Identification of the peptides recognized by the allo-HLA–reactive T-cell clones using multidimensional HPLC fractionation and MS. (A) Five non-T2-recognizing allo-HLA–reactive T-cell clones were stimulated for 18 hours with T2 cells loaded with the first dimension HPLC fractions of peptides eluted from HLA-A2 derived from EBV-LCLs, and IFNγ was measured in the supernatant by standard ELISA. The recognized HPLC fractions were subjected to a second fractionation using a water/isopropanol/TFA gradient, loaded on T2 cells, and subsequently tested for recognition by the T-cell clones. The recognized fractions were fractionated a third time, using a water/methanol/formic acid gradient and tested for recognition. After the third fractionation, the peptide masses present in the recognized and in the adjacent not-recognized fractions were analyzed by MS and the sequences of the peptides were identified. The identification of the peptide recognized by clone HSS12, which is representative for the identification of the peptides recognized by the 4 other clones, is shown. (B) The 5 allo-HLA–reactive T-cell clones, for which the recognized peptides were identified, were tested for their affinity for the respective peptides. The T-cell clones were stimulated with T2 cells loaded with titrated concentrations of the peptides for 18 hours, and IFNγ was measured in the supernatant by standard ELISA. (C) By MS it was determined that the peptide recognized by the T2-reactive T-cell clone HSS8 was derived from KRI1. To determine the affinity of the T-cell clone for this peptide, the clone was tested for IFNγ production against Drosophila cells expressing HLA-A2, CD80 and CD54 loaded with titrated concentrations of the peptide. EC₅₀ represents the peptide concentration needed for half of the maximum IFNγ production by the representative T-cell clone.