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关键词： Antigen presentation   Biotherapeutic development   Immunogenicity   MAPPs   MHC II   MHC-associated peptide proteomics   Risk assessment   T cell epitope   Unwanted immunogenicity  

摘要： Biotherapeutics can provoke unwanted immunogenicity, which can affect the safety and/or efficacy of the drug. Consequently, immunogenicity risk assessments are carried out during drug development. Further, immunogenicity is evaluated during clinical trials prior to licensure. Most therapeutic proteins are either purified from plasma or, increasingly, manufactured using recombinant DNA technology. The immune response to these protein therapies involves internalization of the protein into antigen-presenting cells (APCs), enzymatic processing of the protein into peptides, and presentation of peptides on major histocompatibility class II (MHC II) proteins (HLA class II in humans). The MHC II-peptide complex if recognized by T cell receptors (TCRs) engages with CD4+ T helper cells, which ultimately results in formation of anti-drug antibodies (ADAs). The MHC-associated peptide proteomics (MAPPs) is an assay that in principle can identify the peptides derived from the therapeutic protein that are associated with an individual subject's MHC variants. The MAPPs assay is the only assay that provides information about both protein processing in APCs and peptide presentation by MHC proteins. This is important because not all possible peptides from a protein can be generated during protein processing in APCs. Here we describe a highly robust protocol for MHC II MAPPs.

关键词： Glycosylation   Hydrophilic interaction chromatography   Immunotherapy   LC-MS/MS   MHC peptides   Phosphorylation   Titanium dioxide  

摘要： Glycosylation and phosphorylation are two of the most important forms of post-translational modification. Their dysregulation is related to tumorigenesis with phosphorylating enzymes, known as kinases, having been shown to active cancerous signaling pathways, and aberrant glycosylation has been detected in various types of cancers. For immunotherapy using neoantigens, both glycosylated and phosphorylated peptides presented exclusively on tumor cells by the major histocompatibility complex (MHC) are promising candidates due to their specificity to elicit cytotoxic T-cell responses. Unlike other MHC-presented peptides, glycosylated and phosphorylated MHC peptides cannot be predicted by bioinformatic tools, and their identification relies on specific enrichment from the whole immunopeptidome and sensitive detection with mass spectrometry. Herein, we describe a workflow that sequentially enriches glycosylated and phosphorylated MHC peptides from the immunopeptidome with hydrophilic interaction chromatography and titanium dioxide nanoparticles, followed by analysis of enriched peptides by liquid chromatography tandem mass spectrometry and identification of modified MHC peptides by database searching.

关键词： Bioinformatics   Cancer   HLA-binding affinity   Immune microenvironment   Immunomics   MHC   Mice   Neoantigen prediction  

摘要： Neoantigens are mutated peptides arising from tumor genomic alterations, which can be recognized and attacked by the immune system, leading to antitumor immune responses. In the last decades, many immunotherapeutic strategies have been developed, which has increased the interest in neoantigens. This led to the development of computational tools that facilitate neoantigen identification and prioritization, prior to their validation using experimental approaches. This chapter aims at explaining the key steps that need to be conducted to identify potential neoantigens in silico, including an example of the most frequently used tools. This is followed by a description and comparison of the cutting-edge tools and pipelines for neoantigen prediction both for human and mouse. The last aim of this chapter is to depict the technical challenges that limit neoantigen prediction using bioinformatics, as well as the expected improvements, given the current revolution of artificial intelligence, which is implemented in most of the neoantigen-related tools. As exposed in this book chapter, we believe that advances in immunomics and computational biology will be key to implement personalized cancer immunotherapy in the clinical practice, to improve outcomes of cancer patients.

