 37 °C for 20 h. HLA-A*0201-restricted HIV-derived epitope peptide (ILKEPVHGV) was used as the control peptide. ELISPOT assay was performed in triplicate. HIG2-9-4-specific CTL response was defined according to an evaluation tree algorithm [15 (link)].