BackgroundThe human leucocyte antigen (HLA) complex controls adaptive immunity by presenting defined fractions of the intracellular and extracellular protein content to immune cells. Understanding ...the benign HLA ligand repertoire is a prerequisite to define safe T-cell-based immunotherapies against cancer. Due to the poor availability of benign tissues, if available, normal tissue adjacent to the tumor has been used as a benign surrogate when defining tumor-associated antigens. However, this comparison has proven to be insufficient and even resulted in lethal outcomes. In order to match the tumor immunopeptidome with an equivalent counterpart, we created the HLA Ligand Atlas, the first extensive collection of paired HLA-I and HLA-II immunopeptidomes from 227 benign human tissue samples. This dataset facilitates a balanced comparison between tumor and benign tissues on HLA ligand level.MethodsHuman tissue samples were obtained from 16 subjects at autopsy, five thymus samples and two ovary samples originating from living donors. HLA ligands were isolated via immunoaffinity purification and analyzed in over 1200 liquid chromatography mass spectrometry runs. Experimentally and computationally reproducible protocols were employed for data acquisition and processing.ResultsThe initial release covers 51 HLA-I and 86 HLA-II allotypes presenting 90,428 HLA-I- and 142,625 HLA-II ligands. The HLA allotypes are representative for the world population. We observe that immunopeptidomes differ considerably between tissues and individuals on source protein and HLA-ligand level. Moreover, we discover 1407 HLA-I ligands from non-canonical genomic regions. Such peptides were previously described in tumors, peripheral blood mononuclear cells (PBMCs), healthy lung tissues and cell lines. In a case study in glioblastoma, we show that potential on-target off-tumor adverse events in immunotherapy can be avoided by comparing tumor immunopeptidomes to the provided multi-tissue reference.ConclusionGiven that T-cell-based immunotherapies, such as CAR-T cells, affinity-enhanced T cell transfer, cancer vaccines and immune checkpoint inhibition, have significant side effects, the HLA Ligand Atlas is the first step toward defining tumor-associated targets with an improved safety profile. The resource provides insights into basic and applied immune-associated questions in the context of cancer immunotherapy, infection, transplantation, allergy and autoimmunity. It is publicly available and can be browsed in an easy-to-use web interface at https://hla-ligand-atlas.org .
Summary
The entirety of human leukocyte antigen (HLA)‐presented peptides is referred to as the HLA ligandome of a cell or tissue, in tumours often termed immunopeptidome. Mapping the tumour ...immunopeptidome by mass spectrometry (MS) comprehensively views the pathophysiologically relevant antigenic signature of human malignancies. MS is an unbiased approach stringently filtering the candidates to be tested as opposed to epitope prediction algorithms. In the setting of peptide‐specific immunotherapies, MS‐based strategies significantly diminish the risk of lacking clinical benefit, as they yield highly enriched amounts of truly presented peptides. Early immunopeptidomic efforts were severely limited by technical sensitivity and manual spectra interpretation. The technological progress with development of orbitrap mass analysers and enhanced chromatographic performance led to vast improvements in mass accuracy, sensitivity, resolution, and speed. Concomitantly, bioinformatic tools were developed to process MS data, integrate sequencing results, and deconvolute multi‐allelic datasets. This enabled the immense advancement of tumour immunopeptidomics. Studying the HLA‐presented peptide repertoire bears high potential for both answering basic scientific questions and translational application. Mapping the tumour HLA ligandome has started to significantly contribute to target identification for the design of peptide‐specific cancer immunotherapies in clinical trials and compassionate need treatments. In contrast to prediction algorithms, rare HLA allotypes and HLA class II can be adequately addressed when choosing MS‐guided target identification platforms. Herein, we review the identification of tumour HLA ligands focusing on sources, methods, bioinformatic data analysis, translational application, and provide an outlook on future developments.
