Dapsone‐ and nitroso dapsone‐specific activation of T cells from hypersensitive patients expressing the risk allele HLA‐B*13:01

BACKGROUND:Research into drug hypersensitivity associated with 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 (1) explore whether the parent drug and/or nitroso metabolite activates T-cells and (2) determine whether HLA-B*13:01 is involved in the response. METHODS:PBMC from 6 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:PBMC from 6 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 crossreactive and strongly cross reactive. Nitroso dapsone formed dimers in culture and was reduced to dapsone, providing a rationale for the crossreactivity. 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. This article is protected by copyright. All rights reserved

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades

Influence of reduced glutathione on the proliferative response of sulfamethoxazole-specific and sulfamethoxazole-metabolite-specific human CD4+ T-cells

1. Hypersensitivity to the drug sulfamethoxazole (SMX) is thought to be a consequence of bioactivation to the hydroxylamine metabolite (SMX-NHOH) and further oxidation to the ultimate reactive metabolite, nitroso-sulfamethoxazole (SMX-NO). SMX-NO covalently modifies self proteins which in turn might be recognized as neo-antigens by T-cells. The antioxidant glutathione (GSH) is known to protect cells from reactive metabolites by conjugation and subsequent dissociation to SMX-NHOH and/or SMX. 2. To study the reactivity of T-cells to SMX metabolites and their respective role in the generation of drug-specific T-cells, we analysed the effect of GSH on the response of PBMC to SMX and its metabolites SMX-NHOH and SMX-NO. Furthermore, we monitored the proliferative response of drug-specific T-cell clones in the presence or absence of GSH. 3. We found that addition of GSH to peripheral blood mononuclear cells had no effect on the SMX-specific response but enhanced the proliferation to SMX-metabolites. The response of SMX-NO-specific T-cell clones was abrogated when GSH was present during the covalent haptenation of antigen presenting cells (APC). Conversely, SMX-specific T-cell clones gained reactivity through the conversion of SMX-NO to the parent drug by GSH. While GSH had no effect on the initial activation of T-cell clones, it prevented covalent binding to APCs, reduced toxicity and thereby led to proliferation of drug-specific T-cells to non-reactive drug metabolites. 4. Our data support the concept that in allergic individuals T-cells recognize the non-covalently bound parent drug rather than APC covalently modified by SMX-NO