Wt1126 134 peptide

2D3 cells were used to analyze the avidity for the cognate peptide and functionality of the TCR after cloning. Briefly, T2 cells were pulsed with WT1_126−134 peptide (JPT Peptide Technologies) at decreasing concentrations of a 10-fold serial dilution for 90 min at room temperature under constant motion. Electroporated 2D3 cells were cultured with peptide-pulsed T2 cells at an effector-target ratio of 2:1 for 5 h. After incubation, cultures were stained with anti-human CD3-PerCP (clone SK7) and CD8-PE mAbs (clone SK1; BD Biosciences) for 30 min at 4°C, washed and resuspended in FACS buffer. Recognition of peptide-pulsed T2 cells was analyzed by TCR activation-mediated EGFP expression using a FACScan flow cytometer (BD Biosciences).

The killing capacity of electroporated human primary resting CD8⁺ T cells against T2 cells was determined using a flow cytometry-based protocol as described previously with minor modifications (51 (link)). Briefly, prior to co-culture tumor cells were stained with PKH67 green fluorescent cell linker dye (Sigma-Aldrich) according to the manufacturer's protocol. PKH67⁺ T2 cells were incubated with WT1₃₇₋₄₅ or WT1_126−134 peptide (JPT Peptide Technologies) in AIM-V medium (Gibco Invitrogen) for 90 min at room temperature under constant motion. Next, T2 cells were cultured alone or with electroporated human primary resting CD8⁺ T cells for 6 h at an effector-target ratio of 20:1. After co-culture, samples were stained with propidium iodide (PI) and APC-labeled annexin V (BD Biosciences). Samples were analyzed using a FACSAria II flow cytometer (BD Biosciences). Cytotoxicity was calculated based on the survival of PKH67⁺ T2 cells using the following equation: