Extending the Mass Spectrometry-Detectable Landscape of MHC Peptides by Use of Restricted Access Material.

Mass spectrometry-based immunopeptidomics enables the comprehensive identification of major histocompatibility complex (MHC) peptides from a cell culture as well as from tissue or tumor samples and is applied for the identification of tumor-specific and viral T-cell epitopes. Although mass spectrometry is generally considered an "unbiased" method for MHC peptide identification, the physicochemical properties of MHC peptides can greatly influence their detectability. Here, we demonstrate that highly hydrophobic peptides are lost during sample preparation when C18 solid-phase extraction (SPE) is used for separating MHC peptides from proteins. To overcome this limitation, we established an optimized protocol involving restricted access material (RAM). Compared to C18-SPE, RAM-SPE improved the overall MHC peptide recovery and extended the landscape of mass spectrometry-detectable MHC peptides toward more hydrophobic peptides.

Loss of p14 diminishes immunogenicity in melanoma via non-canonical Wnt signaling by reducing the peptide surface density.

Immunotherapy has achieved tremendous success in melanoma. However, only around 50% of advanced melanoma patients benefit from immunotherapy. Cyclin-dependent kinase inhibitor 2A (CDKN2A), encoding the two tumor-suppressor proteins p14ARF and p16INK4a, belongs to the most frequently inactivated gene loci in melanoma and leads to decreased T cell infiltration. While the role of p16INK4a has been extensively investigated, knowledge about p14ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished their recognition and killing by melanoma differentiation antigen (MDA)-specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. Immunopeptidomic analyses revealed that antigen presentation via human leukocyte antigen class I (HLA-I) molecules was enhanced upon p14ARF downregulation in general, but absolute and relative expression of cognate peptides was decreased. However, this phenotype is associated with a favorable outcome for melanoma patients. Limiting Wnt5a signaling reverted this phenotype, suggesting an involvement of non-canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA-specific T cell responses by decreasing both absolute and relative MDA-peptide presentation in melanoma.