T Cell Receptor Characterization via Tetramer Staining

STCL were generated as above using a single “parental” 15-mer peptide. To examine optimal peptide length and their HLA restriction, 721.221 or C1R cells transfected with a single HLA class I allele were pulsed with short peptides and used as antigen-presenting cells for stimulation of STCL effectors in an ICS assay, which also confirmed that the responding T cells were CD8⁺ [62 (link)]. For tetramer reactivity, 10⁶ STCL cells were stained with pre-titrated amounts of PE-conjugated tetramer (NIH Tetramer Facility, Emory University Vaccine Center, Atlanta, GA, USA) in FACS tubes at room temperature for 10 min. For both HLA and tetramer procedures, a mix of anti-CD8 FITC, anti-CD4 PE, anti-CD3 PE-CF594, anti-IFN-γ V450 (BD Biosciences, Wokingham, UK), and anti-CD3 ECD (Beckman-Coulter, High Wycombe, UK) monoclonal antibodies (mAbs), and LIVE/DEAD fixable cell stain Aqua were added and the tubes incubated for a further 20 min at room temperature. The cells were washed with FACS buffer, fixed with 1% paraformaldehyde prior to analysis, acquired by a Fortessa flow cytometer (Becton-Dickinson, Franklin lakes, NJ, USA) and analyzed using the FlowJo software (Tree Star).