3420 2apt 10

Cryopreserved PBMC were stimulated with peptides (individual or peptide pools) at a final concentration of 1µg/mL per peptide in RPMI supplemented with 8% human serum (Biowest, Nuaillé, Maine-et-Loire, France), Penicillin/Streptomycin (BioConcept, Allschwil, Basel-Country, Switzerland) and beta-mercaptoethanol (Gibco). Recombinant human IL-2 (Proleukin, Clinigen, Yardley, Pennsylvania, United States) was added to the culture after 48 h at a final concentration of 100 U/mL for 12 days. At day 12 of the IVS, IFNγ Enzyme-Linked ImmunoSpot (ELISpot) assays were performed using pre-coated 96-well ELISpot plates (3420-2APT-10 Mabtech, Stockholm, Södermanland and Uppland, Sweden) [37 (link)]. Then, 100,000 cells were re-challenged (in duplicate) with peptides for 16–18 h, and then plates were washed according to manufacturer’s instructions and counted with the AID-Spot Robot ELISpot reader (AutoImmun Diagnostika GMBH, Straßberg, Baden-Württemberg, Germany). SEB (Staphylococcal Enterotoxin B, final concentration of 250ng/mL, Sigma-S4881) and anti-CD3 antibody (final concentration of 1 µg/mL, clone UCHT1, Mabtech) were used as positive control and unstimulated PBMCs as background control.

At day 12 of the IVS, IFNγ ELISpot assays were performed using pre-coated 96-well ELISpot plates (Mabtech #3420-2APT-10) [37 (link)]. Then, 100,000 cells were re-challenged either with the mutated or wild type sequence of cognate peptides used at different concentrations (2 µM, 1 µM, 0.1 µM, and 0.001 µM) for 16–18 h. Plates were then washed according to manufacturer’s instructions and counted with the AID-Spot Robot ELISpot reader (AutoImmun Diagnostika GMBH).