关键词： Glycosylation   Human leucocyte antigen   LC-MS/MS   Major histocompatibility complex  

摘要： Killing of cancer cells by cytotoxic Tcells requires efficient presentation of tumor antigens by human leukocyte antigen (HLA) molecules and patient-specific HLA genotypes also influence response to immune checkpoint inhibitors. Genotyping for a specific HLA allele can be used as biomarker to predict the immunotherapy outcome of a patient. Traditionally, HLA genotyping is achieved by sequencing the genome or transcriptome extracted from whole cells. Unfortunately, this approach does not reflect the actual abundance of each expressed HLA allele that presents antigen on the cell surface or provide any information on the post-translational modification of HLA proteins. To tackle this problem, we present an integrated immunopeptidomics and proteomics approach to comprehensively characterize the HLA proteins. HLA proteins are captured from traditional immunopeptidomics analysis and subjected to digestion, glycopeptides enrichment, and mass spectrometric analysis. Database with the mass spectrometric data will provide the relative expression level of each HLA allele and its pattern of glycosylation, which improves the understanding of antigen presentation.

关键词： Psoriasis   biologic therapy   TNF inhibitors   interleukin inhibitors   IL-23  

摘要： IntroductionPsoriasis (PsO) is a common chronic, inflammatory, immune-mediated disease. In 2023, a 4.4% prevalence of PsO was reported in Portugal. Currently, Tumor Necrosis Factor inhibitors (TNFi) are the recommended first-line (1 L) biologic agents in Portugal given their lower cost. However, TNFi may not be suitable for several patients. In these patients, interleukin inhibitors (ILi) should be considered as they provide more effective outcomes and a better safety *** interviews with PsO experts were conducted to identify PsO biologic treatment needs, resulting in an online survey to explore clinical cases focused on subpopulations of PsO. A delphi study evaluated consensus on clinical criteria to initiate non-TNFi therapy in seven predefined subpopulations of *** study highlights the benefit of starting non-TNFi therapy in all PsO predefined subpopulations. Patients with infection risk, mild heart failure and associated comorbidities, autoimmune diseases and family history of demyelinating disease consensually benefit from starting non-TNFi therapy in 1 L. Several risks associated with latent tuberculosis, advanced age and oncological disease were also *** the existence of various risks associated with TNFi usage, this clinical perspective overview of Portuguese experts in PsO treatment emphasizes the need for a tailored therapeutic framework in the management of PsO.

关键词： Cancer   clinical trials   cytokine   High-dose IL-2 (HD IL-2)   IL-2 receptor (IL-2R)   IL-2R agonist   immunotherapy   Interleukin-2 (IL-2)  

摘要： Immune checkpoint blockade (ICB) has significantly improved the survival for many patients with advanced malignancy. However, fewer than 50% of patients benefit from ICB, highlighting the need for more effective immunotherapy options. High-dose interleukin-2 (HD IL-2) immunotherapy, which is approved for patients with metastatic melanoma and renal cell carcinoma, stimulates CD8+ T cells and NK cells and can generate durable responses in a subset of patients. Moreover, HD IL-2 may have potential efficacy in patients whose disease has progressed following ICB and plays a vital role in expanding tumor-infiltrating lymphocyte (TIL) in TIL therapy. Despite its potential, the use of HD IL-2 is limited by severe toxicities such as hypotension and vascular leak syndrome. Additionally, only a few patients achieve a good outcome after HD IL-2 therapy. To address these challenges, numerous next-generation IL-2 receptor (IL-2 R) agonists have been developed to exhibit treatment effects while minimizing adverse events. This review will explore IL-2 biology, the clinical application of HD IL-2 therapy, and the development of novel IL-2 R agonists for cancer immunotherapy.

关键词： Fusobacterium   novel species   pangenome   comparative genomics   intestinal microbiota   colorectal cancer  

