After 2 h of incubation at room temperature, bound biotinylated peptide-MHC complexes were detected colorimetrically, as indicated

previously 31. TEPITOPE is a T-cell epitope prediction software that enables the identification of ligands binding in a promiscuous or allele-specific mode to HLA-DR molecules 32. We set the TEPITOPE prediction threshold to 10% to select all potential peptide binders to HLA-DR*0401, DR*0404, DR*0101 (which includes weak putative binders and may yield false positives, i.e. peptides not able to bind to these alleles; see 32). HLA-DR*0401-Tg mice were previously described 33 and were purchased from Taconics Farm (Germantown, NY, USA). Mice BI 2536 mw were bred and kept under specific pathogen-free conditions. Mice were immunized subcutaneously with 3 nmole hnRNP-A2 peptide emulsified in CFA containing H37Ra Mycobacteria (Difco, Becton Dickinson). Eight days later, cells from the draining lymph nodes were cultured (6×105 cells/well) in 96-well culture plates (Costar) with 30 μM of C646 hnRNP-A2 peptides in synthetic HL-1 medium (BioWhittaker) supplemented with 2 mM L-glutamine and 50 μg/mL gentamicin (Sigma). PPD was used

as positive control for each culture at a final concentration of 10 μg/mL. Cultures were incubated at 37°C and supernatants were harvested after 20 h (for the detection of IL-2) or 60 h (for the detection of IFN-γ). All animal procedures were performed according to Austrian laws (BGBI. I Nr 162/2005) and approved by the local ethical committee. IFN-γ and

IL-2 were detected by ELISA as previously described 34, using for capture anti-IFN-γ mAb Suplatast tosilate Ph551216 (PharMingen) or anti-IL-2 mAb JES6-1A12 (PharMingen), and for detection biotin-conjugated AN 18.17.24 mAb (kindly provided by Dr. Edgar Schmidt, Mainz) or biotinylated anti-IL-2 mAb JES6-5H4. The cytokine detection limit was 30 pg/mL. ELISPOT plates (Millipore Multiscreen-HA MAIP), pre-incubated with 70% ethanol and washed with distilled water, were coated overnight at 4°C with 10 μg/mL capture mAb (anti-IFN-γ mAb, BMS228ESCA/1, Bender Med Systems, Vienna, Austria) dissolved in 0.1 M NaHCO3 pH 9.5. The plates were then washed once with 200 μL sterile PBS and blocked with 200 μL/well complete RPMI medium (RPMI 1640, 1 mM sodium pyruvate, 200 μM L glutamine, MEM with nonessential amino acids, 10% heat-inactivated AB human serum, all purchased from PAA GmbH, and β2-mercaptoethanol from Gibco) for 2 h at 37°C. The blocking medium was removed and freshly isolated human PBMC (8×105 cells) were cultured with recombinant hnRNP-A2 protein (10 μg/mL), hnRNP-A2 peptides (10 μM), PPD of tuberculin (10 μg/mL), TT (10 μg/mL), or PHA (1/50), in a final volume of 200 μL complete RPMI medium. Control wells contained PBMC with medium alone. After 18- to 24-h incubation at 37°C, cells were removed, plates were washed with PBS/0.