HLA-DRA/DRB Tetramer Production and T Cell Analysis

HLA-DRA and HLA-DRB chains were produced as previously described (42 (link)). For tetramer production, HLA-DRA and HLA-DRB chains were mixed in a 1:1.5 ratio and incubated in 3 μM peptides in PBS (pH 7.4) with 20% glycerol and 0.1% Pluronic F68 for 96 h at 18°C. The resulting monomers were buffer changed into PBS with 5% glycerol and concentrated on 10kD Vivaspin (Satorius) and quantitated by Luminescent Oxygen Channeling Immunoassay (LOCI)-driven assay (42 (link)). The resulting monomers were tetramerized by addition of fluorochrome labeled Streptavidin (Streptavidin-phycoerythrin (SA-PE) or Streptavidin-allophycocyanin (SA-APC); both from BioLegend) sequentially over 60 min at a 1:4 molar ratio of streptavidin to monomer. For T cell analysis, pellets of 4 × 10⁵ cells obtained from in vitro peptide stimulated cell cultures, were re-suspended in a 40 μl tetramer diluted in media to a final concentration of ca. 30 nM, and incubated for 1 h at 37°C, followed by 30 min incubation with fluorochrome conjugated anti-CD3, -CD8, -CD38, and -HLA-DR antibodies. The cells were analyzed by flowcytometry (Fortessa or LSR-II, BD Biosciences) using Diva software.