Background Research into drug hypersensitivity associated with the 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 (a) explore whether the parent drug and/or nitroso metabolite activate T cells and (b) determine whether HLA‐B*13:01 is involved in the response. Methods Peripheral blood mononuclear cells (PBMC) from six 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 Peripheral blood mononuclear cells from six patients and cloned T cells proliferated and secreted Th1/2/22 cytokines when stimulated with dapsone (clones: n = 395; 80% CD4+ CXCR3hiCCR4hi, 20% CD8+CXCR3hiCCR4hiCCR6hiCCR9hiCCR10hi) 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 cross‐reactive, and strongly cross‐reactive. Nitroso dapsone formed dimers in culture and was reduced to dapsone, providing a rationale for the cross‐reactivity. 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.

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Previous studies have shown that cysteine-reactive drug metabolites bind covalently with protein to activate patient T cells. However, the nature of the antigenic determinants that interact with HLA and whether T cell stimulatory peptides contain the bound drug metabolite has not been defined. Because susceptibility to dapsone hypersensitivity is associated with the expression of HLA-B*13:01, we have designed and synthesized nitroso dapsone–modified, HLA-B*13:01 binding peptides and explored their immunogenicity using T cells from hypersensitive human patients. Cysteine-containing 9-mer peptides with high binding affinity to HLA-B*13:01 were designed (AQDCEAAAL [Pep1], AQDACEAAL [Pep2], and AQDAEACAL [Pep3]), and the cysteine residue was modified with nitroso dapsone. CD8+ T cell clones were generated and characterized in terms of phenotype, function, and cross-reactivity. Autologous APCs and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. Mass spectrometry confirmed that nitroso dapsone–peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. APC HLA-B*13:01–restricted nitroso dapsone–modified Pep1- (n = 124) and Pep3-responsive (n = 48) CD8+ clones were generated. Clones proliferated and secreted effector molecules with graded concentrations of nitroso dapsone–modified Pep1 or Pep3. They also displayed reactivity against soluble nitroso dapsone, which forms adducts in situ, but not with the unmodified peptide or dapsone. Cross-reactivity was observed between nitroso dapsone–modified peptides with cysteine residues in different positions in the peptide sequence. These data characterize a drug metabolite hapten CD8+ T cell response in an HLA risk allele–restricted form of drug hypersensitivity and provide a framework for structural analysis of hapten HLA binding interactions.