title: Natural CD8+ T-cell responses against MHC class I epitopes of the HER-2/ neu oncoprotein in patients with epithelial tumors.
abstract: HER-2/ neu is an immunogenic protein eliciting both humoral and cellular immune responses in patients with HER-2/ neu-positive ((+)) tumors. Preexisting cytotoxic T lymphocyte (CTL) immunity to HER-2/ neu has so far been mainly evaluated in terms of detection of CTL precursor (CTLp) frequencies to the immunogenic HLA-A2-binding nona-peptide 369-377 (HER-2(9(369))). In the present study, we examined patients with HER-2/ neu(+) breast, ovarian, lung, colorectal, and prostate cancers for preexisting CTL immunity to four recently described HER-2/ neu-derived and HLA-A2-restricted "cytotoxic" peptides and to a novel one spanning amino acids 777-785 also with HLA-A2-binding motif. We utilized enzyme-linked immunosorbent spot (ELISpot) assay, which allows a quantitative and functional assessment of T cells directed against specific peptides after only brief in vitro incubation. CTL reactivity was determined with an interferon gamma (IFN-gamma) ELISpot assay detecting T cells at the single cell level secreting IFN-gamma. CTLp were defined as peptide-specific precursors per 10(6) peripheral blood mononuclear cells (PBMCs). Patients' PBMCs with increased CTLp were also tested against autologous tumor targets and peptide-pulsed dendritic cells (DCs) in cytotoxicity assays. We also studied patients with HER-2/ neu-negative ((-)) tumors and healthy individuals. Of the HER-2/neu(+) patients examined, 31% had increased CTLp to HER-2(9(952)), 19% to HER-2(9(665)), 16% to HER-2(9(689)), and 12.5% HER-2(9(435)), whereas only 2 of 32 patients (6%) responded to HER-2(9(777)). The CTLp recognizing HER-2(9(952)) were extremely high in two patients with breast cancer, one with lung cancer, and one with prostate cancer. None of the HER-2/neu(-) patients or healthy donors exhibited increased CTLp to any of these peptides. Besides IFN-gamma production, preexisting CTL immunity to all five HER-2/ neu peptides was also shown in cytotoxicity assays where patients' PBMCs with increased CTLp specifically lysed autologous tumor targets and autologous peptide-pulsed DCs. Our results demonstrate for the first time that (1) preexisting immunity to peptides HER-2(9(435)), HER-2(9(952)), HER-2(9(689)), HER-2(9(665)), and HER-2(9(777)) is present in patients with HER-2/ neu(+) tumors of distinct histology, (2) HER-2(9(777)) is a naturally processed peptide expressed on the surface of HER-2/ neu(+) tumors, as are the other four peptides, and (3) HER-2/ neu(+) prostate tumor cells can be recognized and lysed by autologous HER-2 peptide-specific CTL. Our findings broaden the potential application of HER-2/ neu-based immunotherapy.
mesh_terms: D000328:Adult; D000368:Aged; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002455:Cell Division; D003713:Dendritic Cells / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D005260:Female; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D007114:Immunization; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D000939:Epitopes; D015789:HLA-A2 Antigen; D007371:Interferon-gamma; D010446:Peptide Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases