Antigen Presentation Assay Protocol

Ag presentation assays were performed in 96-well round-bottomed plates 17 (link). Type A T-cell hybridoma 3A9 and type B T-cell hybridoma 11A10 were from Professor Emil Unanue (Washington, USA). Briefly, BMDCs were seeded at 3 × 10⁴ cells/well. Cells were exposed to HEL Ag or peptide (HEL_46–61), with or without PAMP stimulants/S. Typhimurium for 18 h in 100 μl volume. The following concentrations of stimulants were used; HKST (50:1 ratio of bacteria:cells), 100 ng/mL PAM3CSK4, 100 ng/mL Poly(I:C), 1 μg/mL LPS, 100 ng/mL flagellin, 100 ng/mL MALP-2, 5 μg /mL imiquimod, 5 μg /mL CpG, 0.5 μg /mL profilin, 100 μg /mL curdlan, and 100 μg /mL zymosan. DCs were washed three times and 5 × 10⁴ washed T hybridoma cells (type-B 11A10 or type A 3A9) were added per well. Culture supernatants were harvested after 24 h and frozen at −80°C. Released IL-2 was quantified by ELISA using a mouse IL-2 Ready-SET-Go kit (eBioscience).