Mass spectrometry (MS) views the pathophysiologically relevant antigenic signature of human malignancies in an unbiased and comprehensive way. This article reviews sources and biochemical methods to isolate tumour human leukocyte antigen (HLA) ligands, state‐of‐the art bioinformatic analysis of MS data, and HLA ligand categories, such as mutated, cryptic, and post‐translationally modified peptides. The review provides an overview of strategies for MS‐guided target definition, clinical application in personalised cancer immunotherapies, and an outlook on future directions in the field of tumour immunopeptidomics.
Radiation-induced bystander effects (RIBE) are discussed as relevant processes during radiotherapy. Irradiated cells are suggested to release growth-inhibitory/DNA-damaging factors transported to ...non-irradiated cells. However, the molecular nature of this phenomenon has not yet been resolved. We aimed at identifying the growth-inhibitory factor(s) transmitted to non-irradiated cells.
RIBE-competent PC3 cells were used to produce conditioned medium (CM) after exposure to ionizing radiation. Indicator cells were incubated with CM and clonogenic survival as well as cell proliferation were determined as endpoints. A549 indicator cells exhibited a bystander effect upon incubation with CM from irradiated PC3 cells. This bystander effect was not due to DNA-damaging factors, but a radiation-triggered reduction of mitogenic/clonogenic activity present in CM.
Several tumor cells, but not normal fibroblasts secrete this factor, whose release is reduced by irradiation. We identified L-Plastin to be responsible for the mitogenic/clonogenic activity. Removal of L-Plastin from CM by immunoprecipitation or siRNA-mediated knockdown of L-Plastin expression resulted in loss or reduction of mitogenic/clonogenic activity transmitted via CM, respectively. Exosome-transported L-Plastin was constitutively Ser5-phosphorylated, indicative of its bioactive conformation.
In summary, we observed production and exosomal secretion of L-Plastin by cancer cells. Via exosome-transmitted L-Plastin, tumors induce clonogenic and mitogenic activity in cancer and normal cells of the tumor microenvironment. Irradiation inhibits L-Plastin production targeting both cancer cells and the tumor niche and may explain the high impact of radiotherapy in tumor control.
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•Bystander cells have reduced clonogenic survival, but no increase in DNA damage.•L-Plastin exerts strong clonogenic activity in an autocrine and paracrine manner.•Different malignant cells, but not normal fibroblasts secrete clonogenic L-Plastin.•Reduced exosomal L-Plastin is responsible for the observed bystander effect.•Exosomal L-Plastin is constitutively phosphorylated at Ser5.
Although mutated HLA ligands are considered ideal cancer-specific immunotherapy targets, evidence for their presentation is lacking in hepatocellular carcinomas (HCCs). Employing a unique multi-omics ...approach comprising a neoepitope identification pipeline, we assessed exome-derived mutations naturally presented as HLA class I ligands in HCCs.
In-depth multi-omics analyses included whole exome and transcriptome sequencing to define individual patient-specific search spaces of neoepitope candidates. Evidence for the natural presentation of mutated HLA ligands was investigated through an in silico pipeline integrating proteome and HLA ligandome profiling data.
The approach was successfully validated in a state-of-the-art dataset from malignant melanoma, and despite multi-omics evidence for somatic mutations, mutated naturally presented HLA ligands remained elusive in HCCs. An analysis of extensive cancer datasets confirmed fundamental differences of tumor mutational burden in HCC and malignant melanoma, challenging the notion that exome-derived mutations contribute relevantly to the expectable neoepitope pool in malignancies with only few mutations.
This study suggests that exome-derived mutated HLA ligands appear to be rarely presented in HCCs, inter alia resulting from a low mutational burden as compared to other malignancies such as malignant melanoma. Our results therefore demand widening the target scope for personalized immunotherapy beyond this limited range of mutated neoepitopes, particularly for malignancies with similar or lower mutational burden.
