Characterization of p-Phenylenediamine–Albumin Binding Sites and T-Cell Responses to Hapten-Modified Protein

Abstract

Exposure to p-phenylenediamine (PPD) is associated with the development of T-cell-mediated allergic contact dermatitis. The purpose of this study was to define the nature of the interaction of PPD with the protein and the antigenic determinant that stimulates T cells. Mass spectrometry was employed to show that PPD oxidation products bind irreversibly to cysteine (Cys, position 34) in human serum albumin (HSA). A modified tryptic peptide was characterized with an increase in mass of 106 Da, corresponding to the addition of PPD and not to the secondary products of self conjugation. Lymphocytes from 10 PPD-allergic patients, but not tolerant/naive individuals, were stimulated with PPD and PPD-modified HSA. A total of 70 PPD-specific and 10 PPD-HSA-specific CD4+, CD8+, and CD4+CD8+, Th2-secreting T-cell clones were generated from three allergic patients. In total, 40 clones were stimulated with both PPD and PPD-modified HSA. PPD-modified HSA triggered T-cell responses through a classical hapten mechanism involving processing. Presentation of PPD to several clones was dependent on protein complex formation (42 out of 48) and processing (32 out of 68); however, 12% of clones were triggered with PPD directly. These data identify Cys as the single target for PPD-HSA binding, and show that PPD protein adducts are antigenic determinants in patients with contact dermatitis.

Identification of PDL-1 as a novel biomarker of sensitizer exposure in dendritic-like cells

“…Sakaguchi et al (2006) also observed variable responses depending on both the sensitizer and cell type used suggesting that sensitizers act differently depending on the cellular system (Sakaguchi et al, 2006). Recent work has indicated that the cell responsiveness to sensitizers is affected by external 'danger signals' (Lavergne et al, 2009) and hapten:protein interactions (Jenkinson et al, 2009) hours (Goldstein et al, 1996;Pype et al, 1999). Therefore, with regards to moDC MCP-1 secretion, optimisation of the time point for maximal detection may be required.…”

Mass Spectrometric Characterization of Circulating and Functional Antigens Derived from Piperacillin in Patients with Cystic Fibrosis

“…We have recently developed and employed MS methods to qualify the site(s) of drug-protein conjugation (20)(21)(22)(23). Drugs and drug metabolites bind in a dose-dependent manner to proteins such as albumin and can display different preferences for the sites of modification.…”

“…For Ag formation, the b-lactam ring is targeted by nucleophilic lysine residues, leading to ring opening and binding of the penicilloyl group (17)(18)(19). We have developed novel mass spectrometric techniques to define unequivocally the chemistry of drug-protein conjugation in patients under physiological conditions (20)(21)(22)(23). In this study, we report on the methods we have developed to detect and fully characterize circulating Ags derived from piperacillin and its metabolite in patients undergoing therapy.…”

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

“…Table 2 summarizes the studies that have used protocols for priming of naive T cells with chemicals applied as shown in Fig. 2 and studies that have tested haptenated self-proteins as antigens for T-cell lines and clones from allergic patients [52][53][54]. Most protocols have used haptenmodified DC as APC and naive T cells as responder cells.…”