Dapsone and Nitroso Dapsone Activation of ... - ACS Publications

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Dapsone and Nitroso Dapsone Activation of Naı ̈ve T‑Cells from Healthy Donors Abdulaziz Alzahrani,† Monday Ogese,‡ Xiaoli Meng,† James C. Waddington,† Arun Tailor,† John Farrell,† James L. Maggs,† Catherine Betts,‡ B. Kevin Park,† and Dean Naisbitt*,† †

MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool L69 3GE, United Kingdom ‡ Pathology Sciences, Drug Safety and Metabolism, AstraZeneca R&D, Darwin Building 310, Cambridge Science Park, Milton Rd, Cambridge CB4 0WG, United Kingdom S Supporting Information *

ABSTRACT: Dapsone (DDS) causes hypersensitivity reactions in 0.5−3.6% of patients. Although clinical diagnosis is indicative of a hypersensitivity reaction, studies have not been performed to define whether dapsone or a metabolite activates specific T-cells. Thus, the aims of this study were to explore the immunogenicity DDS and nitroso DDS (DDS-NO) using peripheral blood mononuclear cells from healthy donors and splenocytes from mice and generate human T-cell clones to characterize mechanisms of T-cell activation. DDS-NO was synthesized from DDS-hydroxylamine and shown to bind to the thiol group of glutathione and human and mouse albumin through sulfonamide and N̈ T-cell priming to DDS and DDS-NO hydroxyl sulphonamide adducts. Naive was successful in three human donors. DDS-specific CD4+ T-cell clones were stimulated to proliferate in response to drug via a MHC class II restricted direct binding interaction. Cross reactivity with DDS-NO, DDS-analogues, and sulfonamides was not observed. DDS-NO clones were CD4+ and CD8+, MHC class II and I restricted, respectively, and activated via a pathway dependent on covalent binding and antigen processing. DDS and DDS-NO-specific clones secreted a mixture of Th1 and Th2 cytokines, but not granzyme-B. Splenocytes from mice immunized with DDS-NO were stimulated to proliferate in vitro with the nitroso metabolite, but not DDS. In contrast, immunization with DDS did not activate T-cells. These data show that DDS- and DDS-NO-specific T-cell responses are readily detectable.

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INTRODUCTION Drug-specific T-lymphocytes participate in the pathogenesis of drug hypersensitivity reactions through a variety of means including the secretion of cytokines and cytolytic mediators. Despite significant advances in recent years, definition of the pathway(s) of drug antigen presentation that result in T-cell activation remains a subject of debate. Drugs can activate T-cells via a hapten pathway involving the formation of protein adducts.1−3 The hapten response is dependent on protein processing within antigen presenting cells and the generation of an antigen that comprises of the MHC binding peptide and the drug moiety bound covalently to a specific nucleophilic amino acid. Drugs might also covalently modify MHC molecules or peptides embedded within MHC molecules on the surface of antigen presenting cells to activate T-cells. In addition to this, drugs have been shown to interact with MHC molecules and Tcells directly via readily reversible bonds (pharmacological interaction pathway).4−7 Although the precise nature of the pharmacological interaction remains ill-defined, several drugs have been shown to stimulate T-cells via this pathway.8−10 Finally, the nucleoside reverse transcriptase inhibitor abacavir activates T-cells by binding deep within the peptide binding groove of one specific MHC molecule, HLA-B*57:01, altering © 2017 American Chemical Society

the three-dimensional space and hence the nature of the peptides that subsequently bind.11−13 It has been proposed, although not yet proven, that these altered peptides, and not the drug per se, activate the T-cells involved in abacavir hypersensitivity. Knowledge of the drug MHC binding interaction is complicated by the fact that most drugs undergo extensive metabolism, and one or more metabolites might activate T-cells via any of the pathways described above. Furthermore, our ability to study drug metabolite-specific T-cell responses is limited by (1) the almost complete absence of synthetic drug metabolites for functional studies; and (2) a disconnect between the drug protein binding interactions that occur in patients and in vitro in cell culture systems. To date, researchers have shown that T-cells isolated from patients with (1) allopurinol-induced hypersensitivity are preferentially activated with the active metabolite oxypurinol through a direct binding interaction with HLAB*58:01;14 and (2) sulfamethoxazole-induced hypersensitivity reactions are activated with the cysteine-reactive nitroso metabolite through a hapten mechanism.15,16 Received: September 22, 2017 Published: October 18, 2017 2174

DOI: 10.1021/acs.chemrestox.7b00263 Chem. Res. Toxicol. 2017, 30, 2174−2186

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Chemical Research in Toxicology

using Lymphoprep (Stemcell Technologies, Cambridge, UK). Layered blood was centrifuged at 2000 rpm for 25 min at room temperature. The buffy layer containing PBMC was removed and washed twice using Hanks balanced salt solution (HBSS). Cells were then counted using a Neubauer hemocytometer (Sigma-Aldrich) under a Leica DME microscope (Leica Microsystems, Milton Keynes) prior to magnetic bead selection of various subsets. Separation of CD14+ Monocytes and Various T-Cell Subsets. CD14+ monocytes and T-cell populations were separated using magnetic beads and columns according to the manufacturer’s instructions (Miltenyi Biotech; Bisley, UK). CD14+ cells were positively ̈ and memory T-cells were negatively selected. selected, while naive CD3+ cells were then subjected to positive selection for Treg (CD25+) and memory cells (CD45RO+). The different cell subsets were stored at −150 °C prior to use. ̈ Human T-Cell Priming Assay. CD14+ monocytes were Naive incubated in culture medium supplemented with GM-CSF (800 U/mL) and IL-4 (800 U/mL) at 37 °C (5% CO2) to generate dendritic cells. On day 7, TNF-α (25 ng/mL) and LPS (1 μg/mL) maturation factors were added to the culture. Dendritic cells characterized previously for phenotypic markers30 were plated (0.8 × 105 cells per well) and cultured ̈ CD3+ T-cells (2.5 × 106 cells per well; 48 well plate) in the with naive presence of either DDS (1 mM) or DDS-NO (40 μM) for 8 days. Primed T-cells were restimulated with a fresh batch of autologous dendritic cells in the presence and absence of the relevant drug antigen. Drug-specific responses were then analyzed for proliferation and cytokine release. Readouts for the Detection of Drug-Specific T-Cell Responses. To measure proliferation, [3H]-thymidine (0.5 μCi/well) was added 48 h after restimulation of the primed T-cells. Incorporated [3H] thymidine was counted after a further 16 h incubation using a MicroBeta TriLux 1450 LSC β-counter (PerkinElmer, Cambridge, UK). The release of IFN-γ was measured using ELISot. On completion of the 48 h culture period, plates were processed according to the manufacturers instructions. Images were viewed using an ELISpot reader (Cadama Medical, Stourbridge, UK). Generation of EBV-Transformed B-Cells. EBV-transformed Bcell lines were generated from PBMC using supernatant from B-958 cells. The B-cell lines were used as antigen-presenting cells in experiments with TCC. Generation and Characterization of Drug-Specific Human TCell Clones. TCC were generated from the DDS and DDS-NO priming experiments by serial dilution. Briefly, T-cells were suspended in culture medium at 0.3−3 cells/well and subjected to PHA-driven expansion (5 μg/mL). Medium was supplemented with IL-2 to maintain the proliferative response. After 14 days, the process was repeated, and well growing clones were selected for specificity testing with DDS or DDS-NO. TCC (5 × 104 cells, 50 μL) were cultured with irradiated autologous EBV-transformed B-cells (1 × 104 cells, 50 μL) in the presence of DDS (1−2 mM) or DDS-NO (20−40 μM) for 48 h in duplicate wells of a U-bottom 96-well plate. [3H]-thymidine (0.5 μCi) was added for the final 16 h of the incubation to measure T-cell proliferation. TCC with a stimulation index (proliferation in test incubations with drug/proliferation in control wells) of 2 or above were expanded further for dose−response studies; assays with structurally related compounds (sulfamethoxazole, nitroso sulfamethoxazole, nitroso benzene, sulfamerazine, sulfadiazine, sulfachloropyridazine, sulfadoxin, sulphanilamide, 4,4 thiodianiline, 4,4 oxyaniline, 3,3 sulfonyldianiline, and the mono and diacetylated forms of DDS), the profile of secreted cytokines by ELISpot (IFN-γ, IL-5, IL-13 and granzyme-B), and the mechanistic studies detailed below. Flow cytometry was used to characterize the phenotype of the TCC. Cells were stained with CD4-PE (3 μL) and CD8-APC (3 μL) antibodies and incubated at 4 °C for 20 min in the dark prior to analysis using a FACS Canto II system. A total of 10,000 events were acquired from each sample. Characterization of the Pathways of Drug Presentation to Human T-Cells. The following strategy was employed to measure pathway of DDS and DDS-NO presentation to T-cells. First, antigen presenting cells were omitted from the T-cell assay. Second, antigen presenting cells were fixed with glutaraldehyde to prevent protein

Given the limited availability of synthetic reactive metabolites, the aim of this study was to focus on dapsone (DDS) and the nitroso (DDS-NO) metabolite to characterize the precise nature of DDS-NO protein binding interaction. Furthermore, the synthetic metabolite was used to investigate the molecular mechanism(s) of antigen-specific T-cell activation using ̈ peripheral blood mononuclear cells (PBMC) from drug-naive healthy donors and a mouse model of drug immunogenicity. DDS is used for the treatment of leprosy because of its antibiotic and anti-inflammatory properties.17,18 N-acetyltransferase enzymes catalyze the conversion of DDS to stable mono and diacetylated derivatives, while cytochrome P-450, flavin monooxygenase, and peroxidase-mediated N-hydroxylation result in the formation DDS hydroxylamine.19−22 DDS hydroxylamine is susceptible to auto-oxidation, and the derived nitroso species has been shown to bind covalently to protein.20,23,24 Despite this, the nature of the binding interaction has not been defined. Although DDS is usually well tolerated and suitable for longterm treatment, a severe hypersensitivity syndrome with a mortality rate of 9.9% develops in 0.5−3.6% of patients.25 Reactions are characterized by a delayed onset (usually 4−6 weeks) with a long latent period.25−27 Zhang et al. found that HLA-B*13:01 expression is a predictor of the hypersensitivity syndrome in Chinese patients with leprosy.28 These genetic data suggest that the adaptive immune system may be involved in the disease pathogenesis; however, the activation of T-cells with dapsone and dapsone metabolites has not been studied.

