Antigen-specific T-cell Clone Generation

Melan-A-specific and gp100-specific T-cell clones were generated by limiting dilution from pre-sensitized antigen (Ag)-specific T-cell lines, as described [23 (link)], and periodically re-stimulated in RPMI 1640 supplemented with 10% human serum (HS, noncommercial, prepared from healthy donors), 1 μg/ml PHA (Roche, 11,082,132,001), 25 U/ml recombinant IL-2 (rIL-2) (Miltenyi Biotec, 130–097-746), and 1 × 10⁶/ml irradiated allogenic PBMC as feeder cells. T-cell clones were used 13–15 d after stimulation. Ag-specificity of T-cell clones was assessed and periodically confirmed using anti-CD8-FITC and HLA-A*0201/PE-Melan-A (ELAGIGILTV) and HLA-A*0201/PE-gp100 (ITDQVPFSV) tetramer staining (see Table S2), performed for 30 min at room temperature (RT). Dead cells were excluded using propidium iodide staining (MP Biomedicals, 195,458). For intracellular staining of lytic molecules, Melan-A-specific and gp100-specific (n = 24) T-cell clones, isolated from 5 melanoma patients, were co-cultured with related (Melan-A⁺gp100⁺, Mel3) or unrelated (Melan-A⁻gp100⁻, Mel2) HLA/Ag melanoma cell lines, for 4–5 h at 37°C in the presence of protein transport inhibitor GolgiStop (BD, 554,724). T-cell clones were collected, and intracellular staining was performed using Intrasure kit (BD, 641,778) according to the manufacturer's instruction. Cells were then incubated for 30 min at RT with anti- PerCP-Cy5.5- tumor necrosis factor (TNF)-α, PE-Cy7-interferon (IFN)-γ and Alexa Fluor647-granzyme (GrzB) mAbs (see Table S2). Lytic activity was assessed in a standard 4-h ⁵¹Cr release assay. Mel2 and Mel3 melanoma cell lines were used as target cells and cytotoxicity was performed by incubating ⁵¹Cr-labeled target cells with effector cells (n = 26) at an effector: ratio (E:T) ratio of 20:1.