Activation of human CD8+ T-cells with nitroso dapsone-modified HLA-B*13:01-binding peptides()

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. Since 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 9mer 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 antigen presenting cells and C1R cells expressing HLA-B*13:01 were used to determine HLA restriction. HPLC/LCMS confirmed that nitroso dapsone-peptides were modified at the appropriate site and were free of soluble dapsone and nitroso dapsone. Antigen presenting cell HLA-B*13:01-restricted nitroso dapsone-modified Pep1 (n=124) and Pep3 (n=48) responsive 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 characterise 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.