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EXPERIMENTAL PROCEDURES

Chemicals and Reagents. DMSO, DDS, phytohemagglutinin (PHA), and all the DDS analogues used in this study were purchased from Sigma-Aldrich (UK). DDS hydroxylamine (C10H9N3O4S) was purchased from Dalton Chemical Laboratories Inc. (Toronto, Canada). Tritiated thymidine-(methyl-3H) was obtained from Moravek (California, USA). Analytical grade (Analar) acetonitrile, HPLC grade distilled water, and HPLC grade methanol were acquired from Fisher Scientific (Loughborough, UK). All other reagents were purchased from Sigma-Aldrich (UK). Synthesis of Nitroso Dapsone. DDS-NO was synthesized from DDS hydroxylamine using iron(III) chloride (Figure S1A) according to the method of Naisbitt et al.29 Briefly, DDS hydroxylamine (60 mg, 0.227 mmol) was dissolved in absolute ethanol (10 mL) and added to a stirred solution of iron(III) chloride hexahydrate (0.5 g, 1.85 mmol) in distilled water (10 mL) over a period of 5−10 min. The reaction mixture was stirred at room temperature for 5 min, and a yellow precipitate was formed. The mixture was filtered under vacuum, and the yellow solid product was analyzed for purity using LC-MS. A solution for analysis was prepared immediately by 10-fold dilution with acetonitrile. The LCMS equipment was a 1260 Infinity LC system (Agilent Technologies, Waldbronn, Germany) connected to a 4000 Qtrap (Sciex, Warrington, United Kingdom). An aliquot (3 μL) was eluted from an Agilent Eclipse XDB-C18 column (3.5-μm; 150 × 4.6 mm) at 0.5 mL/min with ACNformic acid (0.1%, v/v) in formic acid (0.1%, v/v): 25% to 75% over 10 min; 75% for 5 min; 75% to 25% over 0.1 min. Full scanning positive-ion mass spectra were acquired over 5 s between m/z 100 and 1000. The ionspray voltage was 5.5 kV, desolvation potential was 120 V, and source temperature was 500 °C. Culture Medium. T-lymphocytes were grown in RPMI-1640 medium supplemented with 10% human AB serum, 25 mM HEPES buffer, 2 mM L-glutamine, penicillin (1000 U/ml), streptomycin (0.1 mg/mL), and transferrin (25 μg/mL). IL-2 was added to maintain T-cell clones (TCC). Dendritic cells were grown in RPMI-1640 medium supplemented with the constituents above and GM-CSF (800 U/ml) and IL-4 (800 U/ml). Isolation of Peripheral Blood Mononuclear Cells from Healthy Volunteers. PBMC were isolated from venous blood from ̈ healthy donors collected in heparinized vacutainer tubes drug-naive 2175


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Figure 1. LC-MS characterization of nitroso dapsone glutathione adducts. (A) Scheme shows all possible adducts formed by nitroso dapsone and glutathione. (B) Mass spectrometric characterization of DDS-NO glutathione adducts. MS/MS spectra confirm the formation of sulphonamide (m/z 586.3) and N-hydroxysulphonamide (m/z 602.3). processing.31 Third, antigen presenting cells were pulsed with DDS (2 mM) or DDS-NO (40 μM) for 16 h, prior to washing to remove unbound drug. The pulsed antigen presenting cells were then irradiated and used to activate TCC in the absence of soluble drug. Finally, MHC restriction was assessed by culturing the TCC, antigen presenting cells, and drugs together with antihuman MHC I (HLA-A, -B, -C) or antihuman MHC II (HLA-DP, -DQ and -DR) antibodies. T-cell proliferative responses and/or IFN-γ release were measured using [3H]thymidine and ELISpot, respectively. Immunization of Mice with Dapsone and Nitroso Dapsone. We have previously used rodent models to explore the immunogenicity of sulfamethoxazole, an aromatic amine-containing drug that is converted to a nitroso metabolite similar to DDS.32,33 The same method was used herein to study DDS- and DDS-NO-specific T-cell responses, with nitroso sulfamethoxazole as a positive control. Briefly, female C57BL/6 mice (6−9 weeks, 20−30 g) purchased from Charles

River UK Ltd. (Kent, UK) were immunized with DDS or sulfamethoxazole (50 mg mL−1) and DDS-NO or nitroso sulfamethoxazole (1 mg kg−1) in DMSO i.p. 4 times weekly for 2 weeks. On completion of the dosing protocol, mice were sacrificed, and the spleen removed using aseptic technique. Red cell-depleted splenocytes isolated by density centrifugation using Lymphoprep were incubated (1.5 × 105) with DDS (0.1−1 mM), sulfamethoxazole (0.1−1 mM), DDS-NO (5− 20 μM), nitroso sulfamethoxazole (5−20 μM), and nitroso benzene (20 μM) at 37 °C, 5% CO2. After 3 days, proliferation was measured by the addition of [3H]thymidine for the final 16 h of culture. Mass Spectrometric Analysis of Nitroso Dapsone and Nitroso Sulfamethoxazole Binding to Glutathione and Mouse Serum Albumin. DDS-NO (10 mM) was incubated with glutathione (10 mM) in potassium phosphate buffer (10 mM, pH 7.4) at 37 °C for 16 h. The resulting crude products were diluted with 0.1% formic acid (1:10 dilution) and analyzed by LC-MS/MS as described previously.34 2176


