2.6. Assessment of tumour-antigen-specific T cell responses

PBMCs from HLA-A*02 patients or healthy donors were thawed at 37°C and rested overnight in complete-RPMI (cRPMI: RPMI-1640 + 10% FBS + 1% penicillin/streptomycin; Gibco, USA) in a humidified incubator at 37°C, 5% CO₂. 1-2 x 10⁶ PBMCs were cultured in cRPMI with tumour antigen epitopes presented by the common HLA-A*02:01 molecule: survivin (Sur1 M2, LMLGEFLKL), telomerase (hTERT-540, ILAKFLHWL), MAGE-A3 (MAGE-A3_271-279, FLWGPRALV), NY-ESO-1 (NY-ESO-1_157-165, SLLMWITQC) (all 10 µg/mL), and CytoStim™ (Miltenyi Biotec, Germany) or a CEF pool (2 µg/mL/peptide) and a mock condition (CRPMI + 0.5% DMSO) as positive and negative controls. PBMCs were cultured for 8 hours with 1µg/mL GolgiPlug™ (BD Biosciences, USA) and CD107a (AF488, 1:400, BD biosciences, USA), then kept at 4°C overnight prior to antibody staining and flow cytometry assessment. Cells were stained with LIVE/DEAD Aqua™ (1:1000, ThermoFisher, USA) for 20 minutes at room temperature (RT). Cell membrane staining was performed using CD3 (BUV395, 1:200, BD Biosciences), CD4 (BUV737, 1:150, BD Biosciences), and CD8 (BV785, 1:200, BD Biosciences) for 30 minutes at RT. Cells were fixed and permeabilised using CytoFix/CytoPerm^® (BD Biosciences) according to manufacturer’s instructions. Intracellular staining was then performed for IFNγ (BV421, 1:100, BD Biosciences), TNFα (PE, 1:50, BD Biosciences), and IL-2 (BV650, 1:40, BD Biosciences) for 30 minutes at 4°C. Data acquisition was performed on a LSRFortessa X20 cytometer (BD Biosciences). Downstream analyses were performed using FlowJo™ software (version ≥ 10.2, Tree Star Inc., USA). Due to limitations in available PBMC numbers, only one biological replicate was able to be performed.