RESUMEN
Human leukocyte antigen (HLA) is a hallmark genetic marker for the prediction of certain immune-mediated adverse drug reactions (ADRs). Numerous basic and clinical research studies have provided the evidence base to push forward the clinical implementation of HLA testing for the prevention of such ADRs in susceptible patients. This review explores current translational progress in using HLA as a key susceptibility factor for immune ADRs and highlights gaps in our knowledge. Furthermore, relevant findings of HLA-mediated drug-specific T cell activation are covered, focusing on cellular approaches to link genetic associations to drug-HLA binding as a complementary approach to understand disease pathogenesis.
Asunto(s)
Hipersensibilidad a las Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Alelos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Antígenos HLA/genética , Humanos , FarmacogenéticaRESUMEN
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. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01-restricted nitroso dapsone-modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone-modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone-modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele-restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.
Asunto(s)
Dapsona , Hipersensibilidad a las Drogas , Humanos , Cisteína , Linfocitos T CD8-positivos , Antígenos HLA-B , Péptidos , HaptenosRESUMEN
BACKGROUND: Vancomycin, a glycopeptide antibiotic used for Gram-positive bacterial infections, has been linked with drug reaction with eosinophilia and systemic symptoms (DRESS) in HLA-A*32:01-expressing individuals. This is associated with activation of T lymphocytes, for which glycolysis has been isolated as a fuel pathway following antigenic stimulation. However, the metabolic processes that underpin drug-reactive T-cell activation are currently undefined and may shed light on the energetic conditions needed for the elicitation of drug hypersensitivity or tolerogenic pathways. Here, we sought to characterise the immunological and metabolic pathways involved in drug-specific T-cell activation within the context of DRESS pathogenesis using vancomycin as model compound and drug-reactive T-cell clones (TCCs) generated from healthy donors and vancomycin-hypersensitive patients. METHODS: CD4+ and CD8+ vancomycin-responsive TCCs were generated by serial dilution. The Seahorse XFe96 Analyzer was used to measure the extracellular acidification rate (ECAR) as an indicator of glycolytic function. Additionally, T-cell proliferation and cytokine release (IFN-γ) assay were utilised to correlate the bioenergetic characteristics of T-cell activation with in vitro assays. RESULTS: Model T-cell stimulants induced non-specific T-cell activation, characterised by immediate augmentation of ECAR and rate of ATP production (JATPglyc). There was a dose-dependent and drug-specific glycolytic shift when vancomycin-reactive TCCs were exposed to the drug. Vancomycin-reactive TCCs did not exhibit T-cell cross-reactivity with structurally similar compounds within proliferative and cytokine readouts. However, cross-reactivity was observed when analysing energetic responses; TCCs with prior specificity for vancomycin were also found to exhibit glycolytic switching after exposure to teicoplanin. Glycolytic activation of TCC was HLA restricted, as exposure to HLA blockade attenuated the glycolytic induction. CONCLUSION: These studies describe the glycolytic shift of CD4+ and CD8+ T cells following vancomycin exposure. Since similar glycolytic switching is observed with teicoplanin, which did not activate T cells, it is possible the master switch for T-cell activation is located upstream of metabolic signalling.
