Phenotyping T Cell Responses

PBMCs for TCR profiling were cultured in TCRPMI as described above and reported previously (12 (link)). For TCR overexpression experiments we used AIM V media. PBMCs were washed with PBS two times and once with media, subsequently resuspended at 5 × 10⁵ cells/100 µL and aliquoted in a 96-well plate for a 12-h rest. Then, cells were stimulated with 20 µg/mL of antigenic peptide and 2 µg/mL of CD28/49d in 100 µL of media for 24 h. PBMCs were then washed with wash buffer as described above, but RNAsin Plus inhibitor was excluded. PBMCs were then stained with CD3-APCCy7 (Thermo Fisher, cat. no. 47-0036-42), CD8a-PE (Thermo Fisher, cat. no. 12-0088-42), CD4-PECy7, and CD137-APC (Biolegend, cat. no. 309810) antibody for 20 min. Subsequently, cells were washed, resuspended in wash buffer and 7-aminoactinomycin D (7-AAD) (BD, cat. no. 559925) or DAPI was added immediately prior to FACS analysis or sorting. Multimer staining was performed with tetramers as previously described, and MART-1 (ELAGIGILTV) HLA-A2 tetramer was made in-house (12 (link)). Tetramers for NY-ESO-1 (MBL, cat. no. TB-M011-1), CMV pp65 (MBL, cat. no. TB-0010-2), and EBV BMLF1 (MBL, cat. no. TB-M011-2) were purchased.