Ii-MHC II vaccines present novel MHC II peptides that are tumor specific and immunogenic

摘要

We have generated cell-based cancer vaccines to activate patients' CD4~(+) T lymphocytes to facilitate tumor immunity. The vaccines are based on the hypothesis that invariant chain (Ii) negative, major histocompatibility complex class II (MHC II) positive, CD80 positive tumor cells present novel MHC II peptides and circumvent patients' tolerance to their cancer. Using the human breast cancer cell line MCF10 and retroviruses encoding CD80, HLA-DR7, the Class II Transactivator (CIITA), Ii, and siRNA for Ii, we have made HLA-DR~(+)CD80~(+) MHCII vaccines with and without Ii. Our purpose is to compare the MHC II peptide repertoires of Ii~(-) and Ii~(+) vaccine tumor cells, and confirm that the absence of Ii facilitates the presentation of novel immunogenic tumor peptides. Ii~(-) vaccines (MCF10/DR7/CD80 and MCF10/CIITA/CD80/Ii siRNA) and Ii~(+) (MCF10/DR7/CD80/Ii and MCF10/CIITA/CD80) cells were expanded in culture, and 1X10~(9) cells of each line were harvested for each mass spectrometry analysis. Cells were lysed, MHC II molecules purified by affinity chromatography, and peptides released at low pH. Peptide samples were analyzed using a nano LC-MS/MS system. For each vaccine cell line two affinity purifications were performed, and for each affinity purification two LC-MS/MS runs were conducted. Peptides identified by standard bioinformatics approaches in both affinity purifications were further probed. One hundred and sixteen peptides were identified for MCF10/DR7/CD80 and 228 peptides for MCF10/DR7/CD80/Ii. Fifty-two peptides were present in both cell lines. One hundred and eight peptides were identified for MCF10/CIITA/CD80 and 28 peptides for MCF10/CIITA/CD80/Ii siRNA cells, with six peptides common to both cell lines. These findings confirm our hypothesis that Ii~(+) and Ii~(-) MHC II vaccines present distinct peptide repertoires and agree with our previous in vitro studies in which human MHC II Ii~(-) vaccines activated a population of CD4~(+) T cells that is distinct from the population activated by Ii~(+) cells. To identify peptides with the highest MHC II binding affinity, peptides were analyzed using artificial neural networks (ANN) trained on the HLA-DR7 MHC II binding motif. Seven peptides identified by ANN analysis were synthesized. Five peptides unique to Ii~(-) vaccines were tested for their ability to activate tumor-specific CD4~(+) T-cells. These five peptides activated CD4~(+) T-cells to approximately the same extent as the established breast cancer peptide Her2 p776, as measured by IFN(gamma) production. This is the first study that compares the MHC II peptide repertoire in the absence or presence of Ii in human tumor cells and identifies novel immunogenic MHC II-restricted peptides that could be potential therapeutic reagents for cancer patients.