Identification of the peptides recognized by allo-HLA–reactive T-cell clone HSS11. (A) Clone HSS11 recognized 2 fractions after the first HPLC fractionation of peptides eluted from HLA-A2 derived from EBV-LCLs. These 2 recognized fractions were subjected to a second and a third fractionation. The peptide masses present in the fractions recognized by clone HSS11 after the third fractionation as well as in the adjacent not recognized fractions were determined by MS. Sequence analysis by tandem MS was performed on those peptide masses that correlated with the recognition pattern of the T-cell clone. (B) To determine the affinity of clone HSS11 for the 2 identified peptides derived from HLA-DRA and THRAP4, the clone was stimulated with T2 cells loaded with titrated concentrations of the 2 peptides. EC₅₀ represents the peptide concentration needed for half of the maximum IFNγ production by the T-cell clone. (C) To determine whether the 4 amino acids identical between the 2 peptides recognized by clone HSS11, at position P1, P4, P6, and P7, were involved in the TCR interaction, these amino acids were substituted by alanine residues. EC₅₀ of the wt peptides and the modified peptides was determined by stimulating clone HSS11 with T2 cells loaded with titrated concentrations of the different peptides. The EC₅₀ of the modified peptides was divided by the EC₅₀ of the corresponding wt peptide.