Hypoxia promotes tumor immune evasion by suppressing MHC-I expression and antigen presentation.

Hypoxia is a common feature of solid tumors that has previously been linked to resistance to radiotherapy and chemotherapy, and more recently to immunotherapy. In particular, hypoxic tumors exclude T cells and inhibit their activity, suggesting that tumor cells acquire a mechanism to evade T-cell recognition and killing. Our analysis of hypoxic tumors indicates that hypoxia downregulates the expression of MHC class I and its bound peptides (i.

MARS an improved de novo peptide candidate selection method for non-canonical antigen target discovery in cancer.

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS.

Activation of Human CD8+ T Cells with Nitroso Dapsone-Modified HLA-B*13:01-Binding Peptides.

Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone-modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients.

Shared Clavulanate and Tazobactam Antigenic Determinants Activate T-Cells from Hypersensitive Patients.

β-Lactamase inhibitors such as clavulanic acid and tazobactam were developed to overcome β-lactam antibiotic resistance. Hypersensitivity reactions to these drugs have not been studied in detail, and the antigenic determinants that activate T-cells have not been defined. The objectives of this study were to (i) characterize clavulanate- and tazobactam-responsive T-cells from hypersensitive patients, (ii) explore clavulanate and tazobactam T-cell crossreactivity, and (iii) define the antigenic determinants that contribute to T-cell reactivity.

Ionizing Radiation Drives Key Regulators of Antigen Presentation and a Global Expansion of the Immunopeptidome.

Little is known about the pathways regulating MHC antigen presentation and the identity of treatment-specific T cell antigens induced by ionizing radiation. For this reason, we investigated the radiation-specific changes in the colorectal tumor cell proteome. We found an increase in DDX58 and ZBP1 protein expression, two nucleic acid sensing molecules likely involved in induction of the dominant interferon response signature observed after genotoxic insult.

The Choice of Search Engine Affects Sequencing Depth and HLA Class I Allele-Specific Peptide Repertoires.

Standardization of immunopeptidomics experiments across laboratories is a pressing issue within the field, and currently a variety of different methods for sample preparation and data analysis tools are applied. Here, we compared different software packages to interrogate immunopeptidomics datasets and found that Peaks reproducibly reports substantially more peptide sequences (~30-70%) compared with Maxquant, Comet, and MS-GF+ at a global false discovery rate (FDR) of <1%. We noted that these differences are driven by search space and spectral ranking.

HLA Class-II‒Restricted CD8 T Cells Contribute to the Promiscuous Immune Response in Dapsone-Hypersensitive Patients.

HLA-B∗13:01 is associated with dapsone (DDS)-induced hypersensitivity, and it has been shown that CD4+ and CD8+ T cells are activated by DDS and its nitroso metabolite (nitroso dapsone [DDS-NO]). However, there is a need to define the importance of the HLA association in the disease pathogenesis. Thus, DDS- and DDS-NO‒specific CD8+ T-cell clones (TCCs) were generated from hypersensitive patients expressing HLA-B∗13:01 and were assessed for phenotype and function, HLA allele restriction, and killing of target cells.

CDDO-imidazolide Targets Multiple Amino Acid Residues on the Nrf2 Adaptor, Keap1.

Synthetic triterpenoids including CDDO, its methyl ester (CDDO-Me, bardoxolone methyl), and its imidazolide (CDDO-Im) enhance Nrf2-mediated antioxidant and anti-inflammatory activity in many diseases by reacting with thiols on the adaptor protein, Keap1. Unlike monofunctional CDDO-Me, the bifunctional analog, CDDO-Im, has a second reactive site (imidazolide) and can covalently bind to amino acids other than cysteine on target proteins such as glutathione S-transferase pi (GSTP), serum albumin, or Keap1. Here we show for the first time that bifunctional CDDO-Im (in contrast to CDDO-Me), as low as 50 nM, can covalently transacylate arginine and serine residues in GSTP and cross-link them to adjacent cysteine residues.

HLA DRB1*15:01-DQB1*06:02-Restricted Human CD4+ T Cells Are Selectively Activated With Amoxicillin-Peptide Adducts.

Amoxicillin-clavulanate is the most common cause of idiosyncratic drug-induced liver injury (DILI). Drug-specific CD4+ T cells have been detected in patients with DILI, suggestive of an immune etiology. Furthermore, genetic associations including the human leucocyte antigen (HLA) DRB1*15:01-DQB1*06:02 haplotype influence susceptibility.

Identification of Flucloxacillin-Haptenated HLA-B*57:01 Ligands: Evidence of Antigen Processing and Presentation.

Flucloxacillin is a β-lactam antibiotic associated with a high incidence of drug-induced liver reactions. Although expression of human leukocyte antigen (HLA)-B*57:01 increases susceptibility, little is known of the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the modification of peptides that are presented by the risk allele.