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̈ T-cells to DDS and DDS-NO. Naive T-cells from 3 donors were co-cultured with monocyte-derived dendritic cells in the Figure 2. Priming of naive presence of either DDS (500 μM) or DDS-NO (40 μM) for 8 days. The primed T-cells were then restimulated with a second batch of autologous dendritic cells and either DDS-NO (10−40 μM) or DDS (125−1000 μM). (A, C) Plates were incubated at 37 °C, 5% CO2 for 48 h. [3H] Thymidine was added for the final 16 h of experiment and drug-induced T-cell proliferation was evaluated by scintillation counting. (B, D) INF-γ release was measured by ELISpot according to manufacturer’s instructions. Samples were delivered into a 4000 QTRAP (AB Sciex, Framingham, MA) coupled with a 1260 Infinity Quaternary Pump HPLC system (Agilent Technologies, Santa Clara, CA) and Kinetex C18 column (2.6 μm C18, 100 mm × 2.1 mm, Phenomenex, Macclesfield, Cheshire, UK). A gradient from 1% acetonitrile/0.1% formic acid (v/v) to 50% acetonitrile/0.1% formic acid (v/v) in 12 min was applied at a flow rate of 150 μL/min. Data were analyzed using Analyst software, version 1.5.1 (AB Sciex). To investigate the covalent binding of DDS-NO to mouse serum albumin (MSA), DDS-NO freshly dissolved in DMSO (1 mM) was incubated with MSA (0.1 mM, 50 μL) in potassium phosphate buffer (10 mM, pH 7.4) at 37 °C for 16 h. The molar ratios of drug to protein were 0.1:1, 1:1, and 10:1. Protein was purified by ultrafiltration (3K cutoff) to remove the free drug and then reduced with 10 mM

dithiothreitol (15 min) and alkylated with 55 mM iodoacetamide (15 min) at room temperature. The protein was reconstituted in 100 μL 50 mM ammonium hydrogen carbonate, and 165 μg (1.25 nmol) of protein was digested with 1.6 μg trypsin overnight at 37 °C. The tryptic MSA peptides were analyzed by a Q-TOF mass spectrometer as described previously.35 Briefly, samples were delivered into a Triple TOF 5600 mass spectrometer (AB Sciex) by automated in-line reversed phase liquid chromatography, using an Eksigent NanoUltra cHiPLC System (AB Sciex) mounted with a microfluidic trap and analytical column (15 cm × 75 μm) packed with ChromXP C18−CL 3 μm. A NanoSpray III source was fitted with a 10 μm inner diameter PicoTip emitter (New Objective). Samples were loaded in 0.1% formic acid onto the trap, which was then washed with 2% ACN/0.1% FA for 10 min at 2 μL/min before switching in-line with the analytical column. A gradient of 2−50% 2177


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Figure 3. Generation of DDS- and DDS-NO-specific T-cell clones. Drug-specific TCC were generated by serial dilution method and repetitive mitogen stimulation. TCC were co-cultured with irradiated autologous EBV-transformed B cells and DDS or DDS-NO; culture medium was used as negative control. The plates were incubated at 37 °C, 5% CO2 for 48 h. [3H]Thymidine was added the last 16 h of the experiment to measure proliferative responses. TCC with stimulation index >2 were selected and expanded for further functional assays. (A) DDS- and (B) DDS-NO TCC generated from 2 donors. (C) Dose-dependent proliferation of DDS- and DDS-NO-specific TCC and assessment of cross-reactivity. significant values are presented as follows: ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** ≤ 0.005.

(v/v) ACN/0.1% (v/v) FA over 90 min was applied to the column at a flow rate of 300 nL/min. Spectra were acquired automatically in positive ion mode using information-dependent acquisition, using mass ranges of 400−1600 amu in MS and 100−1400 amu in MS/MS. Up to 25 MS/ MS spectra were acquired per cycle (approximately 10 Hz) using a threshold of 100 counts per s, with dynamic exclusion for 12 s and rolling collision energy. Sequence coverage was determined using ProteinPilot software, v4.0 and the most recent version of the SwissProt database. Modified peptides were identified by filtering for specific fragment ions in PeakView 1.2.0.3 (AB Sciex) and manual inspection of the spectra. Statistical Analysis. Graphs were produced using GraphPad Prism 5 software (GraphPad Software Inc., La Jolla, CA, USA). Data analysis and statistical tests were conducted using GraphPad Prism 5. The t test was used to determine statistical significance when comparing two treatment groups. In experiments for which more than two treatment groups were compared, a one-way ANOVA was employed. Statistically

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RESULTS

LC-MS Characterization of Nitroso Dapsone. A solution of DDS-NO (Figure S1A) was prepared and analyzed immediately by HPLC and mass spectrometry. The purity of DDS-NO is 95% based on HPLC analysis (Figure S1B). DDSNO (retention time, 11.6 min) was detected as [M + H]+ at m/z 263 (Figure S1B). Abundant and characteristic fragment ions were seen at m/z 233 ([M + H − NO]+) and m/z 93 ([PhO]+). The material, typically for an arylnitroso preparation, contained a small amount of the azoxy derivative ([M + H]+ at m/z 509; retention time, 12.5 min), which does not bind covalently to 2178


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Figure 4. Characterization of the DDS-NO-specific T-cell response. DDS-NO-specific TCC were cultured with irradiated autologous EBV-transformed B-cells (APC) and DDS-NO and T-cell proliferative responses and IFN-γ release were assessed using [3H]thymidine and ELISpot, respectively. (A) Comparison of the proliferative responses of TCC with DDS-NO, nitroso sulfamethoxazole, and nitroso benzene. (B) Comparison of the proliferative responses of TCC with DDS-NO in the presence and absence of autologous EBV-transformed B-cells. (C) Comparison of the proliferative responses of TCC with soluble DDS-NO and antigen presenting cells pulsed with DDS-NO for 16 h. (D) Time-dependent proliferative responses and IFN-γ release from TCC incubated with antigen presenting cells pulsed with DDS-NO for 1−24 h. (E) Comparison of the proliferative responses of TCC with DDSNO in the presence of irradiated and fixed autologous EBV-transformed B-cells. (F) Comparison of the proliferative responses of TCC with DDS-NO in the presence and absence of glutathione. Figure shows results from one DDS-NO-responsive clone.

586.3) and [3O] N−S conjugates (m/z 602.3) (Figure 1B). The product ion spectra shown in Figure 1B provided confirmative evidence for the formation of the proposed sulfonamide and Nhydroxysulfonamide adducts. ̈ T-Cells to Dapsone and Nitroso Priming of Naive Dapsone. To investigate the immunogenicity of DDS and DDS̈ T-cells isolated from 3 DDS naive ̈ healthy donors NO, naive

protein. The reactivity of DDS-NO with the low molecular weight thiol glutathione was also confirmed by mass spectrometry (Figure 1A). Direct addition of glutathione to DDS-NO resulted in a sulfinamide product, which can be further oxidized to form a sulfonamide and an N-hydroxysulfonamide adduct. Only two of the three putative DDS-NO/glutathione reaction products were detected by LC-MS/MS, namely the [2O] (m/z 2179


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Figure 5. Characterization of DDS-specific T-cell response. DDS-specific TCC were cultured with irradiated autologous EBV-transformed B-cells (APC) and DDS and T-cell proliferative responses and IFN-γ release were assessed using [3H]thymidine incorporation and ELISpot, respectively. (A) Comparison of the proliferative response of TCC with DDS in the presence and absence of autologous EBV-transformed B-cells. (B) Comparison of IFN-γ release from TCC with soluble DDS and antigen presenting cells in the presence of MHC class I and II blocking antibodies. (C) Comparison of IFN-γ release from TCC with soluble DDS and antigen presenting cells pulsed with DDS for 16 h. (D) Comparison of IFN-γ release from TCC with DDS in the presence of irradiated and fixed autologous EBV-transformed B-cells. (E) Comparison of the proliferative responses of TCC with DDS in the presence and absence of glutathione. Figure shows results from one DDS-responsive clone.

were cultured with autologous dendritic cells in the presence of ̈ T-cells from each either DDS or DDS-NO for 7 days. Naive donor were successfully primed with both DDS and DDS-NO (Figure 2). Restimulation of the DDS and DDS-NO primed Tcells with DDS and DDS-NO, respectively, resulted in a

proliferative response and the secretion of IFN-γ (Figure 2A,B, DDS-primed cells; Figure 2C,D, DDS-NO-primed cells). Interestingly, T-cells from one DDS and DDS-NO primed donor (donor 3) displayed cross-reactivity with the alternative form of the drug antigen (i.e., DDS-primed T-cells were 2180


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Figure 6. Reactivity of DDS-specific T-cell clones with closely related structures, sulfonamides and DDS metabolites. DDS-specific clones were incubated with irradiated autologous EBV-transformed B-cells in the presence of DDS: (A) closely related analogues, (B) sulfonamides, or (C) DDS metabolites in a U-bottomed 96-well plate at 37 °C, 5% CO2 for 48 h. T-cell activation was then determined using [3H] thymidine incorporation. (D) Structures of closely related analogues, sulfonamides, and DDS metabolites.

stimulated to proliferate and secrete IFN-γ with DDS-NO and vice versa). Generation of Drug-Specific T-Cell Clones. DDS- and DDS-NO-specific TCC were generated from previously primed T-cells of 2 out of 3 donors to investigate pathways of T-cell activation. Initial testing revealed that 9 out of the 92 expanded TCC from DDS-primed T-cells were stimulated to proliferate with DDS (i.e., stimulation index of 2 or more; Figure 3A), and 302 TCC were expanded from the DDS-NO-primed cells. Of these, 24 were stimulated to proliferate with DDS-NO (Figure 3B). The drug and drug metabolite-responsive TCC were expanded for subsequent testing. TCC were found to proliferate in a concentration-dependent manner in the presence of DDS or DDS-NO and displayed no cross-reactivity (Figure 3C). Nitroso Dapsone-Responsive T-Cell Clones Are Activated via a Hapten Pathway. The DDS-NO-specific CD4+ and CD8+ TCC were not activated with nitroso sulfamethoxazole or nitroso benzene (Figure 4A). Furthermore, they were not stimulated to proliferate when incubated with DDS-NO in the absence of antigen presenting cells (Figure 4B). The TCC were however activated when cultured with antigen presenting cells pulsed with DDS-NO. The response to DDS-NO-pulsed antigen presenting cells was time dependent, with optimal responses detected after a 24 h incubation period (Figure 4C,D). DDS-pulsed antigen presenting cells did not activate the clones (results not shown). In contrast to the DDS-specific TCC (discussed below), glutaraldehyde fixation of antigen presenting cells (Figure 4E) and the addition of glutathione to culture