摘要： Cancerous tissue is a largely unexplored microbial niche that provides a unique environment for the colonization and growth of specific bacterial communities, and with it, the opportunity to identify novel bacterial species. Here, we report distinct features of a novel Fusobacterium species, F. sphaericum sp. nov. (Fs), isolated from primary colon adenocarcinoma tissue. We acquire the complete closed genome and associated methylome of this organism and phylogenetically confirm its classification into the Fusobacterium genus, with F. perfoetens as its closest neighbor. Fs is phenotypically and genetically distinct, with morphological analysis revealing its coccoid shape, that while similar to F. perfoetens is rare for most Fusobacterium members. Fs displays a metabolic profile and antibiotic resistance repertoire consistent with other Fusobacterium species. In vitro, Fs has adherent and immunomodulatory capabilities, as it intimately associates with human colon cancer epithelial cells and promotes IL-8 secretion. An analysis of the prevalence and abundance of Fs in > 20,000 human metagenomic samples shows that it is a rarely detected member within human stool with variable relative abundance, found in both healthy controls and patients with colorectal cancer (CRC). Our study sheds light on a novel bacterial species isolated directly from the human CRC tumor niche and given its in vitro interaction with cancer epithelial cells suggests that its role in human health and disease warrants further investigation.

关键词： Myocardial ischemia/reperfusion injury   caspase-1   IL-1 beta   pyroptosis   mitochondrial homeostasis   cardiomyocyte injury  

摘要： BackgroundPyroptosis is a novel kind of programmed cell death and Caspase-1 plays key roles in driving pyroptosis. The current study aims to elucidate the molecular mechanism affecting cardiomyocyte pyroptosis in myocardial ischemia/reperfusion (I/R) injury, both in vivo and in *** murine model of myocardial I/R injury was established and then treated with lentivirus-mediated shRNA targeting Caspase-1 to evaluate the effect of Caspase-1 on myocardial I/R injury. Further, Caspase-1 was silenced in the cardiomyocytes following hypoxia-reoxygenation (H/R) to detect the function of Caspase-1 in mitochondrial homeostasis and cardiomyocyte *** of Caspase-1 inhibited the secretion of interleukin-1 beta (IL-1 beta), improved cardiac dysfunction and decreased pyroptosis in vivo. The cardio-protective effect was verified in the H/R-induced cardiomyocyte model. Recombinant IL-1 beta protein reversed the inhibitory effect of Caspase-1 knockdown on ***, activating the Caspase-1/IL-1 beta axis by myocardial I/R injury causes mitochondrial homeostasis imbalance, pyroptosis, and the consequent cardiomyocyte injury.

关键词： italic>Escherichia coliChlamydia   inhibition   lymphocyte   IFN gamma  

摘要： Pathogens frequently produce proteins to evade or inhibit host immune responses. One such protein is lymphostatin from attaching and effacing Escherichia coli (also known as lymphocyte inhibitory factor A;LifA), which influences intestinal colonization and inhibits mitogen- and antigen-activated proliferation of T lymphocytes and pro-inflammatory cytokine synthesis. Here, we report the cloning, purification and characterization of a LifA homologue from Chlamydia pecorum. The predicted 382 KDa protein (CPE2_0552) exhibited 36% identity and 55% similarity over 3171 amino acids to lymphostatin from enteropathogenic E. coli strain E2348/69. CPE2_0552 shares glycosyltransferase and cysteine protease motifs required for lymphostatin activity, including similarity in the tertiary structure of these domains predicted by AlphaFold 3. Purified CPE2_0552 did not share the L-shaped globular structure of lymphostatin when analyzed by transmission electron microscopy. CPE2_0552 inhibited concanavalin A-stimulated proliferation of bovine T cells in a concentration-dependent manner, with an inhibitory dose 50 (ID50) of 812 pg/mL. This was 38-fold higher than the ID50 of E. coli E2348/69 lymphostatin tested in parallel on T cells from the same donors (21 pg/mL), but was similar to another LifA homologue from E. coli O157:H7 (ToxB). Moreover, CPE2_0552 inhibited the secretion of interferon gamma (IFN gamma), a key cytokine that influences the outcome of Chlamydia infections. At the concentrations at which CPE2_0552 inhibited T lymphocyte proliferation and IFN gamma secretion, negligible cytotoxicity was observed after 72 h of stimulation. Our study indicates that E. coli lymphostatin belongs to a wider family of lymphocyte-inhibitory molecules that exist in distantly related bacterial pathogens.