Characterization of the Antigen Specificity of T-Cell Clones from Piperacillin-Hypersensitive Patients with Cystic Fibrosis

Abstract

␤-Lactam antibiotics provide the cornerstone of treatment and reduce the rate of decline in lung function in patients with cystic fibrosis, but their use is limited by a high frequency of delayedtype allergic reactions. The objective of this study was to use cloned T-cells expressing a single T-cell receptor from five piperacillin-hypersensitive patients to characterize both the cellular pathophysiology of the reaction and antigen specificity to define the mechanism of activation of T-cells by piperacillin. More than 400 piperacillin-responsive CD4ϩ, CD4ϩCD8ϩ, or CD8ϩ T-cell clones were generated from lymphocyte transformation test and ELIspot-positive patients. The T-cell response (proliferation, T helper 2 cytokine secretion, and cytotoxicity) to piperacillin was concentration-dependent and highly specific. Enzyme-linked immunosorbent assay, gel electrophoresis, and mass spectrometry revealed that piperacillin bound exclusively to albumin in T-cell culture. Irreversible piperacillin binding at Lys 190, 195, 199, 432, and 541 on albumin and the stimulation of T-cells depended on incubation time. A synthetic piperacillin albumin conjugate stimulated T-cell receptors via a major histocompatibility complex-and processing-dependent pathway. Flucloxacillin competes for the same Lys residues on albumin as piperacillin, but the resulting conjugate does not stimulate T-cells, indicating that binding of the ␤-lactam hapten in peptide conjugates confers structural specificity on the activation of the T-cell receptors expressed on drug-specific clones. Collectively, these data describe the cellular processes that underlie the structural specificity of piperacillin antigen binding in hypersensitive patients with cystic fibrosis.

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

“…Interestingly, the b-lactam antibiotics piperacillin, amoxicillin, and penicillin G, which bind to similar albumin lysine residues, also stimulated the flucloxacillinresponsive clones. The detection of broadly crossreactive T cells is in contrast to our recent studies describing highly drug-specific T cells in piperacillin-hypersensitive patients, 14 and suggests that MHC binding peptides and the core penicilloyl structure provide the binding energy to drive T-cell responses in patients with 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.…”

“…The separated cells were then cloned by serial dilution. 14 Epstein-Barr virus (EBV) transformed B-cell lines were created from PBMC by transformation with supernatant from the virus-producing cell line B9.58. Lines were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen, Paisley, UK), 100 mM L-glutamine, 100 lg/mL penicillin, 100 U/ mL streptomycin, and used as a source of autologous antigen-presenting cells.…”

“…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.…”