Combined Analysis of Antigen Presentation and T-cell Recognition Reveals Restricted Immune Responses in Melanoma

Abstract

The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities between metastases from the same patient. Furthermore, we reveal that two neoantigen-specific clonotypes killed 90% of autologous melanoma cells, both and, showing that a limited set of neoantigen-specific T cells may play a central role in melanoma tumor rejection. Our findings indicate that combining HLA peptidomics with neoantigen predictions allows robust identification of targetable neoantigens, which could successfully guide personalized cancer immunotherapies. As neoantigen targeting is becoming more established as a powerful therapeutic approach, investigating these molecules has taken center stage. Here, we show that a limited set of neoantigen-specific T cells mediates tumor rejection, suggesting that identifying just a few antigens and their corresponding T-cell clones could guide personalized immunotherapy. .

Immunoproteasome expression is associated with better prognosis and response to checkpoint therapies in melanoma

“…To test this, we employed HLA peptidomics to profile HLA-Ibound antigens in the three cellular systems noted above, as previously described [23][24][25] . In total, we identified 17,501 unique HLA peptides: 8418 peptides in 108T cells, 6205 peptides in 12T cells and 5575 peptides in A375 cells.…”

“…These TAAs were derived from 49 108T proteins, 45 12T proteins, and 32 A375 proteins, and derived from known cancer/testis and previously described melanoma antigens [26][27][28] . Both neo-antigens derived from missense mutations: DANSFLQSV from a P677S mutation in the mediator complex subunit 15 (MED15) gene, and KLFEDRVGTIK from a S123L mutation in the TPD52 like 2 (TPD52L2) gene 23,25 . We validated the identification of the neo-antigens by comparing their MS/MS spectra with that of synthetic peptides ( Supplementary Fig.…”

“…For the HLA peptidomics analysis we used three experimental replicates per each cell line and each condition (empty vector/ overexpression and treated/ non-treated cells). Samples were processed as described previously 23,25,36 . Briefly, cell pellets were lysed with lysis buffer containing 0.25% sodium deoxycholate, 0.2 mM iodoacetamide, 1 mM EDTA, 1:200 protease inhibitors cocktail (Sigma-Aldrich), 1 mM PMSF and 1%octyl-b-D glucopyranoside in PBS, and then incubated at 4°C for 1 h. The lysates were cleared by centrifugation at 4°C and 48,000g for 60 min, and then passed through a preclearing column containing Protein-A Sepharose beads.…”

Flexible machine learning prediction of antigen presentation for rare and common HLA-I alleles

“…The representation space can also be useful to reveal new features even in the mono-allelic case. A compelling example is provided by HLA-B*51:01-restricted peptides derived from a clinical sample [26,27] (see below for details about how restriction was predicted). Projection of HLA-B*51:01-specific sequences onto a single RBM feature reveals a double-peaked histogram, corresponding to two structurally alternative binding modes, which were validated in [12] (Fig.…”

Streamlined selection of cancer antigens for vaccine development through integrative multi-omics and high-content cell imaging

“…In particular, MHC class-І-restricted peptides (i.e., CTL epitopes) recognized by CD8 + CTLs play a key role in attacking cancer cells by promoting the activation and proliferation of antigen-specific CTLs 22 . To develop effective CTL-associated peptide vaccines, many methods use bioinformatics algorithms and proteomics approaches to identify multiple HLA-class-bound peptides 23,24 . Recently, proteomics approaches associated with mass spectrometry (MS) 25 and specifically LC-MS/MS, were used for HLA-peptidomic analysis to directly facilitate the discovery of numerous natural peptides in complex with specific HLA molecules expressed on cell surfaces 26,27 .…”