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Identification of drug- and drug-metabolite immune responses originating from both naive and memory T cells

2017; Elsevier BV; Volume: 140; Issue: 2 Linguagem: Inglês

10.1016/j.jaci.2016.11.032

1097-6825

Andrew Gibson, Lee Faulkner, Sally Wood, B. Kevin Park, Dean J. Naisbitt,

Mast cells and histamine

T-cell–mediated drug hypersensitivity remains a clinical problem because reactions are difficult to predict and diagnose. This is partly because mechanisms of T-cell activation involving drug-derived antigens have not been fully defined. Here, we focus on sulfamethoxazole (SMX), a sulphonamide antibiotic used for the treatment of opportunistic infections, to investigate the origin of drug and drug-metabolite–specific T cells. T cells isolated from the blood of patients with SMX hypersensitivity are generally CD4+ and secrete TH1 and TH2 cytokines in response to both SMX and the downstream metabolite nitroso sulfamethoxazole (SMX-NO). CD8+ T cells that display cytotoxicity are detected in lower numbers.1Nassif A. Bensussan A. Boumsell L. Deniaud A. Moslehi H. Wolkenstein P. et al.Toxic epidermal necrolysis: effector cells are drug-specific cytotoxic T cells.J Allergy Clin Immunol. 2004; 114: 1209-1215Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 2Castrejon J.L. Berry N. El-Ghaiesh S. Gerber B. Pichler W.J. Park B.K. et al.Stimulation of human T cells with sulfonamides and sulfonamide metabolites.J Allergy Clin Immunol. 2010; 125: 411-418Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar SMX also generates nontoxic acetylated metabolites that have been reported to stimulate a proportion of patient T-cell clones.3Schnyder B. Burkhart C. Schnyder-Frutig K. von Greyerz S. Naisbitt D.J. Pirmohamed M. et al.Recognition of sulfamethoxazole and its reactive metabolites by drug-specific CD4+ T cells from allergic individuals.J Immunol. 2000; 164: 6647-6654Crossref PubMed Google Scholar Although such stimulation likely stems from cross-reactivity to the core sulphonamide structure, acetylated SMX antigens represent a possible source for immune activation during cell culture with SMX. SMX is chemically stable and able to stimulate T cells via (1) a processing-independent direct MHC-TCR interaction and (2) a processing-dependent pathway involving metabolism in antigen presenting cells (APCs). Meanwhile, SMX-NO binds directly to the cysteine residues of proteins to generate a number of potentially immune-activating neo-antigens.4Callan H.E. Jenkins R.E. Maggs J.L. Lavergne S.N. Clarke S.E. Naisbitt D.J. et al.Multiple adduction reactions of nitroso sulfamethoxazole with cysteinyl residues of peptides and proteins: implications for hapten formation.Chem Res Toxicol. 2009; 22: 937-948Crossref PubMed Scopus (75) Google Scholar Animal models poorly reflect the human response as administration of SMX-NO to rats results in the formation of anti–SMX-hapten IgG antibodies and SMX-NO–specific T cells, whereas no such effect was identified for SMX.5Gill H.J. Hough S.J. Naisbitt D.J. Maggs J.L. Kitteringham N.R. Pirmohamed M. et al.The relationship between the disposition and immunogenicity of sulfamethoxazole in the rat.J Pharmacol Exp Ther. 1997; 282: 795-801PubMed Google Scholar Previous studies using PBMCs or whole T-cell populations from healthy human donors have shown that SMX-NO activates T cells from all individuals, whereas SMX stimulates T cells in approximately 30% of individuals.6Engler O.B. Strasser I. Naisbitt D.J. Cerny A. Pichler W.J. A chemically inert drug can stimulate T cells in vitro by their T cell receptor in non-sensitised individuals.Toxicology. 2004; 197: 47-56Crossref PubMed Scopus (47) Google Scholar In contrast, both drug and metabolite are able to promote the maturation of healthy donor-derived dendritic cells (DCs), a critical step in the efficient presentation of drug-antigens on APCs to naive T cells.7Sanderson J.P. Naisbitt D.J. Farrell J. Ashby C.A. Tucker M.J. Rieder M.J. et al.Sulfamethoxazole and its metabolite nitroso sulfamethoxazole stimulate dendritic cell costimulatory signaling.J Immunol. 2007; 178: 5533-5542Crossref PubMed Scopus (105) Google Scholar Despite intensive research, the origin of drug and drug-metabolite–responsive T cells has not been defined. Moreover, it is not known whether previously identified SMX-NO–responsive T cells in healthy donors originate from the naive or memory T-cell compartment. Determining the presence of memory T cells in drug-naive donors that respond to SMX-derived antigens is crucial for understanding whether hypersensitivity is partially mediated by heterologous immunity, where drug-antigens cross-react with T cells initially primed to non–SMX-derived peptides. Thus, we sought to investigate the activation of naive and memory T cells from healthy donors to both SMX and SMX-NO. Furthermore, both SMX- and SMX-NO–specific T cells were cloned to assess cross-reactivity to SMX-NO and SMX, respectively. We used an established in vitro T-cell priming assay8Faulkner L. Martinsson K. Santoyo-Castelazo A. Cederbrant K. Schuppe-Koistinen I. Powell H. et al.The development of in vitro culture methods to characterize primary T-cell responses to drugs.Toxicol Sci. 2012; 127: 150-158Crossref PubMed Scopus (53) Google Scholar (study design and results summarized in Fig E1 in this article's Online Repository at www.jacionline.org) where PBMCs were isolated from peripheral blood, before further isolation of CD14+ cells and naive and memory T cells by magnetic bead sorting. T cells were then combined with autologous, fully matured monocyte-derived DCs, and drug or metabolite for 1 week. T cells were harvested and cultured with a fresh batch of mature DCs and tested for antigen-specificity using [3H] thymidine incorporation and ELISpot to analyze proliferation and cytokine secretion, respectively. IL-13 was used because previous studies using drug-specific T cells from hypersensitive patients have shown that the cytokine is secreted in high levels. T-cell cloning was performed to generate SMX and SMX-NO–responsive clones and to explore cross-reactivity, cytokine release, and MHC restriction. For details, see this article's Methods section in the Online Repository at www.jacionline.org. Both SMX-NO–primed naive T cells and SMX-NO–exposed memory T cells proliferated when restimulated with SMX-NO in all drug-naive donors (n = 11; Fig 1, A). In contrast, the priming of naive T cells or the culture of memory T cells with SMX failed to induce an antigen-specific proliferative response in all 11 donors (Fig 1, B). However, on analysis of IL-13 secretion, both SMX-NO– and SMX-responsive T cells were detected in 2 of 3 donors (Fig 1, C and D, respectively). From one donor, we obtained SMX-responsive cells derived from both the naive and memory T-cell cultures (Fig 1, D), whereas another donor showed a marginal SMX-specific response after naive T-cell priming. Both SMX and SMX-NO–primed cultures did not cross-react with the alternative antigen (Fig E1). Although it is difficult to determine the nature of the APC used during hypersensitivity reactions in patients, it is likely that DCs are responsible for naive T-cell priming because they are the only APC capable of doing so in vitro (see Fig E2 in this article's Online Repository at www.jacionline.org). SMX-NO– and SMX-specific T-cell clones were generated from both the naive and memory compartments of SMX-responsive donors (Fig 2, A). Both SMX- and SMX-NO–responsive CD4+ and CD8+ clones generated from naive T-cell priming were found to be highly specific and did not cross-react with SMX-NO or SMX, respectively (Fig 2, B and C), mirroring the majority of T-cell clones isolated from hypersensitive patients (see Fig E3 in this article's Online Repository at www.jacionline.org). More than 95% of SMX and SMX-NO–responsive clones from the memory T-cell compartment were CD4+, possibly because most memory CD8+ T cells are tissue resident. Three of 5 SMX-responsive T-cell clones from the memory compartment proliferated in the presence of SMX-NO, though these responses were much weaker than in the presence of SMX. Furthermore, 1 SMX-NO–responsive memory T-cell clone responded strongly to both SMX and SMX-NO. SMX and SMX-NO–specific T-cell clones secreted a similar panel of cytokines including IFN-γ, IL-13, and IL-5. Interestingly, the cytolytic molecules granzyme B and perforin were also released (Fig 2, D and E), thus reflecting the predominant cytotoxic phenotype of T cells isolated from the blister fluid of SMX-hypersensitive patients. The secretion of IL-17 was not detected from either drug- or metabolite-responsive clones. As expected, CD8+ and CD4+ SMX-responsive clones were MHC class I–restricted and class II–restricted, respectively (see Fig E4 in this article's Online Repository at www.jacionline.org). In summary, we have uncovered the origins of drug-antigen–responsive T cells. We have shown that SMX and SMX-NO activate both naive and memory T cells. Although all donors responded to SMX-NO, SMX-activated naive and memory T cells were detected in only 2 donors. The low levels of cross-reactivity with naive T cells suggests that clones are activated with the parent drug and nitroso metabolite via different mechanisms; however, it is also possible that intracellular metabolism of the parent drug generates novel drug-metabolite antigens. The inability of the thymidine proliferation assay to detect SMX-responsive cells from the initial priming cultures highlights a known lack of sensitivity in comparison to the ELISpot, but also that SMX is a less potent immunogen than SMX-NO in drug-naive donors. It is likely that in individuals with specific but as yet undetermined predisposing genetic factors the frequency of T cells responsive to SMX-derived antigens is enhanced, rising above a threshold for overwhelming immune activation and hypersensitivity. Similar to patients, SMX-responsive cells derived from drug-naive donors were a mixture of CD4+ and CD8+ T cells.1Nassif A. Bensussan A. Boumsell L. Deniaud A. Moslehi H. Wolkenstein P. et al.Toxic epidermal necrolysis: effector cells are drug-specific cytotoxic T cells.J Allergy Clin Immunol. 2004; 114: 1209-1215Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 2Castrejon J.L. Berry N. El-Ghaiesh S. Gerber B. Pichler W.J. Park B.K. et al.Stimulation of human T cells with sulfonamides and sulfonamide metabolites.J Allergy Clin Immunol. 2010; 125: 411-418Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 3Schnyder B. Burkhart C. Schnyder-Frutig K. von Greyerz S. Naisbitt D.J. Pirmohamed M. et al.Recognition of sulfamethoxazole and its reactive metabolites by drug-specific CD4+ T cells from allergic individuals.J Immunol. 2000; 164: 6647-6654Crossref PubMed Google Scholar Because SMX- and SMX-NO–responsive CD4+ T cells deriving from the memory pool were detected in healthy donors, the data raise the possibility that peptide-specific T cells are present within the general population, which are reactive against drug-associated peptides through some form of molecular mimicry within the MHC antigen-binding cleft. Indeed, heterologous immunity has been previously proposed to be the result of pathogen-primed T cells as viral-specific T cells have the propensity to cross-react with MHC-restricted drug-antigen.9Lucas A. Lucas M. Strhyn A. Keane N.M. McKinnon E. Pavlos R. et al.Abacavir-reactive memory T cells are present in drug naïve individuals.PLoS One. 2015; 10: e0117160Google Scholar Furthermore, in comparison to T-cell clones derived from naive T-cell priming which were highly antigen-specific, those generated from SMX- and SMX-NO–exposed memory cultures displayed the ability to cross-react with SMX-NO and SMX, respectively. Thus, our data indicate that the cross-reactive T cells observed in hypersensitive patients are likely derived from preexisting memory T cells. Although ongoing studies are required to provide data more representative of the generalized population, using an initial small cohort of donors we conclude that not only the priming of naive T cells but also the activation of preexisting memory T cells by SMX-derived antigens may play a role in the onset of SMX hypersensitivity. Therefore, determining the precise nature of the peptides responsible for the initial priming of naive T cells is crucial to understanding drug-induced hypersensitivity reactions and is thus key to the future safe development of drugs. More work will be needed to determine whether a detailed analysis of a patient's memory T-cell response could be used at a clinical level to evaluate allergic risk. We thank the CDSS nurses who helped to collect samples, as well as the patients who participated in the project. Venous blood samples were taken from SMX-hypersensitive patients and SMX-naive healthy volunteers. Study approval was granted by the Liverpool local research ethics committee, and all blood donors gave informed written consent. Extraction of PBMCs from whole blood was performed by density gradient separation using Lymphoprep (Axis-shield, Dundee, United Kingdom [UK]). Magnetic bead separation columns were then used to separate distinct cell populations as devised by the manufacturer (Miltenyi Biotec, Bisley, UK). First, positive selection was used to separate CD14+ monocytes from total PBMCs. Second, a negative selection was performed on the non-CD14 population, which used an anti–T-cell antibody cocktail, to extract a CD3+ pan T-cell population. Last, 2 consecutive isolations were carried out on the pan T-cell population: positive selection of CD25+ T cells to identify and remove regulatory T cells, followed by positive selection of CD45RO on the CD25− population to separate naive (CD45RO−) and memory (CD45RO+) T cells. All cells were frozen in 10% dimethyl sulfoxide (Sigma-Aldrich Ltd, Gillingham, UK) and kept at −150°C before use. DCs and EBV-transformed B cells were used as APCs for in vitro priming and T-cell cloning assays, respectively. DC generation required an 8-day culture of CD14+ monocytes (3-5 × 106/well; 6-well plate; total volume, 6 mL; 37°C/5% CO2) in R9 medium (RPMI 1640, 100 μg/mL penicillin, 100 U/mL streptomycin, 25 μg/mL transferrin, 10% human AB serum [Innovative Research], 25 mM HEPES buffer, 2 mM l-glutamine) supplemented with GM-CSF- and IL-4 (800 U/mL) (PeproTech Ltd). To produce functionally mature DCs, 25 ng/mL TNF-α (PeproTech Ltd) and 1 μg/mL LPS (Sigma-Aldrich Ltd, Gillingham, UK, E coli 0111:B4) were added on the penultimate day of culture. Autologous EBV-transformed B cells were generated as previously described.E1Naisbitt D.J. Britschgi M. Wong G. Farrell J. Depta J.P.H. Chadwick D.W. et al.Hypersensitivity reactions to carbamazepine: characterization of the specificity, phenotype, and cytokine profile of drug-specific T cell clones.Mol Pharmacol. 2003; 63: 732-741Crossref PubMed Scopus (196) Google Scholar Mature DCs (0.8 × 105/well) were cultured with autologous naive or memory T cells (2.5 × 106/well; 24-well plate; total volume, 2 mL) and either SMX (1 mM) or SMX-NO (50 μM) for 8 days. To assess antigen-specific responses, drug-antigen–primed naive or memory T cells were then harvested and re-plated (1 × 106/well; 96-well plate; total volume, 200 μL) with autologous mature DCs (4 × 103/well) and SMX (1-2 mM), SMX-NO (20-50 μM), or PHA (20 μg/mL) as a positive control. Analysis of cytokine/cytolytic molecule secretion was performed using precoated ELISpot plates (duplicate cultures) and proliferative capacity by [3H] thymidine incorporation (triplicate cultures) after a 48-hour incubation (37°C/5% CO2). The secretion of IL-13 was determined according to the manufacturer's ELISpot protocol (Mabtech, Nacka Strand, Sweden). [3H] thymidine (0.5 μCi/well) was added to the proliferation plate and then incubated for an additional 16 hours before analysis of incorporated radioactivity using a Microbeta Trilux 1450 LSC beta counter (PerkinElmer, Cambridge, UK). Both naive and memory T-cell cultures were subject to well-documented serial dilution and mitogen-driven expansion protocolsE2Mauri-Hellweg D. Bettens F. Mauri D. Brander C. Hunziker T. Pichler W.J. Activation of drug-specific CD4+ and CD8+ T cells in individuals allergic to sulfonamides, phenytoin, and carbamazepine.J Immunol. 1995; 155: 462-472PubMed Google Scholar, E3Schnyder B. Burkhart C. Schnyder-Frutig K. von Greyerz S. Naisbitt D.J. Pirmohamed M. et al.Recognition of sulfamethoxazole and its reactive metabolites by drug-specific CD4+ T cells from allergic individuals.J Immunol. 2000; 164: 6647-6654Crossref PubMed Scopus (182) Google Scholar to generate T-cell clones. To assess functionality, T-cell clones (5 × 104/well; 96-well plate; total volume, 200 μL) were cultured with autologous irradiated EBV-transformed B cells (1 × 104/well) in the presence or absence of SMX (1 mM) or SMX-NO (40 μM) for 48 hours (37°C/5% CO2). Antigen-specificity was validated by [3H] thymidine incorporation for an additional 16 hours before analysis by scintillation counting to measure cellular proliferation. T-cell clones with a stimulation index (mean cpm drug-treated wells/mean cpm of control wells) of more than 2 were expanded by repetitive stimulation with allogeneic PBMCs (5 × 104/well; 96-well plate; total volume, 200 μL) in R9 medium supplemented with PHA (5 μg/mL) and IL-2. Further analyses were performed similarly, including the measurement of IFN-γ, IL-5, IL-13, IL-17, perforin, and granzyme B by ELISpot (duplicate cultures), and using [3H] thymidine incorporation assays to analyze MHC restriction by adding MHC I– (IgG1, 10 μg/mL) and MHC II–blocking (IgG2a, 10 μg/mL) antibodies to particular wells alongside respective isotype controls (triplicate cultures). Flow cytometry was used to detail the phenotype of individual T-cell clones. A FACS Canto II flow cytometer was used to acquire T cells on the basis of forward/side scatter characteristics. Collected data were analyzed using associated FACS DIVA or Cyflogic software. Proliferation data from the in vitro priming of naive and memory T cells were analyzed in triplicate cultures and on 3 or more separate donors. Data are presented as the mean of the triplicate values with associated SD. A paired t test was implemented for statistical analysis (Sigmaplot 13 software).Fig E2Comparative ability of DCs and B cells to induce naive and memory T-cell responses to drug-antigen in vitro. Naive and memory T cells from drug-naive donors were exposed to SMX-NO and SMX for 8 days in the presence of EBV-transformed B cells (A) or DCs (B), and then recall responses tested by [3H]-thymidine incorporation. Error bars indicate SD of triplicate wells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Antigen cross-reactivity of T-cell clones derived from priming in drug-naive donors and hypersensitive patients. Naive and memory T cells from drug-naive donors were exposed to SMX and SMX-NO for 8 days and then recall responses tested. Cross-reactivity to SMX-NO and SMX was measured by [3H]-thymidine incorporation and/or ELISpot after exposure of naive and memory T cells to SMX-NO (A, n = 3) and SMX (B, n = 3), or exposure of representative SMX-NO– (C) and SMX-responsive (D) T-cell clones from hypersensitive patients. Error bars indicate SD of triplicate wells (*P ≤ .05, **P ≤ .005, and ***P < .001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4MHC I and II restriction of SMX-responsive T-cell clones derived from drug-naive donors. Naive or memory-derived SMX-responsive T-cell clones from drug-naive donors were cultured with autologous EBV-transformed B cells and SMX, with or without MHC I– or II–blocking antibodies for 48 hours. Proliferative responses were measured by [3H]-thymidine incorporation for a further 16 hours. Error bars indicate SD of triplicate wells (*P ≤ .05, **P ≤ .005, and ***P < .001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)