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

Abstract

A mechanistic understanding of the relationship between the chemistry of drug antigen formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating antigens derived from piperacillin in patients undergoing therapy and the nature of the drug derived-epitopes on protein which can function as an antigen to stimulate T-cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time- and concentration-dependent, with selective modification of Lys541 observed at low concentrations, whereas at higher concentrations up to 13/59 lysine residues were modified, four of which (Lys190, 195, 432 and 541) were detected in patients’ plasma. Piperacillin-specific T-lymphocyte responses (proliferation, cytokines and granzyme-B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T-cells with characterized synthetic conjugates. Analysis of minimally-modified T-cell stimulatory albumin conjugates revealed peptide sequences incorporating Lys190, 432 and 541 as principal functional epitopes for T-cells. This study has characterized the multiple haptenic structures on albumin in patients, and showed that they constitute functional antigenic determinants for T-cells.

Human leukocyte antigen (HLA)-B*57:01-restricted activation of drug-specific T cells provides the immunological basis for flucloxacillin-induced liver injury

“…Using mass spectrometric methods, we recently identified albumin as a major circulating protein modified with b-lactam antibiotics including flucloxacillin, defined the profile of drug protein conjugation at specific lysine residues with respect to dose and incubation time, and characterized for the first time the sites of modification associated with the stimulation of a clinically relevant drug-specific T-cell response. 13,14,16 Herein, we show that (1) the stimulation of clones with flucloxacillin-pulsed antigen-presenting cells, and (2) the detection of flucloxacillin haptens on albumin in culture are time-dependent. Furthermore, simultaneous measurement of antigenicity and immune responsiveness revealed that the cumulative level of flucloxacillin protein binding at each timepoint studied correlated directly with the strength of the T-cell proliferative response.…”

“…Proliferation of patient PBMC (0.15 Â 10 6 /well) against flucloxacillin (0.1-2 mM) and tetanus toxoid (5 lg/mL) was measured using the lymphocyte transformation test. 13 interferon-gamma (IFN-c) and granzyme B-secreting PBMCs were visualized using ELISpot (MabTech, Nacka Strand, Sweden) by culturing PBMC (0.5 Â 10 6 /well; 200 lL) with flucloxacillin (1-2 mM) or PHA (5 lg/mL) for 48 hours. Generation of T-Cell Clones from Patients With Flucloxacillin-Induced Liver Injury.…”

“…Dose ranges have been shown to be optimal for the activation of T cells. 6,[13][14][15] Cell phenotyping was performed by flow cytometry on a BD FACSCanto II using CD4, CD8, CDR1, CCR2, CCR3, CCR4, CCR5, CCR8, CCR9, CCR10, CXCR3, CXCR6, and CLA antibodies (BD Biosciences) and the IOtest Beta Mark TCR Vb repertoire kit.…”

“…To identify the key drug-modified lysine residues in albumin, we utilized our recently described mass spectrometry methods. 13,14,16 Flucloxacillin was incubated with EBV-transformed B cells in RPMI 1640 medium containing 10% human AB serum for 1-48 hours. At each timepoint the cells were removed by centrifugation at 450g.…”

Update on Advances in Research on Idiosyncratic Drug-Induced Liver Injury

“…[41][42][43] Using advanced technologies, such as mass spectrometry, it is possible to define the chemistry of drug-protein conjugation in patients and the nature of the drug-derived epitopes, which can function as an antigen to stimulate T cells. [44][45][46][47][48] There is another mechanism underlying drug-specific T-cell activation. This concept, referred to as the "pharmacological interaction of drugs with immune receptors (p-i concept), " states that drugs by themselves act as antigens interacting in a reversible fashion with immunological receptors.…”