Definition of antigenic determinants in drug hypersensitive patients: an integrated clinical, chemical and cellular approach to quantify and characterize the drug signals presented to T-Lymphocytes

Idiosyncratic drug hypersensitivity remains a major challenge as it causes high morbidity and mortality. This is complicated by the multiple risk factors implicated and the inability to predict these reactions during the early stages of drug development. Thus, this study attempted to delineate the molecular pathomechanism(s) involved in sulfamethoxazole (SMX) hypersensitivity. The reactive metabolite, nitroso-SMX (SMX.NO) generated through the hepatic bioactivation of SMX has long been hypothesised as a major trigger of these reactions. SMX hypersensitivity has been used as a paradigm to study the role of drug metabolism in the activation of T-cells as the synthetic nitroso metabolite is available for functional studies. Metabolism of SMX in hepatic tissue has been extensively studied. CYP2C9 and Myeloperoxidase (MPO) are implicated in the formation of SMX.NO. However, it is unclear whether the SMX.NO generated in the liver migrates to the skin; the primary target in SMX hypersensitivity. It is possible that localised SMX metabolism by immune cells resident in the skin are implicated in the observed reactions. ELISA data revealed SMX metabolism in EBV-transformed B-cells used as antigen presenting cells (APCs). SMX-metabolism was significantly inhibited by methimazole. Furthermore, Western blotting and RT-PCR analyses suggested the presence of low concentrations of MPO in EBV-transformed B-cells. Interestingly, RT-PCR revealed mRNA expression of flavine containing monooxygenases (FMO1-5), TPO and LPO but the protein levels of these enzymes were not detected in immune cells. Subsequent experiments involved the generation and LC-MS/MS characterization of SMX.NO-modified MPO adducts. Although SMX.NO formed both the sulphinamide and N-hydroxysulfinamide adducts, drug specific T-cell clones failed to proliferate in response to drug-modified peptides. Since SMX.NO binds to multiple cellular proteins, it is assumed that peptides derived from the modified protein interact with a number of diverse HLA molecules to activate T-cells. However, the HLA molecules that interact with SMX.NO-modified peptides have not been defined. This study therefore examined the HLA molecules that present SMX.NO (derived peptides) to T-cells. T-cell clones (TCCs) were generated from 5 hypersensitive patients with cystic fibrosis. Fast growing TCCs from 2 SMX hypersensitive patients were used for HLA restriction studies. Drug-specific proliferative response, cytokine secretion and cytolytic markers were measured using [3H]-thymidine incorporation and ELIspot assays. Anti-human class I and class II (DR, DP, and DQ) antibodies were used to determine HLA restriction of drug-specific T-cell activation. APCs expressing similar or different HLAs were used to define the alleles involved in the presentation of SMX.NO-derived antigens to T-cells. A total of 1578 clones were tested for SMX.NO reactivity. Seventy-seven CD4+ clones were activated to proliferate and secrete IFN-ϒ, IL-5, IL-13 and granzyme-B by SMX.NO. Only one TCC was CD8+No cross reactivity with SMX was observed. The SMX.NO-specific response of clones was blocked with antibodies against MHC class II and HLA-DQ. Clones from 2 patients (Patient 1: HLA-DQB1*05:01:01G/ DQB1*06:03:01G; Patient 2: HLA-DQB1*02:01:01G/DQB1*02:01:01G) were used to define the DQ alleles involved in the presentation of SMX.NO derived antigens. SMX.NO-specific responses were detected with heterologous APCs expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. Activation of PD-1 on T-cells is thought to inhibit antigen-specific T-cell priming and regulate T-cell differentiation. Thus, this study sought to measure the drug-specific activation of naïve T-cells after perturbation of PD-L1/PD-1 binding and investigate whether PD-1 signalling influences the differentiation of T-cells. Naive T-cells were co-cultured with monocyte-derived dendritic cells in the presence of SMX.NO for a period of 8 days (±PD-1/2 block) and T-cell priming investigated using readouts for proliferation and cytokine secretion. Priming of naïve T-cells against SMX.NO was found to be more effective when PD-L1 signalling was blocked. Drug-specific TCCs generated through priming and from hypersensitive patients were found to secrete IFN-γ, IL-5 and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL22 secreting clones expressed the skin homing receptors CCR4, CCR10 and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the antigen-specific proliferative response or the secretion of cytokines/cytolytic molecules. In conclusion, this study used a variety of in vitro assays to investigate the multiple factors involved in the pathomechanism of SMX hypersensitivity. A clear understanding of mechanisms of drug hypersensitivity will provide insights that aid drug design and reduce the frequency of such reactions

A blinded in vitro analysis of the intrinsic immunogenicity of hepatotoxic drugs: implications for preclinical risk assessment

In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications towards the immunogenicity of drugs and/or the likelihood for idiosyncratic reactions in the clinic. This is because the molecular initiating event in immune DILI is an interaction of the drug-derived antigen with MHC proteins and the T-cell receptor. This study utilised immune cells from drug-naïve donors, recently established immune cell co-culture systems and blinded compounds with and without DILI liabilities to determine whether these new methods offer an improvement over established assessment methods for the prediction of immune-mediated DILI. Ten blinded test compounds (6 with known DILI liabilities; 4 with lower DILI liabilities) and five training compounds, with known T-cell-mediated immune reactions in patients, were investigated. Naïve T-cells were activated with 4/5 of the training compounds (nitroso sulfamethoxazole, vancomycin, Bandrowski's base and carbamazepine) and clones derived from the priming assays were activated with drug in a dose-dependent manner. The test compounds with DILI liabilities did not stimulate T-cell proliferative responses during dendritic cell-T-cell co-culture; however, CD4+ clones displaying reactivity were detected towards 2 compounds (ciprofloxacin and erythromycin) with known liabilities. Drug-responsive T-cells were not detected with the compounds with lower DILI liabilities. This study provides compelling evidence that assessment of intrinsic drug immunogenicity, although complex, can provide valuable information regarding immune liabilities of some compounds prior to clinical studies or when immune reactions are observed in patients

