Quantifying Antigen-Specific CD8+ T Cell Cytotoxicity

The lytic activity of SIINFEKL-specific CD8⁺ T cells in PbA_Ama1OVA-infected animals was determined with an in vivo cytotoxicity assay on day 6 p.i. as described before (42 (link)). Briefly, splenocytes from syngenic donor mice were pulsed with 1 μM of the ovalbumin–derived specific H-2k^b peptide SIINFEKL (PEPscreen/Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37°C and subsequently with 1 μM of 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE, Sigma-Aldrich, St. Louis, MO, USA) for 15 min (CFSE^high, specific target cells). Reference cells were not pulsed with peptide and labeled with 0.1 μM CFSE for 15 min (CFSE^low, reference cells). Cells were washed with 1 X PBS (PAA, Cölbe, Germany) and the cell number was determined. The cell populations were mixed at a 1:1 ratio (CFSE^high/CFSE^low). Each recipient received 5x10⁶ cells of each population diluted in 0.9% NaCl (Braun, Melsungen, Germany) into the tail vein on day 5 p.i.. Mice were sacrificed 18 h later on day 6 p.i. and spleens were isolated to prepare single-cell suspensions as described above. Lysis of peptide-loaded cells was quantified by measuring the ratio of CFSE^high/CFSE^low cells via flow cytometry (Canto II, BD Biosciences). The percentage of specific lysis, termed S8L-specific lysis, was calculated using the following equation: 100 - [(CFSE^high/CFSE^low) _immunized/(CFSE^high/CFSE^low) _naïve] x 100.