T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) ...remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.
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•Time course analysis of HLA-I immunopeptidome in SARS-CoV-2-infected cells•25% of detected HLA-I peptides originated from out-of-frame ORFs in S and N•Some out-of-frame peptides elicited stronger T cell responses than canonical peptides•Early expressed viral proteins dominated HLA-I presentation and immunogenicity
Analysis of the HLA-I peptidome of SARS-CoV-2 infection identifies peptides derived from canonical and out-of-frame ORFs in viral S and N protein that are not captured by current vaccines and yield potent T cell responses in a mouse model as well as individuals with COVID-19.
Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine ...carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.
Severe respiratory illness is the most prominent manifestation of patients infected with SARS-CoV-2, and yet the molecular mechanisms underlying severe lung disease in COVID-19 affected patients ...still require elucidation. Human leukocyte antigen class I (HLA-I) expression is crucial for antigen presentation and the host's response to SARS-CoV-2.
To gain insights into the immune response and molecular pathways involved in severe lung disease, we performed immunopeptidomic and proteomic analyses of lung tissues recovered at four COVID-19 autopsy and six non-COVID-19 transplants.
We found signals of tissue injury and regeneration in lung fibroblast and alveolar type I/II cells, resulting in the production of highly immunogenic self-antigens within the lungs of COVID-19 patients. We also identified immune activation of the M2c macrophage as the primary source of HLA-I presentation and immunogenicity in this context. Additionally, we identified 28 lung signatures that can serve as early plasma markers for predicting infection and severe COVID-19 disease. These protein signatures were predominantly expressed in macrophages and epithelial cells and were associated with complement and coagulation cascades.
Our findings emphasize the significant role of macrophage-mediated immunity in the development of severe lung disease in COVID-19 patients.
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.
Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine ...carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.
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