Dataset Information

Anti-tumor immunity induces the presentation of aberrant peptides in melanoma

ABSTRACT: Many tumors escape by activating multiple cellular pathways that induce immunosuppression. One pivotal immune-suppressive mechanism is the production of tryptophan metabolites along the kynurenine pathway by IFNγ-induced IDO1 enzyme production 4-8. Phase III clinical trials using chemical inhibition of IDO1 in combination with PD1 pathway blockade, however, failed to improve melanoma treatment 9-12. This points at an incomplete understanding of the role of IDO1 and the consequent tryptophan degradation on mRNA translation and cancer progression. Here, we investigated the effects of prolonged IFNγ treatment on mRNA translation in melanoma cells by ribosome profiling. Surprisingly, we observed a massive accumulation of ribosomes ~20 amino acids downstream of tryptophan codons (termed here as W-Bumps) along with their expected stalling at the tryptophan codon itself. This indicated ribosomal bypass of the tryptophan codons in the absence of tryptophan. Detailed examination of W-Bumps position and its corresponding peptide sequences pinpointed towards ribosomal frameshifting events and their effects in the ribosome exit tunnel. In particular, W-Bumps strength was associated with the disorderedness level of potential out-of-frame peptides predicted downstream of tryptophan codons. Indeed, reporter assays demonstrated the induction of ribosomal frameshifting, and the generation of trans-frame proteins and their presentation at the cell surface after IFNγ treatment. Proteomics and immunopeptidomics analyses verified the production of IFNγ-induced trans-frame and out-of-frame aberrant peptides and their presentation on HLA class I molecules. Priming of naïve T cells from healthy donors with aberrant peptides resulted in identification of reactive, peptide-specific T cells. Altogether, our results suggest that IFNγ-induced IDO1-mediated tryptophan depletion plays a role in the immune recognition of melanoma cells by contributing to the diversity of the peptidome landscape, and by inducing the presentation of aberrant peptides.

INSTRUMENT(S): Q Exactive HF-X, Orbitrap Fusion, Q Exactive Plus

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Melanocyte, Cell Culture

DISEASE(S): Melanoma

SUBMITTER: Onno Bleijerveld

LAB HEAD: Reuven Agami

PROVIDER: PXD020224 | Pride | 2020-12-04

REPOSITORIES: Pride

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			Action	DRS
	20190710_HFX_Project_324_2_Remco_s1.msf	Msf
	20190710_HFX_Project_324_2_Remco_s1.raw	Raw
	20190710_HFX_Project_324_2_Remco_s1_IFN.msf	Msf
	20190710_HFX_Project_324_2_Remco_s1_IFN.raw	Raw
	20190710_HFX_Project_324_2_Remco_s1__.msf	Msf

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Anti-tumour immunity induces aberrant peptide presentation in melanoma.

Bartok Osnat O Pataskar Abhijeet A Nagel Remco R Laos Maarja M Goldfarb Eden E Hayoun Deborah D Levy Ronen R Körner Pierre-Rene PR Kreuger Inger Z M IZM Champagne Julien J Zaal Esther A EA Bleijerveld Onno B OB Huang Xinyao X Kenski Juliana J Wargo Jennifer J Brandis Alexander A Levin Yishai Y Mizrahi Orel O Alon Michal M Lebon Sacha S Yang Weiwen W Nielsen Morten M MM Stern-Ginossar Noam N Altelaar Maarten M Berkers Celia R CR Geiger Tamar T Peeper Daniel S DS Olweus Johanna J Samuels Yardena Y Agami Reuven R

Nature 20201216 7845

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy<sup>1,2</sup>. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ<sup>3-5</sup>. Howeve ...[more]

PMID: 33328638

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Project description:Tumor antigens are central to antitumor immunity. Recent evidence suggests that peptides from non-canonical (nonC) aberrantly translated proteins can be presented on HLA-I by tumor cells. Here, we investigated the presentation and immunogenicity of nonC antigens across different cancer types to better understand their contribution to cancer immunosurveillance and to address whether they could be exploited therapeutically. To this end, we employed a proteogenomics pipeline to identify nonC HLA-I ligands derived from off-frame translation of coding sequences and non-coding regions (UTR, ncRNA, intronic and intergenic) in patient-derived tumor cell lines (TCL) of different histological types (4 gynecological cancer, 3 melanoma and 2 head and neck cancer patients). First, peptides bound to HLA-I were isolated and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using state-of-the-art procedures. Amino acid (Aa) sequences were identified through the previously described pipeline Peptide-PRISM, with some modifications. Briefly, for each MS spectrum, the top 10 candidates were first identified by de novo sequencing and later mapped to a database including the 3-frame transcriptome and 6-frame genome. Additionally, whole-exome sequencing (WES) information of each TCL was included to interrogate the presentation of mutated peptides derived cancer-specific NSM. The false-discovery rate (FDR) was calculated independently for each category considering the search space and peptide length in a stratified mixture model as previously described. Next, in order to select nonC peptides preferentially presented by tumor cells, immunopeptidomics data from several healthy tissues and samples available from the HLA ligand atlas was used to filter out peptides presented in healthy tissues. To overcome a potential bias toward frequent alleles, the peptides were excluded at the ORF level rather than the Aa sequence. As a result, we found that from a total of 839 unique nonC peptides detected in our tumor samples, 38.5% were predicted to derive from ORFs also present in healthy tissue (nonC-HL). Hence, 61.6% (n=517) were considered preferentially presented on tumor HLA-I, referred to as non-canonical tumor ligands (nonC-TL). Out results showed that, although nonC-TL constitued the most abundant source of candidate tumor antigens, as compared to neoantigens, cancer-germline or melanoma-associated antigens, pre-existing antitumor T cells in cancer patients preferentially recognized neoantigens rather than nonC-TL. Nonetehless, nonC-TL elicited de novo T-cell responses via in vitro sensitization of donor lymphocytes. We identified TCRs specific to three nonC-TL, two of which mapped to the 5’ UTR regions of HOXC13 and ZKSCAN1, and one mapping to a non-coding spliced variant of the C5orf22C. These immunogenic nonC-TL were expressed across tumor types but were barely or not detected in healthy cells. Our findings predict a limited contribution of nonC-TL to cancer immunosurveillance but demonstrate they are attractive novel targets for widely applicable immunotherapies.