Functional T Cell Assay Protocol

Functional T cell assays were performed as previously described (9 (link)). PBMCs were cultured for 5 days in the presence of 20 μg/ml peptides at a total cell concentration of 1 × 10⁶ cells/well in flat bottom 96-well plates in RPMI 1640 supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10 mM HEPES, and 10% pooled human serum. PBMCs were restimulated with peptides and anti-CD28 (BioLegend) on day 5 for 6 h with 5 μg/ml Brefeldin A (Sigma-Aldrich) being added for the last 4 h. Restimulated cells were treated with LIVE/DEAD^® Fixable Green Dead Cell Stain (Invitrogen) and thereafter stained for surface expression of CD3 (BioLegend), CD4, and CD14 (BD Biosciences). Cells were then stained intracellularly for IFN-γ and IL-17A (BioLegend) as well as CD154 (CD40L) (BD Biosciences) expression using the Cytofix/Cytoperm fixation and permeabilization solution kit (BD Biosciences). Samples were run on a CyAn™ ADP Analyzer (Beckman Coulter), and data were analyzed using FlowJo software, version 7.5.1 or higher (Tree Star). The gating strategy is depicted in Figure S2 in Supplementary Material. In some experiments, blocking antibodies for HLA-DR (clone L243), -DP (clone B7/21), and -DQ (clone SPLV3), all obtained from the Tetramer Core Facility (BRI, Seattle, WA, USA), were added to the cultures.