Mass Spectrometry Based Identification of Novel HLA Class I Restricted Peptides in Merkel Cell Carcinoma.

Highly polymorphic class I human leukocyte antigen (HLA-I) molecules present endogenous peptides to CD8+ T cells, an essential part of immune regulation. This mechanism allows for the presentation of thousands of epitopes per allele and individual. Liquid chromatography-mass spectrometry (LC-MS/MS) is a high throughput method that can be utilized to identify endogenously processed and presented peptides. Merkel cell carcinoma (MCC) is a rare and aggressive form of neuroendocrine skin cancer. A major subtype of this cancer is caused by the Merkel cell Polyomavirus (MCPyV), so MCPyV+ MCC is an informative model system to explore virally derived cancer-specific epitopes. MCC, however, often downregulates or eliminates HLA-I antigen presentation to evade recognition by the immune system. We hypothesized that reversing the mechanisms of immune evasion will allow us to detect novel epitopes. Here, we used optimized LC-MS/MS methods to characterize the immunopeptidome of MCC in a native and perturbed state. First, we compared the immunopeptidome of primary tumor samples and tumor-derived MCC cell lines. Peptide identities presented on primary tumors and tumor-derived cell lines overlapped by 50% or more, indicating that the tumor-derived cell lines retain their innate tumor properties and are a useful model to study immune presentation in MCC. Next, we investigated mechanisms to restore HLA-I presentation in MCC, such as IFNγ treatment and knockout or inhibition of components of the Polycomb repressive complex (PRC1.1). We found that IFNγ treatment increased peptide yield up to 8-fold across seven cell lines while also increasing presentation on HLA-B alleles. Using tandem mass tag labeling, we observed relative quantitative changes in the immunopeptidome of an MCPyV+ cell line when PRC1.1 components were knocked down. We discovered increased HLA-I presentation upon USP7 inhibitor treatment in comparison to the control. In contrast to IFNγ treatment, USP7 inhibition did not alter the fraction of observed peptides assigned to alleles. Finally, use of High Field Asymmetric Ion Mobility Spectrometry (FAIMS) to analyze samples with reinstated HLA presentation enabled identification of a number of novel antigens, including three MCPyV viral epitopes. The immunogenicity of these viral peptides was determined by ELISpot assays with peripheral blood mononuclear cells (PBMCs). These findings can be translated into medical treatments for patients diagnosed with this cancer.