Detection of Hepatic Drug Metabolite-Specific T-Cell Responses Using a Human Hepatocyte, Immune Cell Coculture System

Drug-responsive T-cells are activated with the parent compound or metabolites, often via different pathways (pharmacological interaction and hapten). An obstacle to the investigation of drug hypersensitivity is the scarcity of reactive metabolites for functional studies and the absence of coculture systems to generate metabolites in situ. Thus, the aim of this study was to utilize dapsone metabolite-responsive T-cells from hypersensitive patients, alongside primary human hepatocytes to drive metabolite formation, and subsequent drug-specific T-cell responses. Nitroso dapsone-responsive T-cell clones were generated from hypersensitive patients and characterized in terms of cross-reactivity and pathways of T-cell activation. Primary human hepatocytes, antigen-presenting cells, and T-cell cocultures were established in various formats with the liver and immune cells separated to avoid cell contact. Cultures were exposed to dapsone, and metabolite formation and T-cell activation were measured by LC-MS and proliferation assessment, respectively. Nitroso dapsone-responsive CD4+ T-cell clones from hypersensitive patients were found to proliferate and secrete cytokines in a dose-dependent manner when exposed to the drug metabolite. Clones were activated with nitroso dapsone-pulsed antigen-presenting cells, while fixation of antigen-presenting cells or omission of antigen-presenting cells from the assay abrogated the nitroso dapsone-specific T-cell response. Importantly, clones displayed no cross-reactivity with the parent drug. Nitroso dapsone glutathione conjugates were detected in the supernatant of hepatocyte immune cell cocultures, indicating that hepatocyte-derived metabolites are formed and transferred to the immune cell compartment. Similarly, nitroso dapsone-responsive clones were stimulated to proliferate with dapsone, when hepatocytes were added to the coculture system. Collectively, our study demonstrates the use of hepatocyte immune cell coculture systems to detect in situ metabolite formation and metabolite-specific T-cell responses. Similar systems should be used in future diagnostic and predictive assays to detect metabolite-specific T-cell responses when synthetic metabolites are not available

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 may play a role in immune activation. Herein, primary human hepatocytes were treated with drugs associated with a high incidence of DILI (flucloxacillin, amoxicillin, isoniazid and nitroso-sulfamethoxazole) to characterize the proteins packaged within exosomes that are subsequently transported to dendritic cells for processing. Exosomes measured between 50-100 nm and expressed enriched CD63. LC-MS/MS identified 2109 proteins, with 608 proteins being quantified across all exosome samples. Data are available via ProteomeXchange with identifier PXD010760. Analysis of gene ontologies revealed that exosomes mirrored whole human liver tissue in terms of the families of proteins present, regardless of drug treatment. However, exosomes from nitroso-sulfamethoxazole-treated hepatocytes selectively packaged a specific subset of proteins. LC-MS also revealed the presence of hepatocyte-derived exosomal proteins covalently modified with amoxicillin, flucloxacillin and nitroso-sulfamethoxazole. Uptake of exosomes by monocyte-derived dendritic cells occurred silently, mainly via phagocytosis, and was inhibited by latrunculin A. An, amoxicillin-modified 9-mer peptide derived from the exosomal transcription factor protein SOX30 activated naïve T-cells from HLA-A*02:01 positive human donors. Conclusion. This study shows that exosomes have the potential to transmit drug-specific hepatocyte-derived signals to the immune system and provides a pathway for the induction of drug hapten-specific T-cell responses. This article is protected by copyright. All rights reserved

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations

Investigation of SARS-CoV-2 faecal shedding in the community: a prospective household cohort study (COVID-LIV) in the UK

Background SARS-CoV-2 is frequently shed in the stool of patients hospitalised with COVID-19. The extent of faecal shedding of SARS-CoV-2 among individuals in the community, and its potential to contribute to spread of disease, is unknown. Methods In this prospective, observational cohort study among households in Liverpool, UK, participants underwent weekly nasal/throat swabbing to detect SARS-CoV-2 virus, over a 12-week period from enrolment starting July 2020. Participants that tested positive for SARS-CoV-2 were asked to provide a stool sample three and 14 days later. In addition, in October and November 2020, during a period of high community transmission, stool sampling was undertaken to determine the prevalence of SARS-CoV-2 faecal shedding among all study participants. SARS-CoV-2 RNA was detected using Real-Time PCR. Results A total of 434 participants from 176 households were enrolled. Eighteen participants (4.2%: 95% confidence interval [CI] 2.5–6.5%) tested positive for SARS-CoV-2 virus on nasal/throat swabs and of these, 3/17 (18%: 95% CI 4–43%) had SARS-CoV-2 detected in stool. Two of three participants demonstrated ongoing faecal shedding of SARS-CoV-2, without gastrointestinal symptoms, after testing negative for SARS-CoV-2 in respiratory samples. Among 165/434 participants without SARS-CoV-2 infection and who took part in the prevalence study, none had SARS-CoV-2 in stool. There was no demonstrable household transmission of SARS-CoV-2 among households containing a participant with faecal shedding. Conclusions Faecal shedding of SARS-CoV-2 occurred among community participants with confirmed SARS-CoV-2 infection. However, during a period of high community transmission, faecal shedding of SARS-CoV-2 was not detected among participants without SARS-CoV-2 infection. It is unlikely that the faecal-oral route plays a significant role in household and community transmission of SARS-CoV-2