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CIITA-transduced glioblastoma cells uncover a rich repertoire of clinically relevant tumor-associated HLA-II antigens

ABSTRACT: CD4+ T cell responses are crucial for inducing and maintaining effective anti-cancer immunity, and the identification of HLA-II cancer-specific epitopes is key to the development of potent cancer immunotherapies. In many tumor types, and especially in glioblastoma (GBM), HLA-II complexes are hardly ever naturally expressed. Hence, little is known about immunogenic HLA-II epitopes in GBM. With stable expression of CIITA coupled to a detailed and sensitive mass spectrometry based immunopeptidomics analysis, we here uncovered a remarkable breadth of the HLA-ligandome in HROG02, HROG17 and RA GBM cell lines. The effect of CIITA expression on the induction of the HLA-II presentation machinery was striking in each of the three cell lines, and it was significantly higher compared to IFNɣ treatment. In total, we identified 16,123 unique HLA-I peptides and 32,690 unique HLA-II peptides. In order to genuinely define the identified peptides as true HLA ligands, we carefully characterized their association with the different HLA allotypes. In addition, we identified 138 and 279 HLA-I and HLA-II ligands, respectively, most of which are novel in GBM, derived from known GBM-associated tumor-antigens that have been used as source proteins for a variety of GBM vaccines. Our data further indicate that CIITA-expressing GBM cells acquired an antigen presenting cell-like phenotype as we found that they directly present external proteins as HLA-II ligands. Not only that CIITA-expressing GBM cells are attractive models for antigen discovery endeavors, but such engineered cells have great therapeutic potential through massive presentation of a diverse antigenic repertoire.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

DISEASE(S): Brain Cancer

SUBMITTER: Michal Bassani-Sternberg

LAB HEAD: Michal Bassani-Sternberg

PROVIDER: PXD020079 | Pride | 2021-02-03

REPOSITORIES: Pride

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Project description:Background: Patient derived organoids (PDOs) can be established from colorectal cancers as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFN) or MEK-inhibitors. Methods: Four PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analysed by MS. Results: We identified an average of 9,936 unique peptides per PDO which compares favourably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. Treatment of four PDOs with IFN upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFN-inducible genes. HLA class II presented peptides increased on average 16-fold with IFN treatment. MEK-inhibitor treatment showed no consistent effect on class I or II HLA expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment. Conclusions: Only 3 out of 612 non-synonymous mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.

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.

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CIITA-transduced glioblastoma cells uncover a rich repertoire of clinically relevant tumor-associated HLA-II antigens

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