medium (Figure 4F) decreased the strength of the proliferative response of TCC with DDS-NO. Dapsone-Responsive T-Cell Clones Are Structurally Specific and Activated via a Direct Binding Interaction between the Drug and MHC. Incubation of DDS-specific TCC with DDS in the absence of autologous antigen presenting cells failed to trigger a T-cell proliferative response (Figure 5A). All of the DDS-specific TCC were CD4+ (results not shown), and the drug-specific T-cell response was MHC class II restricted. Antibody blockade of MHC class II resulted in decreased proliferative response and IFN-γ secretion (Figure 5B). To further investigate the pathway of DDS-specific T-cell activation, antigen presenting cells were pulsed with DDS for 24 h, followed by extensive washing to remove the unbound drug. The DDS-pulsed antigen presenting cells failed to induce proliferation of the TCC (Figure 5C). Furthermore, when glutaraldehyde-fixed antigen presenting cells were cultured with DDS and the TCC, the drug-specific T-cell response was not abrogated (Figure 5D). Finally, glutathione did not block the activation of TCC with DDS (Figure 5E). Cross-reactivity was not observed when DDS-specific TCC were exposed to DDS analogues (4,4 thiodianiline, 4,4 oxidianiline and 3,3 sulfonyldianiline; Figure 6A) or sulfonamides (sulfamethoxazole, sulfamerazine, sulfadiazine, sulfachloropyridazine, sulfadoxin and sulphanilamide; Figure 6B). However, a weak proliferative response and IFN-γ release was detected when the DDS-specific TCC were cultured with the mono- and diacetylated forms of DDS (Figure 6C). 2181


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Figure 7. Proliferative response of splenocytes from mice administrated nitroso sulfamethoxazole and DDS-NO and assessment of DDS-NO binding to mouse serum albumin. (A, B) Splenocytes were cultured with DDS-NO, DDS, sulfamethoxazole, nitroso sulfamethoxazole or nitroso benzene for 72 h, and proliferation was measured by incorporation of [3H] thymidine. Incubations were conducted in triplicate. (C) MS/MS spectra showing DDS-NO modified tryptic peptide 34C*SYDEHAK41 with a mass addition of 278 amu, indicating a sulfonamide adduct was formed at Cys34.

Splenocytes from Immunized Mice Are Activated with Nitroso Dapsone, But Not the Parent Drug. Previous studies have shown that splenocytes from mice immunized against nitroso sulfamethoxazole are stimulated to proliferate in vitro in the presence of the nitroso metabolite. In contrast, splenocytes are not activated with the parent drug.32,36 Herein, splenocytes from nitroso sulfamethoxazole-sensitized mice were also shown to be activated with nitroso sulfamethoxazole, and no

cross-reactivity was observed with either DDS-NO, nitroso benzene, or sulfamethoxazole (Figure 7A). Mice were also administered DDS and DDS-NO following the protocol established with nitroso sulfamethoxazole. Splenocytes from 2 DDS-NO immunized mice proliferated vigorously ex vivo in the presence of the nitroso metabolite (Figure 7B). Weaker DDS-NO-specific proliferative responses were detected with splenocytes from mice 3 and 4. Somewhat surprisingly, 2182


