Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4(+) T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii(-) cells may present peptides not presented by Ii(+) cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii(+) cells, but will not be tolerant to novel peptides presented by Ii(-) cells, we generated MHC II vaccines to activate cancer patients' T cells. The vaccines are Ii(-) tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii(+) and Ii(-) MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii(-) cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii(+) and Ii(+) cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii(-) cells are not presented by Ii(-) cells, and are derived from source proteins not used by Ii(+) cells. Peptides from Ii(-) cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7(+) human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II+ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019232, 1457-1467, 2012.