High Immunogenicity of the Human Leukocyte Antigen Peptidomes of Melanoma Tumor Cells
peer-reviewed · Journal of Biological Chemistry · 2012
| Date | 2012-09-01 |
| Type | peer-reviewed |
| Venue | Journal of Biological Chemistry |
| Publisher | American Society for Biochemistry and Molecular Biology |
| Contribution | downstream-application |
| DOI | 10.1074/jbc.M112.358903 |
Abstract
Human leukocyte antigens (HLA) bind peptides generated by limited proteolysis in cells and present them at the cell surfaces for recognition by T cells. Through this antigen presentation function they control the specificity of T cell responses and thereby adaptive immune responses. Knowledge of HLA-bound peptides is thus key to understanding adaptive immunity and to the development of vaccines and other specific immune intervention strategies. To gain insight into the antigenicity of melanomas, peptides were extracted from HLA isolated from the tumor cells, separated by two-dimensional HPLC, and sequenced by mass spectrometry. The spectra were analyzed by database-dependent MASCOT searches and database-independent de novo sequencing and, where required, confirmed with synthetic peptides, which were also used to determine their immunogenicity. Comparing four different melanoma cell lines, little overlap of the HLA-bound peptides was found, suggesting a high degree of individualization of the HLA peptidomes. This notwithstanding, the peptidomes were highly immunogenic in the patients from whom the tumor cells had been established and in unrelated patients. This broad cross-patient immunogenicity was only exceptionally related to individual peptides. The majority of the identified epitopes were derived from low to medium abundance proteins, mostly involved in sensitive cellular processes such as cell cycle control, DNA replication, control of gene expression, tumor suppressor function, and protein metabolism. The peptidomes thus provide insights into processes potentially related to tumorigenesis. Furthermore, analyses of the peptide sequences yield information on the specificity of peptide selection by HLA applicable to the developing prediction algorithms for T cell epitopes.
Methods and tools
- Melanoma HLA peptidome immunogenicity: HLA class I peptidomes of four melanoma cell lines, extracted from isolated HLA, separated by two-dimensional HPLC and sequenced by MALDI post-source-decay MS. Spectra were interpreted twice over, by database-dependent Mascot search and by database-independent de novo sequencing in Sequit!, with synthetic peptides used to confirm assignments and to measure immunogenicity. Overlap between the four peptidomes was small, indicating highly individual HLA peptidomes, yet they were broadly immunogenic both in the patients the lines came from and in unrelated patients, and that cross-patient immunogenicity was only exceptionally attributable to individual peptides. Most epitopes came from low to medium abundance proteins in sensitive processes such as cell cycle control, DNA replication and tumour suppression.
- Sequit: Software for de novo peptide sequencing by MALDI post-source decay mass spectrometry.