NTAmer Staining and Dissociation Kinetic Measurements

NTAmers were synthetized by the Peptide and Tetramer Core Facility, Ludwig Cancer Research, UNIL CHUV (Lausanne, Switzerland) as described previously (36). Dually labeled NTAmers are composed of streptavidin-phycoerythrin (SA-PE) complexed with biotinylated peptides and non-covalently bound to His-tagged HLA-A^*0201 monomers containing Cy5-labeled β2m (36) and were used for dissociation kinetic measurements as described previously (34, 37). Briefly, individual vaccine-induced Melan-A-specific CD8 T-cell clones were stained for 45 min at 4°C in PBS, 0.2% BSA, and 5 mM EDTA with Melan-A-specific NTAmers, in which HLA-A^*0201 molecules were either loaded with the native (EAA) or the analog (ELA) Melan-A₂₆₋₃₅ peptide. When indicated, we also used CD8 binding-deficient HLA-A^*0201 monomers bearing the D227K/T228A mutations in the HLA-α3 domain (38) and loaded with the analog ELA peptide. NTAmer staining was assessed at 4°C on a SORP-LSR II flow cytometer (BD Biosciences). Following 25 s of baseline acquisition, 100 mM of imidazole was added allowing the rapid dissociation of the SA-PE-NTA₄ scaffold and monomeric Cy5 fluorescence was measured during the following 5 min. Data were analyzed using the kinetic module of FlowJo software (v.9.7.6, Tree Star) and modeled (1-phase exponential decay) using Prism software (v.6, GraphPad).