Identification of the HLA-A*0301–associated peptide epitope KLPNSVLGR by mass spectrometry. (A) In vitro epitope reconstitution with naturally processed RCC peptides. Peptides were acid-eluted from immunoaffinity-purified HLA-A*0301 complexes of 2.5 × 10¹⁰ MZ1257-RCC cells and fractionated by RP-HPLC. Aliquots of each fraction corresponding to 8 × 10⁸ cell equivalents were preincubated with HLA-A*0301–transfected T2 (T2-A3) cells and tested for recognition by HLA-A*0301–restricted CTL clone B5 at an E/T ratio of 20:1 in a ⁵¹Cr-release assay. Background lysis on T2-A3 by CTL B5 in the absence of peptides was 2%. Lysis on MZ1257-RCC by CTL B5 was 97% (not shown). ACN indicates acetonitrile. (B) MALDI-TOF mass spectrum from HPLC fraction 29 of the HLA-A*0301 peptide extract that contained the CTL B5 epitope. Data were obtained from 3.8 × 10⁸ MZ1257-RCC cell equivalents and are shown in the 950 m/z to 1300 m/z range. The m/z 983.60 ion is indicated by arrow. (C) CID mass spectrum recorded on candidate peptide ions of m/z 983.60 observed in HPLC fraction 29. Peptide material from HPLC fraction 29 corresponding to 6 × 10⁹ MZ1257-RCC cell equivalents was separated by microcapillary liquid chromatography prior to fragmentation by MALDI-TOF/TOF mass spectrometry. (D) Epitope reconstitution with synthetic peptide KLPNSVLGR. RCC-reactive CTL clone B5 was tested in an IFN-γ ELISPOT assay against T2-A3 cells pulsed with synthetic peptide KLPNSVLGR at indicated concentrations. Results obtained with 5000 CTLs/well are shown. Unpulsed T2-A3 cells and MZ1257-RCC cells were negative and positive controls, respectively. Results shown in panels A to D are representative of at least 2 experiments.