Clone mar 1

CTL apoptosis was analyzed incubating MAGEB2_256-264-specific CTL (1×10⁵ cells/well) for 24h at 37°C with mTECs (1×10⁵ cells/well) expressing either the TT or the CC genotype of AIRE gene in 100 μl final volume of culture medium. Non-adherent cells were, then, harvested, viable cells counted by 7AAD exclusion, and stained with FITC annexin V kit as well as CD8-specific APC-mAb (Biolegend). FACS analysis was then performed using the FACSCanto flow cytometer. A blocking hamster IgG anti-mouse Fas ligand mAb (clone MFL3, eBioscience, San Diego, CA) was used to study the dependency of mTEC-induced CTL apoptosis on Fas-Fas ligand interaction. To this aim, mTEC were co-cultured with CD8⁺MAGEB2_256-264 specific T cells for 24h in the absence or the presence of a hamster IgG anti-mouse Fas ligand mAb (clone MFL3, Ebioscience, New Jersey, USA), blocking Fas-mediated cell death (used at either 0.5 or 2.5 μg/ml concentrations). A unrelated, isotype-matched mAb (clone MAR-1, Ebioscience, New Jersey, USA) was used as control. The analysis of apoptosis on CD8⁺MAGEB2_256-264-specific T cells was performed as above described. The experiments were repeated 3 times in duplicate.