The Journal of Experimental Medicine
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Published 5 November 2001. doi:10.1084/jem.194.9.1313
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© Rockefeller University Press, 0022-1007/2001/11/1313/ $5.00
The Journal of Experimental Medicine, Volume 194, Number 9, November 5, 2001 1313-1324

Original Article

Identification of Cyclin B1 as a Shared Human Epithelial Tumor-Associated Antigen Recognized by T Cells

Henry Kao1, Jarrod A. Marto4, Thomas K. Hoffmann2, Jeffrey Shabanowitz4, Sydney D. Finkelstein3, Theresa L. Whiteside2, Donald F. Hunt4,5 and Olivera J. Finn1,2

1 Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261
2 University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15261
3 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261
4 Department of Chemistry, University of Virginia, Charlottesville, VA 22901
5 Department of Pathology, University of Virginia, Charlottesville, VA 22901

Address correspondence to Olivera J. Finn, University of Pittsburgh School of Medicine, Department of Molecular Genetics and Biochemistry, W1142 Biomedical Science Tower, Terrace and DeSoto Streets, Pittsburgh, PA 15261. Phone: 412-648-9816; Fax: 412-383-8859; E-mail: ojfinn{at}pitt.edu

We eluted peptides from class I molecules of HLA-A2.1+ breast adenocarcinoma and loaded reverse phase high-performance liquid chromatography (HPLC) fractions onto dendritic cells to prime naive CD8+ T cells. Fractions that supported growth of tumor-specific cytotoxic T lymphocytes were analyzed by nano-HPLC micro-ESI tandem mass spectrometry. Six HLA-A2.1-binding peptides, four 9-mers (P1-P4) differing in the COOH-terminal residue, and two 10-mers (P5 and P6) with an additional COOH-terminal alanine, were identified in one fraction. Peptide sequences were homologous to cyclin B1. We primed CD8+ T cells from another HLA-A2.1+ healthy donor with synthetic peptides and generated P4-specific responses. We also detected memory T cells specific for one or more of these peptides in patients with breast cancer and squamous cell carcinomas of the head and neck (SCCHN). T cells from one patient, restimulated once in vitro, could kill the tumor cell line from which the peptides were derived. Immunohistochemical analysis of tumor lines and tissue sections showed cyclin B1 overexpression and aberrant localization in the cytoplasm instead of the nucleus. Sequencing genomic DNA and cDNA corresponding to P1–P6 region showed that differences in COOH-terminal residues were not due to either DNA mutations or errors in transcription, suggesting a high error rate in translation of cyclin B1 protein in tumors.

Key Words: dendritic cells • in vitro priming • tandem mass spectrometry • CTL • ELISPOT


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