Manal Monshi

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

The role of the adaptive immune system in adverse drug reactions that target the liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of human leukocyte antigen (HLA)‐B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, we characterize flucloxacillin‐responsive CD4+ and CD8+ T cells from patients with liver injury and show that naive CD45RA+CD8+ T cells from volunteers expressing HLA‐B*57:01 are activated with flucloxacillin when dendritic cells present the drug antigen. T‐cell clones expressing CCR4 and CCR9 migrated toward CCL17 and CCL 25, and secreted interferon‐gamma (IFN‐γ), T helper (Th)2 cytokines, perforin, granzyme B, and FasL following drug stimulation. Flucloxacillin bound covalently to selective lysine residues on albumin in a time‐dependent manner and the level of binding correlated directly with the stimulation of clones. Activation of CD8+ clones with flucloxacillin was processing‐dependent and restricted by HLA‐B*57:01 and the closely related HLA‐B*58:01. Clones displayed additional reactivity against β‐lactam antibiotics including oxacillin, cloxacillin, and dicloxacillin, but not abacavir or nitroso sulfamethoxazole. Conclusion: This work defines the immune basis for flucloxacillin‐induced liver injury and links the genetic association to the iatrogenic disease. (HEPATOLOGY 2013;)

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

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag 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 out of 59 lysine residues were modified, four of which (Lys190, Lys195, Lys432, and Lys541) 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, Lys432, and Lys541 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.

Characterization of the antigen specificity of T-cell clones from piperacillin hypersensitive patients with cystic fibrosis

β-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 delayed-type 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 CD4+, CD4+CD8+, or CD8+ 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 β-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.

β-Lactam antibiotics form distinct haptenic structures on albumin and activate drug-specific T-lymphocyte responses in multiallergic patients with cystic fibrosis.

β-Lactam antibiotics provide the cornerstone of treatment for respiratory exacerbations in patients with cystic fibrosis. Unfortunately, approximately 20% of patients develop multiple nonimmediate allergic reactions that restrict therapeutic options. The purpose of this study was to explore the chemical and immunological basis of multiple β-lactam allergy through the analysis of human serum albumin (HSA) covalent binding profiles and T-cell responses against 3 commonly prescribed drugs; piperacillin, meropenem, and aztreonam. The chemical structures of the drug haptens were defined by mass spectrometry. Peripheral blood mononuclear cells (PBMC) were isolated from 4 patients with multiple allergic reactions and cultured with piperacillin, meropenem, and aztreonam. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ /IL-13 ELIspot. T-cell clones were generated from drug-stimulated T-cell lines and characterized in terms of phenotype, function, and cross-reactivity. Piperacillin, meropenem, and aztreonam formed complex and structurally distinct haptenic structures with lysine residues on HSA. Each drug modified Lys190 and at least 6 additional lysine residues in a time- and concentration-dependent manner. PBMC proliferative responses and cytokine release were detected with cells from the allergic patients, but not tolerant controls, following exposure to the drugs. 122 CD4+, CD8+, or CD4+CD8+ T-cell clones isolated from the allergic patients were found to proliferate and release cytokines following stimulation with piperacillin, meropenem, or aztreonam. Cross-reactivity with the different drugs was not observed. In conclusion, our data show that piperacillin-, meropenem-, and aztreonam-specific T-cell responses are readily detectable in allergic patients with cystic fibrosis, which indicates that multiple β-lactam allergies are instigated through priming of naïve T-cells against the different drug antigens. Characterization of complex haptenic structures on distinct HSA lysine residues provides a chemical basis for the drug-specific T-cell response.