Reconstitution of the CTL-7A7 mHAg epitope with HPLC-fractionated peptides extracted from HLA-A3 molecules. HLA-A3-associated peptides were purified from 5 × 10¹⁰ mHAg⁺ EBV-LCLs and fractionated by RP-HPLC as described in “Materials and methods.” Aliquots of each fraction corresponding to 4 × 10⁹ cell equivalents in panels A and B and 6 × 10⁹ cell equivalents in panels C and D were preincubated with ⁵¹Cr-labeled donor EBV-LCLs and tested for their ability to sensitize targets to lysis by CTL-7A7 CTLs. An E/T ratio of 10:1 was used. (A) First-dimension separation of extracted peptides was achieved using TFA as the ion-pairing agent. (B) Fractions 61 and 62 from panel A were pooled and rechromatographed using HFBA as the ion-pairing agent. (C) Fractions 70 and 71 from panel B were pooled and rechromatographed on a microcapillary column using TFA as the ion-pairing agent. (D) Determination of candidate peptides via mass spectrometry correlated with ⁵¹Cr release assay. Fractions 52 and 53 from panel C were pooled and rechromatographed using nanoflow effluent splitter technology. Ion abundances of candidate masses within the MS scan window 806-856 were plotted and correlated to the percent specific ⁵¹Cr release in that same region. Background lysis of donor EBV-LCLs by CTL-7A7 CTLs in the absence of peptide was 10% in panel A, 3% in panel B, 16% in panel C, and 10% in panel D. Lysis of recipient EBV-LCLs (positive control) was 54% in panel A, 67% in panel B, 86% in panel C, and 66% in panel D.