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

Abstract

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

Figure 1

Generation of nitroso dapsone-specific clones and assessment of cross-reactivity. (A) PBMCs from a patient with dapsone hypersensitivity were cultured with nitroso dapsone (20–40 μM) for 14 days. Clones were then generated through serial dilution and repeated mitogen stimulation. Clones (0.5 × 10⁵) were cultured with autologous EBV-transformed B-cells (0.1 × 10⁵) and DDS-NO (20 μM) in duplicate for 48 h at 37 °C, 5% CO₂. For the final 16 h of culture, [3H]-thymidine (0.5 μCi/well) was added to assess proliferation. TCCs demonstrating an SI > 2 were expanded for further characterization. (B) ELISpot was used for the detection of IFN-γ, GB, IL-5, perforin, IL-13, IL-17, IL-22, and Fas-L from clones exposed to nitroso dapsone. Drug-specific TCCs (0.5 × 10⁵) were cultured with autologous EBV-transformed B-cells (0.1 × 10⁵) and dapsone or nitroso dapsone in ELISpot plates precoated with the target cytokine antibody for 48 h. Plates were then processed and developed using the relative secondary Abs and the secretion was visualized as spots. PHA was used as a positive control. (C) To identify metabolite-specific T-cell clones which do not respond to the parent drug, clones were cultured with increasing concentration of dapsone and nitroso dapsone in the presence of autologous EBV-transformed B-cells for 48 h at 37 °C, 5% CO₂. [3H]-thymidine was added for the final 16 h to measure proliferation.

Figure 2

Pathway of activation for nitroso-dapsone-specific T-cell clones. (A) Drug-responsive T-cells were cultured for 48 h with nitroso dapsone and glutaraldehyde-fixed or irradiated EBV-transformed B-cells, or in the absence of B-cells. [3H]-thymidine was added for the final 16 h for proliferative assessments. (B) Autologous EBV-transformed B-cells were pulsed with nitroso dapsone for 24 h before washing to remove the unbound drug. These B-cells were then cultured with clones alongside in the absence of soluble drugs for 48 h. Soluble nitroso dapsone served as a positive control. [3H]-thymidine was added for the final 16 h to measure proliferative responses.

Figure 3

Viability of primary human hepatocytes upon dapsone exposure. To assess whether the drug concentrations utilized in the coculture were nontoxic, primary human hepatocytes were cultured in the presence of increasing concentrations of dapsone for 48 h. The CellTiter-Glo Cell Viability Assay, a luminescence-based assay, was used to measure viability of hepatocytes. Blank and untreated control wells were used to calculate a % viability value (i.e., % of mean viable cells in comparison to the untreated control once adjusted for background luminescence). The experiment was repeated on separate occasions with hepatocytes from three donors.

Figure 4

Establishment of the IdMOC hepatocyte immune cell coculture system for metabolite generation and T-cell activation studies. (A) Freshly isolated primary human hepatocytes were plated in the middle two wells of a 96-well chamber with T-cells and EBV-transformed B-cells plated in the outer four wells. The chamber was then loaded with dapsone, covering all cellular components. (i) Seeded hepatocytes after 48 h culture and (ii) undisturbed clones and EBV-transformed B-cells after 48 h culture. Similar chambers were set up with medium only and nitroso dapsone as negative and positive controls (B) after the initial culture with hepatocytes and T-cell clones were moved to 96-well U-bottomed plates and cultured for a further 24 h. [3H]-thymidine was used to measure proliferative responses.

Figure 5

Establishment of a transwell hepatocyte immune cell coculture system for metabolite generation and T-cell activation studies. (A) Primary human hepatocytes were plated in collagen-coated 24-well plates. An insert was fitted on top of the hepatocyte wells and loaded with T-cell clones and EBV-transformed B-cells. Individual wells were then loaded with an overlaying medium containing medium control, dapsone, or nitroso dapsone (as a positive control). (B) After 48 h, T-cells were harvested from the insert, transferred to 96-well U-bottomed plates, and cultured for a further 24 h. [3H]-thymidine was then added to assess proliferative responses. Statistical analysis compares cultures with and without drugs, Student’s t-test (*P < 0.05; ** P < 0.01).

Figure 6

Establishment of an in-house designed and printed hepatocyte immune cell coculture system for metabolite generation and T-cell activation studies. (A) Self-designed plate has a larger surface area to seed hepatocytes and immune cells. Clones and EBV-transformed B-cells were separated from hepatocytes as indicated. Medium, dapsone, or nitroso dapsone (positive control) were added to the cells for 48 h. T-cells were then harvested, transferred to 96-well U-bottomed plates, and cultured for a further 24 h. [3H]-thymidine was then added to assess proliferative responses.

Figure 7

Mass spectrometric analysis of dapsone metabolites formed in the hepatocyte immune cell cocultures. (A) MS/MS spectra show that a sulfonamide glutathione adduct was formed by direct incubation of the synthetic nitroso dapsone with glutathione without hepatocytes. (B) Similar adduct was detected when dapsone was cultured with hepatocytes in the presence of glutathione. (C) Extracted ion chromatograms demonstrate that the glutathione adduct formed in the hepatocyte culture (black trace) has the same retention time as the synthetic adduct (blue trace). (D–F) MS/MS spectra show that additional dapsone metabolites including dapsone hydroxylamine (D), mono-acetyl dapsone (E), and azoxy dapsone (F) were also detected in hepatocyte immune cell cocultures. All dapsone metabolites detected in the hepatocyte immune cell cocultures are listed in (G).