Cd8 clone rpa t8

Cytotoxicity assays were conducted as previously described (Kurioka et al., 2015 (link)). In brief, appropriately HLA-matched peptide-loaded B cell lines were used as target cells and labeled with CFSE according to the manufacturer’s protocol. Cells were pulsed with peptide at the indicated concentrations for 1 h followed by three washes with R10. Nonpeptide-pulsed cells were labeled with Cell Trace violet (CTV; Molecular Probes). Pulsed and unpulsed cells were mixed in a 1:1 ratio and incubated with peptide-specific short-term T cell lines for 4 h at an effector/target ratio of 10:1 in duplicates. CD107a (clone H4A3) was added at the start of the stimulation. After incubation, cells were stained with near IR viability marker, CD3 (clone UCHT1; eBioscience), CD8 (clone RPA-T8; eBioscience), and CD19 (clone LT19; Miltenyi Biotec). The mean percent survival of CFSE-labeled cells in wells containing no effectors was used to calculate the expected frequency of target cells in each well: expected ratio (ER) was calculated as %CFSE⁺/%CTV⁺. The specific killing was then calculated as: % Specific killing = 100 × [(ER × %CTV⁺ cells) − %CFSE⁺ cells]/(ER × %CTV⁺ cells).