Asunto(s)
Teicoplanina , Vancomicina , Humanos , Vancomicina/efectos adversos , Linfocitos T CD8-positivos , Activación de Linfocitos , Citocinas , GlucólisisRESUMEN
BACKGROUND: Exposure to nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen (IBU) and naproxen (NAP) is associated with idiosyncratic drug-induced liver injury (DILI). Carboxylate bioactivation into reactive metabolites (e.g., acyl glucuronides, AG) and resulting T-cell activation is hypothesized as causal for this adverse event. However, conclusive evidence supporting this is lacking. METHODS: In this work, we identify CD4+ and CD8+ T-cell hepatic infiltration in a biopsy from an IBU DILI patient. Lymphocyte transformation test and IFN-γ ELIspot, conducted on peripheral blood mononuclear cells (PBMCs) of patients with NAP-DILI, were used to explore drug-specific T-cell activation. T-cell clones (TCC) were generated and tested for drug specificity, phenotype/function, and pathways of T-cell activation. Cells were exposed to NAP, its oxidative metabolite 6-O-desmethyl NAP (DM-NAP), its AG or synthesized NAP-AG human-serum albumin adducts (NAP-AG adduct). RESULTS: CD4+ and CD8+ T-cells from patients expressing a range of different Vß receptors were stimulated to proliferate and secrete IFN-γ and IL-22 when exposed to DM-NAP, but not NAP, NAP-AG or the NAP-AG adduct. Activation of the CD4+ TCC was HLA-DQ-restricted and dependent on antigen presenting cells (APC); most TCC were activated with DM-NAP-pulsed APC, while fixation of APC blocked the T-cell response. Cross-reactivity was not observed with structurally-related drugs. CONCLUSION: Our results confirm hepatic T-cell infiltrations in NSAID-induced DILI, and show a T-cell memory response toward DM-NAP indicating an immune-mediated basis for the adverse event. Whilst bioactivation at the carboxylate group is widely hypothesized to be pathogenic for NSAID associated DILI, we found no evidence of this with NAP.
Asunto(s)
Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Naproxeno , Humanos , Naproxeno/efectos adversos , Naproxeno/metabolismo , Glucurónidos/metabolismo , Linfocitos T CD8-positivos , Leucocitos Mononucleares/metabolismo , Antiinflamatorios no Esteroideos , Ibuprofeno , Estrés Oxidativo , Activación de LinfocitosRESUMEN
With the rapid expansion in the development and clinical utility of immune checkpoint inhibitors (ICIs) for oncology, the continual evaluation of the safety profile of such agents is imperative. The safety profile of ICIs as monotherapy is dominated by immune-related adverse events, which can be considered as an extension of the mechanism of action of these immunomodulatory drugs. Further to this, an emerging theme is that ICI treatment can significantly impact upon the tolerability of coadministered medications. Numerous reports in literature indicate that ICIs may alter the immunological perception of coadministered drugs, resulting in undesirable reactions to a variety of concomitant medications. These reactions can be severe in manifestation, including hepatotoxicity and Stevens-Johnson Syndrome (SJS)/toxic epidermal necrolysis (TEN), but may also have detrimental impact on malignancy control. To minimize the impact of such drug-drug interactions on patients, it is imperative to identify medications that may cause these reactions, understand the underlying mechanisms, consider the timing and dosing of comedication, and explore alternative medications with comparable efficacies. Improving our understanding of how concomitant medications affect the safety and efficacy of ICIs can allow for potential culprit drugs to be identified/removed/desensitized. This approach will allow the continuation of ICI therapy that may have been discontinued otherwise, thereby improving malignant control and patient and drug development outcomes.
Asunto(s)
Interacciones Farmacológicas , Inhibidores de Puntos de Control Inmunológico , Humanos , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunologíaRESUMEN
Epigallocatechin-3-O-gallate (EGCG) is the major component of green tea extract, commonly found in dietary supplements, and has been associated with immune-mediated liver injury. The purpose of this study was to investigate the immunogenicity of EGCG in healthy donors expressing HLA-B*35:01, and characterize EGCG responsive T-cell clones. We have shown that EGCG can prime peripheral blood mononuclear cells and T-cells from donors with and without the HLA-B*35:01 allele. T-cell clones were CD4+ve and capable of secreting Th1, Th2, and cytolytic molecules. These data demonstrate that EGCG can activate T-cells in vitro, suggesting a significant role in the pathogenesis of green tea extract induced liver injury.
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Catequina , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Humanos , Leucocitos Mononucleares , Antioxidantes , Té , Antígenos HLA-B/genética , Extractos Vegetales/farmacología , Catequina/farmacologíaRESUMEN
Carbamazepine (CBZ) is an aromatic anticonvulsant known to cause drug hypersensitivity reactions, which range in severity from relatively mild maculopapular exanthema to potentially fatal Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS-TEN). These reactions are known to be associated with human leukocyte antigen (HLA) class I alleles, and CBZ interacts preferentially with the related HLA proteins to activate CD8+ T-cells. This study aimed to evaluate the contribution of HLA class II in the effector mechanism(s) of CBZ hypersensitivity. CBZ-specific T-cells clones were generated from two healthy donors and two hypersensitive patients with high-risk HLA class I markers. Phenotype, function, HLA allele restriction, response pathways, and cross-reactivity of CBZ-specific T-cells were assessed using flow cytometry, proliferation analysis, enzyme-linked immunosorbent spot, and enzyme-linked immunosorbent assay. The association between HLA class II allele restriction and CBZ hypersensitivity was reviewed using Allele Frequency Net Database. Forty-four polyclonal CD4+ CBZ-specific T-cell clones were generated and found to be restricted to HLA-DR, particularly HLA-DRB1*07:01. This CD4+-mediated response proceeded through a direct pharmacological interaction between CBZ and HLA-DR molecules. Similar to the CD8+ response, CBZ-stimulated CD4+ clones secreted granulysin, a key mediator of SJS-TEN. Our database review revealed an association between HLA-DRB1*07:01 and CBZ-induced SJS-TEN. These findings implicate HLA class II antigen presentation as an additional pathogenic factor for CBZ hypersensitivity reactions. Both HLA class II molecules and drug-responsive CD4+ T-cells should be evaluated further to gain better insights into the pathogenesis of drug hypersensitivity reactions.
Asunto(s)
Hipersensibilidad a las Drogas , Síndrome de Stevens-Johnson , Humanos , Linfocitos T CD8-positivos , Cadenas HLA-DRB1/genética , Carbamazepina/efectos adversos , Anticonvulsivantes/efectos adversos , Hipersensibilidad a las Drogas/genética , Antígenos HLA , Síndrome de Stevens-Johnson/genética , Linfocitos T CD4-Positivos , Antígenos HLA-BRESUMEN
Epidermal growth factor receptor inhibitors (EGFRIs) are widely used to treat various types of malignancies. One of the common adverse reactions is cutaneous toxicity, mostly presenting as acneiform eruptions, paronychia and xerosis. Erosive pustular dermatosis of the scalp (EPDS) is a rare cutaneous adverse reaction that develops during treatment with EGFRIs. The pathogenesis of EGFRI-induced EPDS is poorly understood. Here we present three cases of EPDS induced by EGFRIs. The proteins LTA4H (leukotriene A-4 hydrolase), METAP1 (methionine aminopeptidase 1), BID (BH3-interacting domain death agonist), SMAD1 (mothers against decapentaplegic homologue), PRKRA (interferon-inducible double-stranded RNA-dependent protein kinase activator A), YES1 (tyrosine-protein kinase Yes) and EGFL7 (epidermal growth factor-like protein 7) were significantly upregulated in EGFRI-stimulated peripheral blood mononuclear cell cultures, and validated in the lesions. All of the proteins colocalized with CD4+ and CD8+ T-cell expression. Next-generation-based human leucocyte antigen (HLA) typing showed all patients carried HLA-C*15:02, and modelling studies showed that afatinib and erlotinib bound well within the E/F binding pockets of HLA-C*15:02. Moreover, T cells were preferentially activated by EGFRIs in individuals carrying HLA-C*15:02. The case series revealed that EGFRI-induced EPDS may be mediated by drug-specific T cells.
Asunto(s)
Exantema , Enfermedades de la Piel , Humanos , Cuero Cabelludo , Antígenos HLA-C , Leucocitos Mononucleares/metabolismo , Receptores ErbB , Aminopeptidasas/metabolismo , Proteínas de Unión al Calcio , Familia de Proteínas EGF/metabolismoRESUMEN
Immune-mediated type IV adverse drug reactions are idiosyncratic in nature, generally not related to the primary or secondary pharmacology of the drug. Due to their complex nature and rarity, these iatrogenic reactions are seldom predicted or encountered during preclinical/early clinical development stages, and often precipitate upon exposure to wider populations (i.e. phase III onwards). They confer a burden on the healthcare sector in both a clinical and financial sense presenting a severe impediment to the drug discovery and development process. Research over the past 50 years has improved our understanding of these reactions markedly as both in vitro and in vivo studies have placed the role of the immune system, in particular; drug-responsive T cells, firmly in the spotlight as the mediators of these reactions. Indeed, the role of different populations of T cells in adverse events and the interaction of drug molecules with HLA proteins expressed on the surface of antigen-presenting cells is of considerable interest. Herein, this review examines the pathways of immune-mediated adverse events including the various T cell subtypes implicated and the mechanisms of T cell activation. Additionally, we address the enigma of immunological tolerance and explore the role tolerance plays in determination of susceptibility to such adverse events even in individuals carrying immunogenic liabilities.
Asunto(s)
Hipersensibilidad a las Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Humanos , Hipersensibilidad a las Drogas/diagnóstico , Tolerancia Inmunológica , Linfocitos T , Activación de LinfocitosRESUMEN
Teicoplanin is a glycopeptide antibiotic deployed to combat Gram-positive bacterial infection and has recently been associated with development of adverse drug reactions, particularly following previous exposure to vancomycin. In this study, we generated teicoplanin-specific monoclonal T-cell populations from healthy volunteers expressing HLA-A*32:01 and defined pathways of T-cell activation and HLA allele restriction. Teicoplanin-responsive T-cells were CD8+, HLA class I-restricted, and cross-reacted with the lipoglycopeptide daptomycin in proliferation and cytokine/cytolytic molecule (granzyme B, Perforin, and FasL) release assays. These data show that teicoplanin activates T-cells, which may play a role in the pathogenesis of teicoplanin-induced adverse events, in HLA-A*32:01 positive donors.
Asunto(s)
Antibacterianos/farmacología , Antígenos HLA-A/biosíntesis , Linfocitos T/efectos de los fármacos , Teicoplanina/farmacología , Antibacterianos/química , Voluntarios Sanos , Humanos , Linfocitos T/metabolismo , Teicoplanina/químicaRESUMEN
Drugs can activate different cells of the immune system and initiate an immune response that can lead to life-threatening diseases collectively known as severe cutaneous adverse reactions (SCARs). Antibiotics, anticonvulsants, and antiretrovirals are involved in the development of SCARs by the activation of αß naïve T-cells. However, other subsets of lymphocytes known as nonconventional T-cells with a limited T-cell receptor repertoire and innate and adaptative functions also recognize drugs and drug-like molecules, but their role in the pathogenesis of SCARs has only just begun to be explored. Despite 30 years of advances in our understanding of the mechanisms in which drugs interact with T-cells and the pathways for tissue injury seen during T-cell activation, at present, the development of useful clinical biomarkers for SCARs or predictive preclinical in vitro assays that could identify immunogenic moieties during drug discovery is an unmet goal. Therefore, the present review focuses on (i) advances in the understanding of the pathogenesis of SCARs reactions, (ii) a description of the interaction of drugs with conventional and nonconventional T-cells, and (iii) the current state of soluble blood circulating biomarker candidates for SCARs.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Linfocitos T , Anticonvulsivantes , Biomarcadores/metabolismo , Cicatriz/complicaciones , Cicatriz/tratamiento farmacológico , Cicatriz/patología , Humanos , Piel/metabolismo , Síndrome de Stevens-Johnson/tratamiento farmacológico , Síndrome de Stevens-Johnson/etiología , Síndrome de Stevens-Johnson/patologíaRESUMEN
Use of the atypical antipsychotic clozapine is associated with life-threatening agranulocytosis. The delayed onset and the association with HLA variants are characteristic of an immunological mechanism. The objective of this study was to generate clozapine-specific T cell clones (TCC) and characterize pathways of T cell activation and cross-reactivity with clozapine metabolites and olanzapine. TCC were established and characterized by culturing PBMCs from healthy donors and patients with a history of clozapine-induced agranulocytosis. Modeling was used to explore the drug-HLA binding interaction. Global TCC protein changes were profiled by mass spectrometry. Six well-growing clozapine-responsive CD4+ and CD8+ TCC were used for experiments; activation of TCC required APC, with clozapine interacting directly at therapeutic concentrations with several HLA-DR molecules. TCC were also activated with N-desmethylclozapine and olanzapine at supratherapeutic concentrations. Marked changes in TCC protein expression profiles were observed when clozapine treatment was compared with olanzapine and the medium control. Docking of the compounds into the HLA-DRB1*15:01 and HLA-DRB1*04:01 binding clefts revealed that clozapine and olanzapine bind in a similar conformation to the P4-P6 peptide binding pockets, whereas clozapine N-oxide, which did not activate the TCC, bound in a different conformation. TCC secreted Th1, Th2, and Th22 cytokines and effector molecules and expressed TCR Vß 5.1, 16, 20, and 22 as well as chemokine receptors CXCR3, CCR6, CCR4, and CCR9. Collectively, these data show that clozapine interacts at therapeutic concentrations with HLA-DR molecules and activates human CD4+ T cells. Olanzapine only activates TCC at supratherapeutic concentrations.
Asunto(s)
Clozapina/inmunología , Linfocitos T/inmunología , Adulto , Células Clonales/inmunología , Clozapina/análogos & derivados , Reacciones Cruzadas/inmunología , Citocinas/inmunología , Femenino , Antígenos HLA-DR/inmunología , Humanos , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana EdadRESUMEN
Drug haptens are formed from the irreversible, covalent binding of drugs to nucleophilic moieties on proteins, which can warrant adverse reactions in the body including severe delayed-type, T-cell mediated, drug hypersensitivity reactions (DHRs). While three main pathways exist for the activation of T-cells in DHRs, namely the hapten model, the pharmacological interaction model and the altered peptide repertoire model, the exact antigenic determinants responsible have not yet been defined. In recent years, progress has been made using advanced mass spectrometry-based proteomic methods to identify protein carriers and characterise the structure of drug-haptenated proteins. Since genome-wide association studies discovered a link between human leukocyte antigens (HLA) and an individual's susceptibility to DHRs, much effort has been made to define the drug-associated HLA ligands driving T-cell activation, including the elution of natural HLA peptides from HLA molecules and the generation of HLA-binding peptides. In this review, we discuss our current methodology used to design and synthesise drug-modified HLA ligands to investigate their immunogenicity using T-cell models, and thus their implication in drug hypersensitivity.
Asunto(s)
Preparaciones Farmacéuticas , Proteómica , Estudio de Asociación del Genoma Completo , Haptenos , Humanos , Linfocitos TRESUMEN
BACKGROUND: Atabecestat is an orally administered BACE inhibitor developed to treat Alzheimer's disease. Elevations in hepatic enzymes were detected in a number of in trial patients, which resulted in termination of the drug development programme. Immunohistochemical characterization of liver tissue from an index case of atabecestat-mediated liver injury revealed an infiltration of T-lymphocytes in areas of hepatocellular damage. This coupled with the fact that liver injury had a delayed onset suggests that the adaptive immune system may be involved in the pathogenesis. The aim of this study was to generate and characterize atabecestat(metabolite)-responsive T-cell clones from patients with liver injury. METHODS: Peripheral blood mononuclear cells were cultured with atabecestat and its metabolites (diaminothiazine [DIAT], N-acetyl DIAT & epoxide) and cloning was attempted in a number of patients. Atabecestat(metabolite)-responsive clones were analysed in terms of T-cell phenotype, function, pathways of T-cell activation and cross-reactivity with structurally related compounds. RESULTS: CD4+ T-cell clones activated with the DIAT metabolite were detected in 5 out of 8 patients (up to 4.5% cloning efficiency). Lower numbers of CD4+ and CD8+ clones displayed reactivity against atabecestat. Clones proliferated and secreted IFN-γ, IL-13 and cytolytic molecules following atabecestat or DIAT stimulation. Certain atabecestat and DIAT-responsive clones cross-reacted with N-acetyl DIAT; however, no cross-reactivity was observed between atabecestat and DIAT. CD4+ clones were activated through a direct, reversible compound-HLA class II interaction with no requirement for protein processing. CONCLUSION: The detection of atabecestat metabolite-responsive T-cell clones activated via a pharmacological interactions pathway in patients with liver injury is indicative of an immune-based mechanism for the observed hepatic enzyme elevations.
Asunto(s)
Preparaciones Farmacéuticas , Linfocitos T , Linfocitos T CD4-Positivos , Células Clonales , Humanos , Leucocitos Mononucleares , Hígado , Activación de Linfocitos , Piridinas , TiazinasRESUMEN
Cutaneous drug-induced reactions are immune-mediated responses that can lead to life-threatening diseases such as drug reaction with eosinophilia and systemic symptoms (DRESS), Stevens-Johnson syndrome, and toxic epidermal necrolysis, collectively known as severe cutaneous adverse reactions (SCARs). Unfortunately, they cannot be predicted during drug development, and, at present, a prognostic biomarker is not available nor are validated in vitro assays for diagnosis. Thus, by using proteomic and microarray miRNA analysis, the cargo of extracellular vesicles obtained from SCARs patients was analyzed and correlated with the severity of the reaction. Confirmatory assays using Western blot and qRT-PCR were performed to validate findings, and bioinformatic tools were used to establish the correlation between protein and miRNAs expression between groups. The proteomic analysis showed an increase in the amount of pro-inflammatory proteins, von Willebrand factor, and C-reactive protein and a decrease in anti-inflammatory and protective proteins in the SCARs group compared with the control group. Additionally, histone protein H2A was enriched in DRESS patients. APO1 and SERPINA4 proteins, highly increased in the control group but absent in the SCARs group, are the target of several overexpressed miRNAs, suggesting that the regulation of these proteins might involve gene silencing and protein repressing mechanisms in the severe patients. According with previous reports showing its presence in plasma and T-cells, microRNA miR-18 was upregulated in extracellular vesicles obtained from the most severe patients. Determination of the unique cargo associated with different disease conditions will help to understand the pathophysiology of these complex reactions and might help to develop novel biomarkers for life-threatening iatrogenic cutaneous disease.
Asunto(s)
Erupciones por Medicamentos/genética , Vesículas Extracelulares/genética , MicroARNs/genética , Erupciones por Medicamentos/diagnóstico , Vesículas Extracelulares/química , Vesículas Extracelulares/patología , Humanos , Proteoma/análisis , Proteoma/genética , Proteómica , TranscriptomaRESUMEN
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. 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 and 100 nm and expressed enriched CD63. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) identified 2,109 proteins, with 608 proteins being quantified across all exosome samples. Data are available through 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-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 through phagocytosis, and was inhibited by latrunculin A. An amoxicillin-modified 9-mer peptide derived from the exosomal transcription factor protein SRY (sex determining region Y)-box 30 activated naïve T cells from human leukocyte antigen 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 provide a pathway for the induction of drug hapten-specific T-cell responses.
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Células Dendríticas/metabolismo , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Hepatocitos/efectos de los fármacos , Sistema Inmunológico/metabolismo , Transporte de Proteínas , Células Cultivadas , Hepatocitos/ultraestructura , HumanosRESUMEN
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. METHODS: Drug-specific T-cell clones were generated from immediate hypersensitive patients´ blood by serial dilution and repetitive mitogen stimulation. Antigen specificity was assessed by measurement of proliferation and cytokine release. CD4+ /CD8+ phenotype and chemokine receptor expression were analyzed by flow cytometry. RESULTS: 110 AX-specific and 96 Clav-specific T-cell clones were generated from seven patients with positive skin test to either AX or Clav. Proliferation of AX- and Clav-specific clones was dose-dependent, and no cross-reactivity was observed. AX- and Clav-specific clones required antigen-presenting cells to proliferate, and drugs were presented to CD4+ and CD8+ T-cells by MHC class-II and I, respectively. A higher secretion of IL-13 and IL-5 was detected in presence of the culprit drug compared with the alternative drug. Clones expressed CD69, CCR4, CXCR3, and CCR10. CONCLUSIONS: Our study details the antigen specificity and phenotype of T-cell clones generated from patients with AX-Clav-induced immediate hypersensitivity diagnosed by positive skin test. AX- and Clav-specific clones were generated from patients irrespective of whether AX or Clav was the culprit, although differences in cytokine secretion were observed.
Asunto(s)
Hipersensibilidad a las Drogas , Hipersensibilidad Inmediata , Amoxicilina/efectos adversos , Linfocitos T CD8-positivos , Ácido Clavulánico/efectos adversos , Células Clonales , Hipersensibilidad a las Drogas/diagnóstico , Humanos , Hipersensibilidad Inmediata/diagnósticoRESUMEN
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. Despite this, many individuals expressing HLA risk alleles do not develop hypersensitivity when exposed to culprit drugs suggesting a nonlinear, multifactorial relationship in which HLA risk alleles are one factor. This has prompted a search for additional susceptibility factors. Herein, we argue that immune regulatory pathways are one key determinant of susceptibility. As expression and activity of these pathways are influenced by disease, environmental and patient factors, it is currently impossible to predict whether drug exposure will result in a health benefit, hypersensitivity or both. Thus, a concerted effort is required to investigate how immune dysregulation influences susceptibility towards drug hypersensitivity.
Asunto(s)
Hipersensibilidad a las Drogas , Hipersensibilidad Tardía , Alelos , Hipersensibilidad a las Drogas/epidemiología , Hipersensibilidad a las Drogas/genética , Humanos , Hipersensibilidad Tardía/inducido químicamente , Hipersensibilidad Tardía/epidemiología , Incidencia , Linfocitos TRESUMEN
BACKGROUND: Abacavir is associated with hypersensitivity reactions in individuals positive for the HLA-B*57:01 allele. The drug binds within the peptide binding groove of HLA-B*57:01 altering peptides displayed on the cell surface. Presentation of these HLA-abacavir-peptide complexes to T-cells is hypothesized to trigger a CD8+ T-cell response underpinning the hypersensitivity. Thus, the aim of this study was to explore the relationship between the structure of abacavir with HLA-B*57:01 binding and the CD8+ T-cell activation. METHODS: Seventeen abacavir analogues were synthesized and cytokine secretion from abacavir/abacavir analogue-responsive CD8+ T-cell clones was measured using IFN-γ ELIspot. In silico docking studies were undertaken to assess the predicted binding poses of the abacavir analogues within the HLA-B*57:01 peptide binding groove. In parallel, the effect of selected abacavir analogues on the repertoire of self-peptides presented by cellular HLA-B*57:01 was characterized using mass spectrometry. RESULTS: Abacavir and ten analogues stimulated CD8+ T-cell IFN-γ release. Molecular docking of analogues that retained antiviral activity demonstrated a relationship between predicted HLA-B*57:01 binding orientations and the ability to induce a T-cell response. Analogues that stimulated T-cells displayed a perturbation of the natural peptides displayed by HLA-B*57:01. The antigen-specific CD8+ T-cell response was dependent on the enantiomeric form of abacavir at both cyclopropyl and cyclopentyl regions. CONCLUSION: Alteration of the chemical constitution of abacavir generates analogues that retain a degree of pharmacological activity, but have variable ability to activate T-cells. Modelling and immunopeptidome analysis delineate how drug HLA-B*57:01 binding and peptide display by antigen presenting cells relate to the activation of CD8+ T-cells.
Asunto(s)
Linfocitos T CD8-positivos , Hipersensibilidad a las Drogas , Didesoxinucleósidos , Antígenos HLA-B/genética , Humanos , Simulación del Acoplamiento Molecular , Relación Estructura-ActividadRESUMEN
Drug hypersensitivity reactions adversely affect treatment outcome, increase the length of patients' hospitalization, and limit the prescription options available to physicians. In addition, late stage drug attrition and the withdrawal of licensed drugs cost the pharmaceutical industry billions of dollars. This significantly increases the overall cost of drug development and by extension the price of licensed drugs. Drug hypersensitivity reactions are characterized by a delayed onset, and reactions tend to be more serious upon re-exposure. The role of drug-specific T-cells in the pathogenesis of drug hypersensitivity reactions and definition of the nature of the binding interaction of drugs with HLA and T-cell receptors continues to be the focus of intensive research, primarily because susceptibility is associated with expression of one or a small number of HLA alleles. This review critically examines the mechanisms of T-cell activation by drugs. Specific examples of drugs that activate T-cells via the hapten, the pharmacological interaction with immune receptors and the altered self-peptide repertoire pathways, are discussed. Furthermore, the impacts of drug metabolism, drug-protein adduct formation, and immune regulation on the development of drug antigen-responsive T-cells are highlighted. The knowledge gained from understanding the pathways of T-cell activation and susceptibility factors for drug hypersensitivity will provide the building blocks for the development of predictive in vitro assays that will prevent or help to minimize the incidence of these reactions in clinic.