Anti cd56 clone b159

Expression of CD107a and production of TNFα and IFNγ were measured as described previously (17 (link)). Briefly, PBMCs were either left unstimulated or were stimulated overnight. The next day, cells were washed and then co-incubated with K562 target cells at an effector-to-target (E:T) ratio of 2:1 for 5 hours. Anti-CD107a (H4A3) was added at the start of culture. Brefeldin A and monensin (both BD Biosciences) were added after 1 hour. The following antibodies were used: anti-CD56 (clone B159), anti-CD3 (SK7), anti-IFNγ (B27), anti-TNFα (MAb11) and fixable viability stain FV575 (all BD Biosciences). Cells were analysed on an BD LSR Fortessa X-20 and using FlowJo Version 10 software (BD Biosciences).

The following mouse anti-human monoclonal antibodies (MAb) were used: anti-PD1/clone EH12.2H7 (BioLegend Inc., San Diego, CA, USA), anti-2B4/clone C1.7 (Beckman Coulter, Fullerton, CA, USA) as wells as anti-Tim3/clone 344823, and anti-human IFNγ/clone 25723 (R&D Systems, Minneapolis, MN, USA). Anti-CD14/clone M5E2, anti-CD19/clone 5J25C1, anti-CD56/clone B159, anti-CD107b/clone H4B4, anti-TNF/clone MAb11, anti-CD8/clone SK1, and mouse IgG isotype controls were all obtained from BD Pharmingen (Becton Dickinson, Heidelberg, Germany). Tetramer and antibody stainings were performed as detailed previously (19 (link)). Cells were acquired using a BD FACSCanto II flow cytometer (Becton Dickinson, Heidelberg, Germany) and analyzed with FlowJo Software (TreeStar Inc., San Diego, CA, USA). A multimer-specific CD8⁺ T cell population was considered detectable if the frequency was at least 0.1% of total CD8⁺ T cells. Exclusion of unspecific events was done using a dump channel (CD14⁺CD19⁺CD56⁺). Respective FMO controls were included to enable gating of cell populations.