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sulfonamide, which was presumably derived from N-hydroxysulfinamide To explore dapsone immunogenicity, we utilized a recently established assay30,39 that involves the culture of dendritic cells ̈ T-cells and either DDS or DDS-NO for 8 with autologous naive days. The primed T-cells were then restimulated with a second batch of dendritic cells and DSS or DDS-NO, and antigenspecific proliferative responses and IFN-γ release were measured. Good to strong responses, according to the classification of Faulkner et al.,30 were detected with DDS in both readouts. ̈ T-cells from all three donors; Similarly, DDS-NO activated naive however, the strength of the drug-metabolite-specific response was significantly lower. Interestingly, DDS- and DDS-NOprimed T-cells from donor 3 alone were also activated with the alternative form of the drug antigen (i.e., DDS-NO-primed Tcells were activated with DDS and vice versa). TCC were subsequently generated from two donors (donors 1 and 3). DDS-specific TCC were CD4+, whereas both CD4+ and CD8+ TCC were activated with DDS-NO. Importantly, the clones did not display cross-reactivity. Ourselves and others have developed and utilized a battery of functional assays to distinguish between hapten and pharmacological pathways of T-cell activation.1,4,16,40−42 The same approach was adopted herein to define pathways of DDS- and DDS-NO-specific activation of TCC. The activation of TCC with both DDS and DDS-NO was dependent on the presence of antigen presenting cells. Antigen presenting cells were therefore pulsed with DDS or DDS-NO, prior to repeated washing to remove unbound drug, to explore whether TCC are activated by DDS(metabolite)-modified proteins. DDS-NO-specific TCC were activated with DDS-NO-, but not DDS-, pulsed antigen presenting cells and the T-cell proliferative response, and IFN-γ release was time-dependent. Antigen presenting cells pulsed with DDS-NO for 1 h activated a suboptimal response, whereas the strength of the response increased after 8 and 24 h incubation periods. In contrast, DDS-specific TCC were not activated with antigen presenting cells pulsed with either DDS or DDS-NO. In the next experiments, glutathione was added to the T-cell proliferation assay. As discussed above, glutathione interacts covalently with DDS-NO, limiting protein binding. Glutathione reduced the activation of TCC with DDS-NO, while it had no effect on the DDS-specific TCC. Collectively, these data indicate that DDS-NO forms a protein adduct within antigen presenting cells to activate TCC, whereas DDS-specific TCC are activated via a pharmacological interaction of the parent drug with MHC and the T-cell receptor. Fixation of antigen presenting cells with glutaraldehyde blocks antigen processing, but not the display of MHC molecules on the cells surface.31 To confirm that the processing of proteins into peptide fragments is involved in the activation of DDS-NO-, but not DDS-, specific TCC, glutaraldehyde-fixed antigen presenting cells were used in the proliferation assay. TCC were not activated with DDS-NO, whereas DDS activated TCC to a similar extent when experiments with fixed and irradiated antigen presenting cells were compared. Sulfones such as DDS are used rarely in pharmacology, when compared with drug classes such as the sulfonamides. For this reason, we chose to explore the reactivity of DDS-specific TCC with DDS analogues and a panel of sulfonamides. TCC were not stimulated to proliferate with 4,4 thiodianiline, 4,4 oxidianiline, or 3,3 sulfonyldianiline, which indicates that the sulfone moiety (OSO) and the position of the amine groups are important for T-cell activation. Similarly, TCC were not activated with the

splenocytes from all 4 mice were also activated with nitroso sulfamethoxazole. DDS and sulfamethoxazole did not activate the T-cells. Splenocytes from mice immunized with DDS were not stimulated to proliferate in the presence of DDS, DDS-NO, sulfamethoxazole, or nitroso sulfamethoxazole (results not shown). Nitroso Sulfamethoxazole and Nitroso Dapsone Form Similar Adducts with Cysteinyl Residues on Mouse Serum Albumin. SMX-NO has previously been shown to undergo multiple adduction reactions with cysteinyl residues of proteins such as human serum albumin and glutathione Stransferease pi. Glutathione S-transferase pi was modified at Cys47 forming sulfinamide, N-hydroxysulfinamide, and Nhydroxysulfonamide adducts. Human serum albumin was modified with nitroso sulfamethoxazole at Cys34 and in contrast to glutathione S-transferase pi, only the [2O] adduct was detected. Herein, DDS-NO and nitroso sulfamethoxazole were incubated with mouse albumin for 16 h, and adduct formation was measured by mass spectrometry. Similar to the [2O] adduct observed with SMX-NO on HSA, stable DDS-NO cysteine adducts on MSA were detected. A representative MS/MS spectrum for a doubly charged ion at m/z 615.72 corresponds to the tryptic peptide 34CSYDEHAK41 with an additional mass of 278 amu, indicating that a [2O] adduct was formed. The peptide sequence was confirmed by the presence of a series of y and b ions. The modification site was confirmed by b2* (m/z 469.1), b3* (m/z 632.2), b6* (m/z 1013.3), and b7* (m/z 1084.3), all with adduction of 278 amu, giving evidence of modification at Cys34 (Figure 7C).

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DISCUSSION A clear understanding of the mechanistic basis of drug hypersensitivity remains a significant challenge as genetic, environmental, and chemical factors impact upon susceptibility and the nature of the adverse event.37 Layered on top of this is the fact that drugs interact with immune receptors in a number of ways to initiate an antigen-specific T-cell response. The dearth of synthetic protein-reactive metabolites for functional studies has complicated the development of research strategies to explore drug antigen presentation to T-cells. Thus, the majority of studies have focused on the interaction of parent drugs with MHC. For this reason, the aim of the current study was to synthesize the reactive nitroso metabolite of dapsone and explore immunogenicity of the parent drug and metabolite in human and murine test systems. Dapsone was selected as the study drug because of the hypersensitivity syndrome developed in dapsoneexposed Chinese patients with leprosy is strongly associated with expression of a single HLA allele, B*13:01.28 DDS-NO was synthesized by oxidation of DDS hydroxylamine using the method established by Naisbitt et al.29 and incubated with glutathione to characterize the adduct structures. Similar to the reactions of SMX-NO, direct addition of glutathione to DDS-NO may have resulted in a sulfinamide product, which was detected after 1 min incubation of SMX-NO with glutathione.38 However, the sulfinamide is unstable and can be further oxidized to form a stable [2O] adduct and an Nhydroxysulfonamide adduct. Indeed, only two stable adducts were detected after 16 h incubation of DDS-NO with glutathione, consistent with the previous findings. The structure of [2O] adduct has been controversial, however, the MS data from this study provided evidence for the formation of 2183


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sites on proteins may allow us to explain the difference observed between MSA and GSTP. Nonetheless, it is worth noting that there are two reactive cysteine residues on MSA, Cys34 and Cys579 (Figure S3), however, modification was only detected on Cys34. It is possible that DDS-NO could modify both residues, however, the tryptic peptide containing modified Cys579 is too short to be detected by the current mass spectrometric method. In summary, our data indicate that DDS-NO can activate ̈ T-cells via a hapten pathway. In human and mouse naive contrast, the parent drug binds to MHC molecules expressed on the surface of antigen presenting cells to activate T-cells; however, these responses are only detected in human donors. It is important to emphasize that our healthy donors did not express the HLA risk allele HLA-B*13:01. Thus, both pathways of T-cell activation are feasible in donors expressing different HLA alleles. In ongoing studies we are recruiting HLA-B*13:01+ donors with a history of DDS hypersensitivity to explore the nature of the drug-specific T-cell response and whether DDS and/or DDS-NO bind preferentially to HLA-B*13:01 to selectively activate CD8+ T-cells.

panel of sulfonamides. Hence, the interaction of DDS with MHC that leads to the T-cell response seems to be highly structurally specific. It is plausible that DDS interacts with MHC peptides through the π−π interactions of the aromatic ring and Hbonding of the amine group, thus positioning the sulfone and aniline moieties toward T cell receptors. Therefore, both the unique structure of DDS, for example, sulfone and aniline moieties, and the amino acids of the MHC binding peptides will determine the specificity of T-cell receptors. Severe cutaneous hypersensitivity reactions caused by allopurinol have been shown to be mediated primarily by Tcells activated with the active metabolite oxypurinol bound directly to HLA-B*58:01,10,14 which is a genetic marker for the iatrogenic disease.43 In addition to N-hydroxylation, which yields DDS hydroxylamine and DDS-NO, DDS is metabolized by Nacetyl transferase enzymes to mono- and diacetylated forms,19 which are presumed to be nontoxic. Certain DDS-specific clones were stimulated to proliferate weakly and secrete low levels of IFN-γ in the presence of both mono- and diacetylated DDS, suggesting that these species also interact with MHC molecules to stimulate T-cells. Further experiments however are ongoing to ̈ Texplore whether the acetylated DDS metabolites prime naive cells. Animal models of drug hypersensitivity reactions are rare, and our understanding of their relevance to the human disease is in its infancy.44,45 Animals have however been used to investigate intrinsic immunogenicity of drugs and chemicals. Focusing on sulfamethoxazole, we were able to use rodent models to reproduce the nitroso sulfamethoxazole (hapten)-specific Tcell responses observed in hypersensitive human patients and explore the relationship between direct metabolite-mediated toxicity and the activation of T-cells.32,36,46 However, the activation of T-cells with sulfamethoxazole via a direct pharmacological pathway was not observed. Mice were immunized against DDS and DDS-NO using the same dosing strategy. Splenocytes from DDS-NO immunized mice were activated ex vivo in the presence of the nitroso metabolite, but not the parent drug. Interestingly, in contrast to nitroso sulfamethoxazoleimmunized mice, mice immunized against DDS-NO displayed a degree of cross-reactivity. In fact, in 3 out of 4 mice, the strength of the recall response to nitroso sulfamethoxazole was stronger than that seen with DDS-NO itself. DDS did not activate T-cells in the mouse model. One possible explanation for this would be that DDS-NO formed protein adducts in immunized mice that activated the splenocytes. These cells may cross-react with nitroso sulfamethoxazole haptenated peptides, which may share a great degree of structural similarity to those DDS-NO peptides. As noted before, both the amino acid sequence of peptides and haptenic structure appeared to form the epitopes that can activate drug specific T-cells. In order to characterize the antigens that stimulated splenocytes from DDS-NO immunized mice, in vitro incubations of DDS-NO with mouse serum albumin (MSA) were performed. DDS-NO was found to covalently modify cysteine residues in MSA. In contrast to DDS-NO glutathione adducts, only one type of cysteine adduct in MSA was detected, corresponding to either a N-hydroxysulfinamide or sulfonamide adduct. This is consistent with previous findings that SMX-NO formed only one stable Cys34 adduct with human serum albumin. Notably, when DDS-NO was incubated with glutathione transferase pi (GSTP), all three types of adducts were detectable, though the sulfinamide and N-hydroxysulfonamide adducts were present at very low levels (Figure S2). Docking of DDS-NO to the binding

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.7b00263. Three supplemental figures (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: (+) 44 151 794 5346. Fax: (+) 44 151 794 5540. ORCID

Dean Naisbitt: 0000-0003-4107-7832 Funding

This work was supported by the Medical Research Council Centre for Drug Safety Science (Grant G0700654). The Saudi Arabian Ministry of Higher Education funded a Ph.D. studentship to A.A. M.O. is an Astra Zeneca postdoctoral research fellow. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the patients who donated blood for this project. ABBREVIATIONS CPM, counts per minute; DDS, dapsone; DMSO, dimethyl sulfoxide; HBSS, Hanks balanced salt solution; DDS-NO, nitroso dapsone; IFN-γ, interferon-γ; IL-5, interleukin-5; PBMC, peripheral blood mononuclear cells; PHA, phytohemagglutinin

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REFERENCES

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