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Identification of cyclin B1 as a shared human epithelial tumor-associated antigen recognized by T cells.

Kao H, Marto JA, Hoffmann TK, Shabanowitz J, Finkelstein SD, Whiteside TL, Hunt DF, Finn OJ - J. Exp. Med. (2001)

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261, USA.

ABSTRACT
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.

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HLA class I–restricted T cell response of a healthy HLA-A2+ donor to cyclin B1 peptides. CD8+ T cells generated in vitro by priming to synthetic cyclin B1 peptides were used in an IFN-γ ELISPOT assay after the fourth restimulation. Number of T cells per well is indicated in parenthesis.
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fig1: HLA class I–restricted T cell response of a healthy HLA-A2+ donor to cyclin B1 peptides. CD8+ T cells generated in vitro by priming to synthetic cyclin B1 peptides were used in an IFN-γ ELISPOT assay after the fourth restimulation. Number of T cells per well is indicated in parenthesis.

Mentions: As the cyclin B1 peptides were derived from a first dimension HPLC fraction that primed tumor-specific CTLs from an HLA-A2.1+ donor, we sought to determine whether these peptides were indeed responsible for the immunostimulatory activity. We used naive CD8+ T cells from another healthy HLA-A2.1+ donor and autologous DCs loaded with the individual synthetic peptides (P1–P6). No T cell responses were detected against the peptides in the absence of in vitro stimulation in either this donor or another HLA-A2.1+ donor (data not shown). However, after four rounds of stimulation, we detected antigen-specific IFN-γ secretion by CD8+ T cells in response to P4, and not to other peptides (P1, P2, P5, P6) or peptide HIV-POL (ILKEPGSHV) that is known to bind HLA-A2.1 and served here as the negative control (Fig. 1) . We were able to block the T cell response to P4 using the anti–class I antibody W6/32, showing that the P4-specific responses were HLA class I restricted. These in vitro primed T cells that specifically recognized P4-loaded DCs were unable to kill the original tumor from which the peptides were derived (data not shown). This was not unexpected considering that these T cells were primed with high concentrations of peptide (50 μM), and are thus expected to be of low affinity and incapable of recognizing the comparatively lower levels of the same HLA-peptide complexes on the tumor.


Identification of cyclin B1 as a shared human epithelial tumor-associated antigen recognized by T cells.

Kao H, Marto JA, Hoffmann TK, Shabanowitz J, Finkelstein SD, Whiteside TL, Hunt DF, Finn OJ - J. Exp. Med. (2001)

HLA class I–restricted T cell response of a healthy HLA-A2+ donor to cyclin B1 peptides. CD8+ T cells generated in vitro by priming to synthetic cyclin B1 peptides were used in an IFN-γ ELISPOT assay after the fourth restimulation. Number of T cells per well is indicated in parenthesis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195974&req=5

fig1: HLA class I–restricted T cell response of a healthy HLA-A2+ donor to cyclin B1 peptides. CD8+ T cells generated in vitro by priming to synthetic cyclin B1 peptides were used in an IFN-γ ELISPOT assay after the fourth restimulation. Number of T cells per well is indicated in parenthesis.
Mentions: As the cyclin B1 peptides were derived from a first dimension HPLC fraction that primed tumor-specific CTLs from an HLA-A2.1+ donor, we sought to determine whether these peptides were indeed responsible for the immunostimulatory activity. We used naive CD8+ T cells from another healthy HLA-A2.1+ donor and autologous DCs loaded with the individual synthetic peptides (P1–P6). No T cell responses were detected against the peptides in the absence of in vitro stimulation in either this donor or another HLA-A2.1+ donor (data not shown). However, after four rounds of stimulation, we detected antigen-specific IFN-γ secretion by CD8+ T cells in response to P4, and not to other peptides (P1, P2, P5, P6) or peptide HIV-POL (ILKEPGSHV) that is known to bind HLA-A2.1 and served here as the negative control (Fig. 1) . We were able to block the T cell response to P4 using the anti–class I antibody W6/32, showing that the P4-specific responses were HLA class I restricted. These in vitro primed T cells that specifically recognized P4-loaded DCs were unable to kill the original tumor from which the peptides were derived (data not shown). This was not unexpected considering that these T cells were primed with high concentrations of peptide (50 μM), and are thus expected to be of low affinity and incapable of recognizing the comparatively lower levels of the same HLA-peptide complexes on the tumor.

Bottom Line: 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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus