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Title: Generation and characterization of dapsone- and nitroso-dapsone-specific T-cells : an insight into the molecular pathomechanism of dapsone hypersensitivity syndrome
Author: Alzahrani, A. A.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Dapsone (DDS) is an antibiotic associated with hypersensitivity reactions in 0.5 to 3.6 % of patients. Although clinical diagnosis is indicative of a drug 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 (i) explore the immunogenicity DDS and nitroso DDS (DDS-NO) using PBMC from healthy donors and splenocytes from mice, (ii) generate human T-cell clones to characterize mechanisms of T-cell activation and (iii) explore how the two antigenic forms of the dapsone interact with HLA-B*13:01, which is associated with the development of the DDS hypersensitivity among patients with leprosy. Naïve T-cell priming to DDS and DDS-NO was successful in three human donors. DDS-specific CD4+ T-cell clones generated from 2 donors were stimulated to proliferate in response to the 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. To explore whether similar T-cell responses are detected in hypersensitive patients expressing HLA-B*13:01, we obtained samples from 6 patients, and evaluated T-cell specificity and the phenotype of drug-specific clones. Lymphocytes from certain patients proliferated in the presence of DDS and DDS-NO. DDS and DDS-NO-specific CD4+ clones were generated: DDS-specific clones were activated by the drug binding directly to HLA molecules on antigen presenting cells, while DDS-NO-specific clones were activated via a hapten mechanism involving formation of drug protein adducts and antigen processing by antigen presenting cells. Mass spectrometry was used to show that DDS-NO modified cysteine residues on mouse serum albumin and human glutathione-S-transferase P1.
Supervisor: Naisbitt, Dean ; Park, Kevin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral