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Aberrantly-active signal transducer and activator of transcription (Stat)3 has a causal role in many human cancers and represents a validated anticancer drug target, though it has posed significant challenge to drug development. A new small molecule, JKB887, was identified through virtual library screening and is predicted to interact with Lys591, Arg609 and Pro63 in the phospho-tyrosine (pTyr)-binding pocket of the Stat3 SH2 domain. JKB887 inhibited Stat3 DNA-binding activity in vitro in a time-dependent manner, with IC50 of 2.2-4.5 µM at 30-60-min incubation. It directly disrupted both the Stat3 binding to the cognate, high-affinity pTyr (pY) peptide, GpYLPQTV-NH2 in fluorescent polarization assay with IC50 of 3.5-5.5 µM at 60-90-min incubation, and to the IL-6 receptor/gp130 or Src in treated malignant cells. Treatment with JKB887 selectively blocked constitutive Stat3 phosphorylation, nuclear translocation and transcriptional activity, Stat3-regulated gene expression, and decreased viable cell numbers, cell growth, colony formation, migration, and survival in human or mouse tumor cells. By contrast, JKB887 had minimal effects on Stat1 activity, pErk1/2MAPK, pShc, pJAK2, pSrc induction, or cells that do not harbor aberrantly-active Stat3. Additionally, JKB887 inhibited growth of human breast cancer xenografts in mice. JKB887 is a Stat3-selective inhibitor with demonstrable antitumor effects against Stat3-dependent human cancers.
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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.
Assuntos
Clozapina/imunologia , Linfócitos T/imunologia , Adulto , Células Clonais/imunologia , Clozapina/análogos & derivados , Reações Cruzadas/imunologia , Citocinas/imunologia , Feminino , Antígenos HLA-DR/imunologia , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Células Dendríticas/metabolismo , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Hepatócitos/efeitos dos fármacos , Sistema Imunitário/metabolismo , Transporte Proteico , Células Cultivadas , Hepatócitos/ultraestrutura , HumanosRESUMO
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.
Assuntos
Hipersensibilidade a Drogas , Hipersensibilidade Imediata , Amoxicilina/efeitos adversos , Linfócitos T CD8-Positivos , Ácido Clavulânico/efeitos adversos , Células Clonais , Hipersensibilidade a Drogas/diagnóstico , Humanos , Hipersensibilidade Imediata/diagnósticoRESUMO
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.
Assuntos
Hipersensibilidade a Drogas , Hipersensibilidade Tardia , Alelos , Hipersensibilidade a Drogas/epidemiologia , Hipersensibilidade a Drogas/genética , Humanos , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/epidemiologia , Incidência , Linfócitos TRESUMO
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.
Assuntos
Hipersensibilidade a Drogas/imunologia , Linfócitos T/imunologia , Animais , Humanos , Imunomodulação , Peso Molecular , Preparações Farmacêuticas/químicaRESUMO
BACKGROUND: Research into drug hypersensitivity associated with the expression of specific HLA alleles has focussed on the interaction between parent drug and the HLA with no attention given to reactive metabolites. For this reason, we have studied HLA-B*13:01-linked dapsone hypersensitivity to (a) explore whether the parent drug and/or nitroso metabolite activate T cells and (b) determine whether HLA-B*13:01 is involved in the response. METHODS: Peripheral blood mononuclear cells (PBMC) from six patients were cultured with dapsone and nitroso dapsone, and proliferative responses and IFN-γ release were measured. Dapsone- and nitroso dapsone-specific T-cell clones were generated and phenotype, function, HLA allele restriction, and cross-reactivity assessed. Dapsone intermediates were characterized by mass spectrometry. RESULTS: Peripheral blood mononuclear cells from six patients and cloned T cells proliferated and secreted Th1/2/22 cytokines when stimulated with dapsone (clones: n = 395; 80% CD4+ CXCR3hi CCR4hi , 20% CD8+CXCR3hi CCR4hi CCR6hi CCR9hi CCR10hi ) and nitroso dapsone (clones: n = 399; 78% CD4+, 22% CD8+ with same chemokine receptor profile). CD4+ and CD8+ clones were HLA class II and class I restricted, respectively, and displayed three patterns of reactivity: compound specific, weakly cross-reactive, and strongly cross-reactive. Nitroso dapsone formed dimers in culture and was reduced to dapsone, providing a rationale for the cross-reactivity. T-cell responses to nitroso dapsone were dependent on the formation of a cysteine-modified protein adduct, while dapsone interacted in a labile manner with antigen-presenting cells. CD8+ clones displayed an HLA-B*13:01-restricted pattern of activation. CONCLUSION: These studies describe the phenotype and function of dapsone- and nitroso dapsone-responsive CD4+ and CD8+ T cells from hypersensitive patients. Discovery of HLA-B*13:01-restricted CD8+ T-cell responses indicates that drugs and their reactive metabolites participate in HLA allele-linked forms of hypersensitivity.
Assuntos
Dapsona/farmacologia , Antígenos HLA-B/genética , Hipersensibilidade/etiologia , Ativação Linfocitária/genética , Compostos Nitrosos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adulto , Reações Cruzadas , Feminino , Expressão Gênica , Antígenos HLA-B/imunologia , Humanos , Hipersensibilidade/diagnóstico , Hipersensibilidade/metabolismo , Imunofenotipagem , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
The workshop on "New Approaches to Investigate Drug-Induced Hypersensitivity" was held on June 5, 2014 at the Foresight Center, University of Liverpool. The aims of the workshop were to (1) discuss our current understanding of the genetic, clinical, and chemical basis of small molecule drug hypersensitivity, (2) highlight the current status of assays that might be developed to predict potential drug immunogenicity, and (3) identify the limitations, knowledge gaps, and challenges that limit the use of these assays and utilize the knowledge gained from the workshop to develop a pathway to establish new and improved assays that better predict drug-induced hypersensitivity reactions during the early stages of drug development. This perspective reviews the clinical and immunological bases of drug hypersensitivity and summarizes various experts' views on the different topics covered during the meeting.
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Hipersensibilidade a Drogas , Animais , Bioensaio , Hipersensibilidade a Drogas/genética , Hipersensibilidade a Drogas/imunologia , Indústria Farmacêutica , Predisposição Genética para Doença , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Fenótipo , Fatores de RiscoAssuntos
Aminopiridinas/efeitos adversos , Benzodioxóis/efeitos adversos , Linfócitos T CD4-Positivos/imunologia , Fibrose Cística/tratamento farmacológico , Hipersensibilidade a Drogas/imunologia , Aminofenóis/uso terapêutico , Aminopiridinas/uso terapêutico , Benzodioxóis/uso terapêutico , Regulador de Condutância Transmembrana em Fibrose Cística , Combinação de Medicamentos , Hipersensibilidade a Drogas/etiologia , Feminino , Humanos , Quinolonas/uso terapêutico , Adulto JovemRESUMO
Although idiosyncratic adverse drug reactions are rare, they are still a major concern to patient safety. Reactive metabolites are widely accepted as playing a pivotal role in the pathogenesis of idiosyncratic adverse drug reactions. While there are today well established strategies for the risk assessment of stable metabolites within the pharmaceutical industry, there is still no consensus on reactive metabolite risk assessment strategies. This is due to the complexity of the mechanisms of these toxicities as well as the difficulty in identifying and quantifying short-lived reactive intermediates such as reactive metabolites. In this review, reactive metabolite risk and hazard assessment approaches are discussed, and their pros and cons highlighted. We also discuss the nature of idiosyncratic adverse drug reactions, using acetaminophen and nefazodone to exemplify the complexity of the underlying mechanisms of reactive metabolite mediated hepatotoxicity. One of the key gaps moving forward is our understanding of and ability to predict the contribution of immune activation in idiosyncratic adverse drug reactions. Sections are included on the clinical phenotypes of immune mediated idiosyncratic adverse drug reactions and on the present understanding of immune activation by reactive metabolites. The advances being made in microphysiological systems have a great potential to transform our ability to risk assess reactive metabolites, and an overview of the key components of these systems is presented. Finally, the potential impact of systems pharmacology approaches in reactive metabolite risk assessments is highlighted.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Preparações Farmacêuticas/metabolismo , Acetaminofen/metabolismo , Acetaminofen/toxicidade , Analgésicos não Narcóticos/metabolismo , Analgésicos não Narcóticos/toxicidade , Animais , Antidepressivos de Segunda Geração/metabolismo , Antidepressivos de Segunda Geração/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Hipersensibilidade a Drogas/diagnóstico , Hipersensibilidade a Drogas/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Piperazinas , Medição de Risco/métodos , Triazóis/metabolismo , Triazóis/toxicidadeRESUMO
Drug hypersensitivity remains a major concern, as it causes high morbidity and mortality. Understanding the mechanistic basis of drug hypersensitivity is complicated by the multiple risk factors implicated. This study utilized sulfamethoxazole (SMX) as a model drug to (1) relate SMX metabolism in antigen presenting cells (APCs) to the activation of T-cells and (2) characterize covalent adducts of SMX and myeloperoxidase, which might represent antigenic determinants for T-cells. The SMX metabolite nitroso-SMX (SMX-NO) was found to bind irreversibly to APCs. Time- and concentration-dependent drug-protein adducts were also detected when APCs were cultured with SMX. Metabolic activation of SMX was significantly reduced by the oxygenase/peroxidase inhibitor methimazole. Similarly, SMX-NO-specific T-cells were activated by APCs pulsed with SMX, and the response was inhibited by pretreatment with methimazole or glutaraldehyde, which blocks antigen processing. Western blotting, real-time polymerase chain reaction (RT-PCR), and mass spectrometry analyses suggested the presence of low concentrations of myeloperoxidase in APCs. RT-PCR revealed mRNA expression for flavin-containing monooxygenases (FMO1-5), thyroid peroxidase, and lactoperoxidase, but the corresponding proteins were not detected. Mass spectrometric characterization of SMX-NO-modified myeloperoxidase revealed the formation of N-hydroxysulfinamide adducts on Cys309 and Cys398. These data show that SMX's metabolism in APCs generates antigenic determinants for T-cells. Peptides derived from SMX-NO-modified myeloperoxidase may represent one form of functional antigen.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Peroxidases/metabolismo , Sulfametoxazol/metabolismo , Linfócitos B/metabolismo , Células HL-60 , Humanos , Oxigenases/metabolismo , Ligação Proteica , Linfócitos T/metabolismoRESUMO
HLA-A*31:01 is associated with carbamazepine (CBZ) hypersensitivity in Caucasian and Japanese populations. Herein, we show that HLA-A*31:01+ restricted the activation of carbamazepine-specific CD8(+) T-cells, which provides an immunological basis for the genetic association. Furthermore, CD4(+) T-cells were activated with carbamazepine in a HLA-DRB1*04:04-restricted manner, indicating that a common HLA haplotype may contribute to the multiclonal T-cell response seen in European patients with CBZ hypersensitivity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Carbamazepina/efeitos adversos , Hipersensibilidade a Drogas/imunologia , Antígenos HLA-A/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Clonais/imunologia , HumanosRESUMO
Pityriasis rubra pilaris (PRP) is an inflammatory papulosquamous dermatosis, characterized by hyperkeratotic follicular papules and erythematous desquamative plaques. The precise pathogenic mechanism underlying PRP remains incompletely understood. Herein, we conduct a case-control study involving a cohort of 102 patients with sporadic PRP and 800 healthy controls of Han Chinese population and identify significant associations (P = 1.73 × 10-6) between PRP and heterozygous mutations in the Keratin 32 gene (KRT32). KRT32 is found to be predominantly localized in basal keratinocytes and exhibits an inhibitory effect on skin inflammation by antagonizing the NF-κB pathway. Mechanistically, KRT32 binds to NEMO, promoting excessive K48-linked polyubiquitination and NEMO degradation, which hinders IKK complex formation. Conversely, loss-of-function mutations in KRT32 among PRP patients result in NF-κB hyperactivation. Importantly, Krt32 knockout mice exhibit a PRP-like dermatitis phenotype, suggesting compromised anti-inflammatory function of keratinocytes in response to external pro-inflammatory stimuli. This study proposes a role for KRT32 in regulating inflammatory immune responses, with damaging variants in KRT32 being an important driver in PRP development. These findings offer insights into the regulation of skin immune homeostasis by keratin and open up the possibility of using KRT32 as a therapeutic target for PRP.
Assuntos
Queratinócitos , Pitiríase Rubra Pilar , Pele , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto Jovem , Estudos de Casos e Controles , Homeostase , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Queratinócitos/imunologia , Queratinócitos/metabolismo , Queratinas/metabolismo , Queratinas/genética , Mutação com Perda de Função , Camundongos Knockout , NF-kappa B/metabolismo , Pitiríase Rubra Pilar/genética , Pitiríase Rubra Pilar/imunologia , Pitiríase Rubra Pilar/patologia , Pitiríase Rubra Pilar/metabolismo , Transdução de Sinais , Pele/patologia , Pele/imunologia , Pele/metabolismo , UbiquitinaçãoRESUMO
In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on the use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications toward the immunogenicity of drugs and/or the likelihood of 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 utilized immune cells from drug-naïve donors, recently established immune cell coculture 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 5 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 coculture; however, CD4+ clones displaying reactivity were detected toward 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.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Hepatócitos , Humanos , Células Cultivadas , Hepatócitos/metabolismo , Técnicas de Cocultura , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Medição de RiscoRESUMO
Cytokine release from dendritic cells in vitro is a useful marker to discriminate between sensitizing and irritant haptenic chemicals. Unfortunately, pro-haptens, which gain reactivity following metabolic/auto activation, yield negative results. To overcome this, we exposed human neutrophils and THP-1 cells to haptens/pro-haptens and measured IL-8 release. Haptenic compounds stimulated IL-8 release in neutrophils and THP-1 cells. In contrast, the pro-haptens eugenol, isoeugenol, and 2-aminophenol stimulated high levels of IL-8 release from neutrophils alone. Neutrophil cytokine release was reduced when glutathione was added. Cyp1A1/1B1/3A4 were not detectable in THP-1 cells or neutrophils; however, neutrophils expressed high levels of myeloperoxidase.
Assuntos
Aminofenóis/imunologia , Eugenol/análogos & derivados , Eugenol/imunologia , Interleucina-8/imunologia , Neutrófilos/imunologia , Linhagem Celular , Células Cultivadas , Haptenos/imunologia , HumanosRESUMO
Drug rash with eosinophilia with systemic symptoms (DRESS) is a serious adverse event associated with use of the glycopeptide antibiotic vancomycin. Vancomycin-induced drug rash with eosinophilia with systemic symptoms is associated with the expression of human leukocyte antigen (HLA)-A*32:01, suggesting that the drug interacts with this HLA to activate CD8+ T cells. The purpose of this study was to utilize peripheral blood mononuclear cell from healthy donors to: (1) investigate whether expression of HLA-A*32:01 is critical for the priming naïve of T cells with vancomycin and (2) generate T-cell clones (TCC) to determine whether vancomycin exclusively activates CD8+ T cells and to define cellular phenotype, pathways of drug presentation and cross-reactivity. Dendritic cells were cultured with naïve T cells and vancomycin for 2 weeks. On day 14, cells were restimulated with vancomycin and T-cell proliferation was assessed by [3H]-thymidine incorporation. Vancomycin-specific TCC were generated by serial dilution and repetitive mitogen stimulation. Naïve T cells from HLA-A*02:01 positive and negative donors were activated with vancomycin; however the strength of the induced response was significantly stronger in donors expressing HLA-A*32:01. Vancomycin-responsive CD4+ and CD8+ TCC from HLA-A*32:01+ donors expressed high levels of CXCR3 and CCR4, and secreted IFN-γ, IL-13, and cytolytic molecules. Activation of CD8+ TCC was HLA class I-restricted and dependent on a direct vancomycin HLA binding interaction with no requirement for processing. Several TCC displayed cross-reactivity with teicoplanin and daptomycin. To conclude, this study provides evidence that vancomycin primes naïve T cells from healthy donors expressing HLA-A*32:01 through a direct pharmacological binding interaction. Cross-reactivity of CD8+ TCC with teicoplanin provides an explanation for the teicoplanin reactions observed in vancomycin hypersensitive patients.
Assuntos
Preparações Farmacêuticas , Vancomicina , Linfócitos T CD8-Positivos , Antígenos HLA-A , Humanos , Interleucina-13 , Leucócitos Mononucleares , Vancomicina/toxicidadeRESUMO
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-NOâspecific 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. CD8+ TCCs were stimulated to proliferate and secrete effector molecules when exposed to DDS and/or DDS-NO. DDS-responsive and several DDS-NOâresponsive TCCs expressing a variety of TCR sequences displayed HLA class-I restriction, with the drug (metabolite) interacting with multiple HLA-B alleles. However, activation of certain DDS-NOâresponsive CD8+ TCCs was inhibited with HLA class-II block, with DDS-NO binding to HLA-DQB1∗05:01. These TCCs were of different origin but expressed TCRs displaying the same amino acid sequences. They were activated through a hapten pathway; displayed CD45RO, CD28, PD-1, and CTLA-4 surface molecules; secreted the same panel of effector molecules as HLA class-Iârestricted TCCs; but displayed a lower capacity to lyse target cells. To conclude, DDS and DDS-NO interact with a number of HLA molecules to activate CD8+ TCCs, with HLA class-IIârestricted CD8+ TCCs that display hybrid CD4âCD8 features also contributing to the promiscuous immune response that develops in patients.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Dapsona/farmacologia , Síndrome de Hipersensibilidade a Medicamentos/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Adulto , Alelos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Citotoxicidade Imunológica , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Adulto JovemRESUMO
The prediction of drug hypersensitivity is difficult due to the lack of appropriate models and known risk factors. In vitro naïve T-cell priming assays that assess immunogenicity have been developed. However, their application is limited due requirements for 2 batches of autologous dendritic cells (DC) and inconsistent results; a consequence of single well readouts when exploring reactions where compound-specific T-cell frequency is undefined. Hence, we aimed to develop an improved, but simplified assay, termed the T-cell multiple well assay (T-MWA), that permits assessment of drug-specific activation of naïve T cells, alongside analysis of the strength of the induced response and the number of cultures that respond. DC naïve T-cell coculture, depleted of regulatory T cells (Tregs), was conducted in up to 48 wells for 2 weeks with model haptens (nitroso sulfamethoxazole [SMX-NO], Bandrowski's base [BB], or piperacillin [PIP]). Cultures were rechallenged with hapten and T-cell proliferation was measured using [3H]-thymidine incorporation. Priming of naïve T cells was observed with SMX-NO, with no requirement for DC during restimulation. Greater than 65% of cultures were activated with SMX-NO; with 8.0%, 30.8%, and 27.2% characterized as weak (stimulation index [SI] =1.5-1.9), moderate (SI = 2-3.9), and strong responses (SI > 4), respectively. The number of responding cultures and strength of the response was reproducible when separate blood donations were compared. Coinhibitory checkpoint blockade increased the strength of the proliferative response, but not the number of responding cultures. Moderate to strong priming responses were detected with BB, whereas PIP stimulated only a small number of cultures to proliferate weakly. In drug-responsive cultures inducible CD4+CD25+FoxP3+CD127low Tregs were also identified. To conclude, the T-MWA offers improvements over existing assays and with development it could be used to study multiple HLA-typed donors in a single plate format.
Assuntos
Células Sanguíneas/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Hipersensibilidade a Drogas/imunologia , Haptenos/toxicidade , Morte Celular Imunogênica/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Testes de Toxicidade/métodos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
It is now apparent that antigen-specific T-cells are activated in certain patients with drug-induced liver injury (DILI). Since cross-talk between hepatocytes and immune cells is likely to be critical in determining the outcome of drug exposure, the aim of this study was to profile the signals released by drug-treated hepatocytes and to characterize the impact of these molecules on dendritic cells. Human hepatocytes were exposed to 3 drugs (flucloxacillin, amoxicillin, and isoniazid) associated with DILI potentially mediated by the adaptive immune system as drug-specific T-cells have been isolated from DILI patients, and the metabolite nitroso-sulfamethoxazole (SMX-NO). Hepatocyte toxicity, cytokine release and activation of oxidative stress pathways were measured. Supernatants were transferred to monocyte-derived dendritic cells and cell phenotype and function were assessed. High-mobility group box 1 protein (HMGB1) and lactate dehydrogenase release as well as adenosine triphosphate depletion occurred in a drug-, time-, and concentration-dependent manner with SMX-NO and flucloxacillin, whereas isoniazid and amoxicillin were nontoxic. Furthermore, drug-induced activation of nuclear factor (erythroid-derived 2)-like 2 marker genes was observed when hepatocytes were exposed to test drugs. The disulfide isoform of HMGB1 stimulated dendritic cell cytokine release and enhanced the priming of naive T-cells. Incubation of dendritic cells with supernatant from drug-treated hepatocytes resulted in 2 distinct cytokine profiles. SMX-NO/flucloxacillin stimulated secretion of TNF-α, IL-6, IL-1α, and IL-1-ß. Isoniazid which did not induce significant hepatocyte toxicity, compared with SMX-NO and flucloxacillin, stimulated the release of a panel of cytokines including the above and IFN-γ, IL-12, IL-17A, IP-10, and IL-10. Collectively, our study identifies drug-specific signaling pathways between hepatocytes and immune cells that could influence whether drug exposure will result in an immune response and tissue injury.