Glioblastoma is the most frequent malignant primary brain tumor. In a hierarchical tumor model, glioblastoma stem-like cells (GSC) play a major role in tumor initiation and maintenance as well as in ...therapy resistance and recurrence. Thus, targeting this cellular subset may be key to effective immunotherapy. Here, we present a mass spectrometry-based analysis of HLA-presented peptidomes of GSC and glioblastoma patient specimens. Based on the analysis of patient samples (
n
= 9) and GSC (
n
= 3), we performed comparative HLA peptidome profiling against a dataset of normal human tissues. Using this immunopeptidome-centric approach we could clearly delineate a subset of naturally presented, GSC-associated HLA ligands, which might serve as highly specific targets for T cell-based immunotherapy. In total, we identified 17 antigens represented by 41 different HLA ligands showing natural and exclusive presentation both on GSC and patient samples. Importantly, in vitro immunogenicity and antigen-specific target cell killing assays suggest these peptides to be epitopes of functional CD8+ T cell responses, thus rendering them prime candidates for antigen-specific immunotherapy of glioblastoma.
Immune checkpoint inhibitors (ICIs) have improved the survival of patients with non-small cell lung cancer (NSCLC) by reinvigorating tumor-specific T cell responses. However, the specificity of such ...T cells and the human leukocyte antigen (HLA)-associated epitopes recognized, remain elusive. In this study, we identified NSCLC T cell epitopes of recently described NSCLC-associated antigens, termed keratinocyte differentiation antigens. Epitopes of these antigens were presented by HLA-A 03:01 and HLA-C 04:01 and were associated with responses to ICI therapy. Patients with CD8
+
T cell responses to these epitopes had improved overall and progression-free survival. T cells specific for such epitopes could eliminate HLA class I-matched NSCLC cells ex vivo and were enriched in patient lung tumors. The identification of novel lung cancer HLA-associated epitopes that correlate with improved ICI-dependent treatment outcomes suggests that keratinocyte-specific proteins are important tumor-associated antigens in NSCLC. These findings improve our understanding of the mechanisms of ICI therapy and may help support the development of vaccination strategies to improve ICI-based treatment of these tumors.
Abstract
We provide a comprehensive analysis of the antigenic landscape of glioblastoma using a multi-omics approach including ligandome mapping of the Human Leukocyte Antigen (HLA) ligandome, next ...generation sequencing (NGS) as well as an in-depth characterization of tumor-infiltrating lymphocytes (TIL) using mass cytometry and ultra-deep sequencing of the T-cell receptor (TCR). Tumor-exclusive HLA class I and class II ligands (immune precipitation and LC-MS/MS) of 24 isocitrate dehydrogenase 1 wild type glioblastoma samples and 10 autologous primary glioblastoma cell lines were defined in comparison to an HLA ligandome normal tissue reference database (n > 418). We found 11,496 glioblastoma exclusive HLA class I ligands (2,064 shared with cell lines; 3,754 on ≥ 2 glioblastoma samples). On the source protein level, 239 glioblastoma exclusive proteins were identified; among them 54 were also found in cell lines. For HLA class II ligands the analysis revealed 11,870 glioblastoma exclusive peptides (444 shared with cell lines; 3,420 on ≥ 2 glioblastoma samples) and 278 glioblastoma exclusive proteins; among which 18 were present also in cell lines. Moreover, whole-exome sequencing and whole RNA sequencing of 13 tumor samples was performed with the aim to predict neoantigens. On average 5,662 somatic missense effects were identified per patient (min: 4,258; max: 7,479). Candidate peptides are grouped into (i) in silico predicted neoepitopes, (ii) tumor-exclusivity on HLA, (iii) gene expression (e.g. cancer testis antigens). Top-ranking candidates from each group will be tested with regards to their immunogenicity in an autologous setting (TIL, peripheral blood mononuclear cells, patient derived tumor cells). Finally, the peptide and immunogenicity data is correlated with the immune phenotype of the TIL compartment as well as the TCR repertoire of the sample.
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