Anti cd4

HLA-I-/II-restriction was determined by ELISpot assays using antibodies blocking either CD4 or CD8. The cells were incubated at a final concentration of 50 μg/mL of an anti-CD4 (OriGene Technologies, Rockville, Maryland, USA) or 625 ng/mL of an anti-CD8 antibody (BD Bioscience, San Jose, CA, USA) for one hour at 37 °C and 7% CO₂. Then, peptides were added directly to the wells at a final concentration of 20 μg/mL. The restricting HLA-I allele was determined using HLA-matched allogeneic B-LCLs from HLA-I-typed blood donors. These target cells were incubated with the respective peptides for one hour and washed twice with 15 mL PBS. Peptide-sensitized target cells and target cells without peptides were then co-incubated with the T-cell lines in a γ-IFN ELISpot assay, using 2 × 10⁵ target cells per well and 2 × 10⁵ T-cells in duplicate.