T-cell transduction, cytokine staining and bioluminescence (BLI)-based killing assay have been described in details elsewhere [9 (link)]. Briefly, primary T-cell transduction with retroviral particles was performed by double spinoculation followed by expansion on CD3/CD28 beads. The staining of the CAR was performed with Biotin-SP-AffiniPure F (ab′)2 Fragment Goat Anti-Mouse IgG (Jackson ImmuResearch, USA), followed by a secondary staining with Streptavidin`-PE (Biolegend, USA). CAR-expressing T cells were co-cultured with cells positive or negative for the target antigen, and TNF-α was detected by flow cytometry using TNF-α-PE (MAb11, BD Biosciences). The samples were run on a BD FACSCanto flow cytometer (BD Biosciences, USA), and data were analyzed using FlowJo software (Treestar Inc., USA). BLI killing assay was also based on a co-culture experiment where target cells permanently expressed firefly Luciferase gene, and killing was correlated to the luminescence signal [10 (link)].