Gilgfvftl

PBMCs derived from buffy coat of healthy donors were co-cultured with K562 cells, pulsed with HLAI-A2 restricted Flu peptide (GILGFVFTL) (10 μg/mL) (Proimmune, Oxford, UK) for 12 days in RPMI+10% FCS in presence of IL-2 (10 U/ml) and IL-15 (10ng/ml). Flu-enriched CD8⁺ T cells were isolated employing the human microbeads CD8 (Miltenyi Biotech) and co-cultured with autologous Tregs (+/- rhMGL-Fc) and/or mDCs pulsed with Flu peptide in the anti-IFN-γ precoated (1:200; Pharmingen San Diego, CA, USA) ELISpot plates (Millipore, Bedford, MA, USA) overnight at 4°C. Cytokine release was detected with biotinylated anti-IFN-γ antibody (Pharmingen; 1:250, 2 hrs at RT) revealed with streptavidin-alkaline phosphatase (Pharmingen) (1:1000, 100 mL/well, 1 h at RT) and chromogen substrate (5-bromo-4- chloro-3-indolylphosphate (BCIP)/nitroblue tetrazolium alkaline phosphatase substrate, Sigma) (50 μL/well). Spots were counted using the ImmunoSpot Image Analyzer (Aelvis).

Human peripheral blood from HLA-A2–specific donors (Volpin et al., 2020 (link)) was separated by Ficoll gradient centrifugation and pre-enriched with anti-human CD8 beads and expanded in the presence of the influenza peptide (GILGFVFTL; ProImmune) for 14 d. The CD8⁻ fraction was irradiated and used as feeder cells for 1 wk until being replaced by irradiated T2 cells. After 1 and 8 d, 100 U/ml IL-2 and 5 ng/ml IL-15 were added. Cells were sorted for influenza specificity by pentamer staining (GILGFVFTL-APC, #P007-0A-E; ProImmune) on day 14 before expanding the cells in medium containing 3,000 U/ml IL-2 and 30 ng/ml anti-CD3 for 14 d. Influenza-specific T cells were frozen until use in subsequent assays.