TRBV4-3⁺ and TRBV13⁺ TRAT-specific T-cell clones display differential pMHC avidity. (A) TRAT-specific T-cell clones were exposed to varying concentrations of synthetic TRAT peptide and then assessed for IFN-γ expression by the use of ICS assays. (B-C) Tetramer dissociation kinetics of TRAT-specific T-cell clones and TRAT-specific T cells in fresh PBMCs. T cells or PBMCs were initially incubated with PE-labeled anti-TRBV antibodies and then incubated with the APC-labeled HLA-Cw*0602-TRAT tetramer at 4°C for 30 minutes. These cells were washed, resuspended in FACS, and then incubated at room temperature for the indicated time points. T cells were then analyzed on a FACSCanto. Data in panel B are based on T-cell clones, whereas panel C shows data from fresh PBMCs.