Characterization of amoxicillin and clavulanic acid specific T-cell clones from patients with immediate drug hypersensitivity.

Background: Betalactam (BL) antibiotics are the most common cause of drug hypersensitivity. Amoxicillin (AX), which is often prescribed alongside clavulanic acid (Clav), is the most common elicitor. The aim of this study was to determine whether AX and Clav-responsive T-cells are detectable in patients with immediate hypersensitivity to AX-Clav, to assess whether these T-cells display the same specificity as that detected in skin and provocation testing, and to explore T-cell activation pathways.

Immune dysregulation increases the incidence of delayed-type drug hypersensitivity reactions.

Delayed-type, T cell-mediated, drug hypersensitivity reactions are a serious unwanted manifestation of drug exposure that develops in a small percentage of the human population. Drugs and drug metabolites are known to interact directly and indirectly (through irreversible protein binding and processing to the derived adducts) with HLA proteins that present the drug-peptide complex to T cells. Multiple forms of drug hypersensitivity are strongly linked to expression of a single HLA allele, and there is increasing evidence that drugs and peptides interact selectively with the protein encoded by the HLA allele.

Definition of Haptens Derived from Sulfamethoxazole: In Vitro and in Vivo.

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides.

Exosomal Transport of Hepatocyte-Derived Drug-Modified Proteins to the Immune System.

Idiosyncratic drug-induced liver injury (DILI) is a rare, often difficult-to-predict adverse reaction with complex pathomechanisms. However, it is now evident that certain forms of DILI are immune-mediated and may involve the activation of drug-specific T cells. Exosomes are cell-derived vesicles that carry RNA, lipids, and protein cargo from their cell of origin to distant cells, and they may play a role in immune activation.

Characterization of Healthy Donor-Derived T-Cell Responses Specific to Telaprevir Diastereomers.

Telaprevir, a protease inhibitor, was used alongside PEGylated interferon-α and ribavirin to treat hepatitis C viral infections. The triple regimen proved successful; however, the appearance of severe skin reactions alongside competition from newer drugs restricted its use. Skin reactions presented with a delayed onset indicative of a T-cell mediated reaction.

HLA-A*33:03-Restricted Activation of Ticlopidine-Specific T-Cells from Human Donors.

The HLA class I allele HLA-A*33:03 is a risk factor for ticlopidine-induced liver injury. Herein, we show HLA class I-restricted ticlopidine-specific CD8+ T-cell activation in healthy donors expressing HLA-A*33:03. Cloned CD8+ T-cells proliferated and secreted IFN-γ in the presence of ticlopidine and autologous antigen presenting cells.

Dapsone and Nitroso Dapsone Activation of Naı̈ve T-Cells from Healthy Donors.

Dapsone (DDS) causes hypersensitivity reactions in 0.5-3.6% of patients.

Mass Spectrometric and Functional Aspects of Drug-Protein Conjugation.

The covalent binding of drugs (metabolites) to proteins to form drug-protein adducts can have an adverse effect on the body. These adducts are thought to be responsible for idiosyncratic drug reactions including severe drug hypersensitivity reactions. Major advances in proteomics technology have allowed for the identification and quantification of target proteins for certain drugs.

Detection of Primary T Cell Responses to Drugs and Chemicals in HLA-Typed Volunteers: Implications for the Prediction of Drug Immunogenicity.

A number of serious adverse drug reactions are caused by T cells. An association with HLA alleles has been identified with certain reactions, which makes it difficult to develop standardized preclinical tests to predict chemical liability. We have recently developed a T cell priming assay using the drug metabolite nitroso sulfamethoxazole (SMX-NO).

Amoxicillin and Clavulanate Form Chemically and Immunologically Distinct Multiple Haptenic Structures in Patients.

Amoxicillin-clavulanate (AC) is one of the most common causes of drug induced liver injury (DILI). The association between AC-DILI and HLA alleles and the detection of drug-specific T cells in patients with AC-DILI indicate that the adaptive immune system is involved in the disease pathogenesis. In this study, mass spectrometric methods were employed to characterize the antigen formed by AC in exposed patients and the antigenic determinants that stimulate T cells.

Characterization of amoxicillin- and clavulanic acid-specific T cells in patients with amoxicillin-clavulanate-induced liver injury.

Unlabelled: Drug-induced liver injury (DILI) frequently has a delayed onset with several human leukocyte antigen (HLA) genotypes affecting susceptibility, indicating a potential role for the adaptive immune system in the disease. The aim of this study was to investigate whether drug-responsive T lymphocytes are detectable in patients who developed DILI with the combination, antimicrobial amoxicillin-clavulanate. Lymphocytes from 6 of 7 patients were found to proliferate and/or secrete interferon-gamma (IFN-γ) when cultured with amoxicillin and/or clavulanic acid.