Mass-spectrometric analysis of naturally processed peptides recognized by ovarian tumor-associated CD8(+) CTL

Abstract

Antigens recognized by cytotoxic T cells (CTL) are expressed as peptides presented by MHC class I molecules. To isolate peptides from the MHC molecule HLA-A2.1 and identify epitopes that define the activity profile of ovarian CD8(+) CTL, peptides were separated by reverse-phase high-pressure liquid chromatography (HPLC), and analyzed by electrospray ionization-tandem mass spectrometry (ES-MS). HLA-A2.1-bound peptides were extracted from the ovarian tumor line SKOV3 transfected with the HLA-A2.1 (clone 1E4) and C1R.A2 cells transfected with HCA-A2.1 and HER-2 (clone HER-2.J) by immunoaffinity chromatography. At least five peaks of distinct retention times (termed 1, 2A, 2B, 2C, and 3) were recognized by an ovarian HER-2(high) (HER-2(hi)) tumor-associated HLA-A2(+), CD8(+) CTL line. ES-MS analysis was performed for peak 2B peptides from both types of cells. In the four consecutive fractions of peak 2B, at least 27 and 16 ion species of mass-to-charge (m/z) ratio between 760-1300 were detected in 1E4 and HER-2.J cells, respectively. The abundance of four 1E4 and six HER-2.J ions believed to be peptides in four consecutive HPLC fractions in this peak matched the CTL activity profile. Of these, two ions with actual m/z ratios 497.3-498.4 and 792.8-793.2, were found in the peak 2B from both types of cells. Since little is known about the tumor Ag recognized in human cancers, characterization of these ions may lead to identification of novel tumor Ag in breast and ovarian cancers. This may also be useful in developing quantitative approaches to the identification of tumor Ag and the determination of epitope density on tumor and normal cells. This may help characterize the relationship between tumor immunity and epitope tolerance in human epithelial cancers.