Psoriasis is an inflammatory skin disease with strong neutrophil (PMN) infiltration and high levels of the antimicrobial peptide, LL37. LL37 in complex with DNA and RNA is thought to initiate disease exacerbation via plasmacytoid dendritic cells. However, the source of nucleic acids supposed to start this initial inflammatory event remains unknown. We show here that primary murine and human PMNs mount a fulminant and self-propagating neutrophil extracellular trap (NET) and cytokine response, but independently of the canonical NET component, DNA. Unexpectedly, RNA, which is abundant in NETs and psoriatic but not healthy skin, in complex with LL37 triggered TLR8/TLR13-mediated cytokine and NET release by PMNs in vitro and in vivo. Transfer of NETs to naive human PMNs prompts additional NET release, promoting further inflammation. Our study thus uncovers a self-propagating vicious cycle contributing to chronic inflammation in psoriasis, and NET-associated RNA (naRNA) as a physiologically relevant NET component.

Background: The 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. Methods: Human 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. Results: The 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. Conclusion: Given 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 .

Immune-checkpoint inhibition provides an unmatched level of durable clinical efficacy in various malignancies. Such therapies promote the activation of antigen-specific T cells, although the precise targets of these T cells remain unknown. Exploiting these targets holds great potential to amplify responses to treatment, such as by combining immune-checkpoint inhibition with therapeutic vaccination or other antigen-directed treatments. In this scenario, the pivotal hurdle remains the definition of valid HLA-restricted tumour antigens, which requires several levels of evidence before targets can be established with sufficient confidence. Suitable antigens might include tumour-specific antigens with alternative or wild-type sequences, tumour-associated antigens and cryptic antigens that exceed exome boundaries. Comprehensive antigen classification is required to enable future clinical development and the definition of innovative treatment strategies. Furthermore, clinical development remains challenging with regard to drug manufacturing and regulation, as well as treatment feasibility. Despite these challenges, treatments based on diligently curated antigens combined with a suitable therapeutic platform have the potential to enable optimal antitumour efficacy in patients, either as monotherapies or in combination with other established immunotherapies. In this Review, we summarize the current state-of-the-art approaches for the identification of candidate tumour antigens and provide a structured terminology based on their underlying characteristics.

Significance: Despite the revolution in cancer therapy initiated by checkpoint inhibitors, durable clinical responses remain sporadic in many types of cancer, including ovarian cancer. Understanding which antigens are essentially presented by tumor cells and further able to be recognized by T cells provides a major step toward novel effective targeted immunotherapies. In this study, we comprehensively analyzed the immunopeptidomic landscape of ovarian carcinoma and compared it to variety of benign sources to identify antigens exclusively presented on tumor cells. With personalized therapies moving into the focus of clinical cancer therapy, we further present insights on how gene-expression analysis and immunohistochemistry can support antigen selection for individualized immunotherapy.

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.

Peritoneal carcinomatosis is a frequent finding in gastric cancer associated with a poor prognosis. The features that enable gastric tumors to disseminate are poorly understood until now. Previously, we showed elevated mRNA levels of phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate‐generating enzyme in the glycolytic pathway, the chemokine receptor 4 (CXCR4), the corresponding chemokine ligand 12 (CXCL12) and β‐catenin in specimens from gastric cancer patients with peritoneal carcinomatosis. In this study, the influence of PGK1 on CXCR4 and β‐catenin was assessed as well as the invasiveness of PGK1 overexpressing cancer cells. In this current study, we found that PGK1 regulates the expression of CXCR4 and β‐catenin at the mRNA and protein levels. On the other hand, CXCR4 regulates the expression of PGK1. Plasmid‐mediated overexpression of PGK1 dramatically increased the invasiveness of gastric cancer cells. Interestingly, inhibition of CXCR4 in cells overexpressing PGK1 produced only a moderate reduction of invasiveness suggesting that, PGK1 itself has a critical role in tumor invasiveness. Immunohistochemistry in specimens from diffuse gastric cancer patients also revealed an overexpression of PGK1 in patients with development of peritoneal carcinomatosis. Therefore, PGK1 may be a crucial enzyme in peritoneal dissemination. Together these findings suggest that the enhanced expression of PGK1 and its signaling targets CXCR4 and β‐catenin in gastric cancer cells promote peritoneal carcinomatosis. Thus, PGK1 may serve as prognostic marker and/or be a potential therapeutic target to prevent dissemination of gastric carcinoma cells into the peritoneum.