title: Characterisation of the cellular basis of beta-lactam induced skin and liver injury
Creators: name: Monshi, Manal
Publisher: name: University of Liverpool Repository
Year: 2014

description: Drug hypersensitivity reactions are a significant cause of patient morbidity and mortality. They are difficult to predict in the clinic and during the drug development process. This is because mechanisms have not been fully elucidated. In fact, recent data using T-cells cloned from hypersensitive human patients have questioned the long-standing hapten concept, which states that a drug must bind irreversibly to protein to initiate a T-cell response. The work described herein was performed to investigate the chemical and cellular mechanisms of β-lactam-induced skin and liver injury and in particular to understand the relationship between drug-protein adduct formation and the activation of antigen-specific T-cells. Β-lactam antibiotics, such as piperacillin, provide the cornerstone of treatment and reduce the rate of decline in lung function in patients with cystic fibrosis, but use is limited by a high frequency of hypersensitivity reactions. Using the lymphocyte transformation test, drug-responsive lymphocytes were found in approximately 70% of clinically diagnosed piperacillin hypersensitive patients. By cloning over 400 antigen-specific CD4+, CD8+ and CD4+CD8+ T-cells, the T-cell proliferative response and cytokine secretion to piperacillin was shown to be concentration-dependent and highly drug-specific. Mass spectrometry revealed irreversible binding of piperacillin to selective lysine residues on albumin and a synthetic albumin conjugate stimulated piperacilin-specific clones via a processing-dependent pathway. These results describe the cellular processes that underlie piperacillin hypersensitivity. Approximately 20% of hypersensitive patients with cystic fibrosis develop multiple reactions that restrict therapeutic options. To explore the mechanistic basis of multiple β-lactam hypersensitivity, albumin binding profiles and T-cell responses against three commonly prescribed drugs; piperacillin, meropenem and aztreonam, were studied. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ ELIspot. Clones were generated and found to proliferate and release cytokines following stimulation with all three drugs. However, crossreactivity with the different drugs was not observed. Each compound formed distinct haptens with lysine residues on albumin, which may explain the highly drug-specific T-cell response. These data indicate that multiple β-lactam reactions are instigated through priming naïve T-cells against the different drugs. The role of the adaptive immune system in reactions that target liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of HLA-B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, flucloxacillin-responsive CD4+ and CD8+ T-cells were characterized from patients with liver injury. Clones expressed the gut-homing chemokine receptors CCR4 and CCR9 and secreted IFN-γ, Th2 cytokines and cytolytic molecules following drug stimulation. In contrast to the piperacillin clones, flucloxacillin clones were activated with several structurally-related β-lactam antibiotics. Furthermore, naïve CD8+ T-cells from volunteers expressing B*57:01 were activated with flucloxacillin when dendritic cells presented the drug antigen. Activation of CD8+ cells from patients and volunteers was processing-dependent and restricted by HLA-B*57:01, which effectively links the genetic association to the iatrogenic disease. In conclusion, the studies described herein provide novel insight into the way in which β-lactam antibiotics interact with protein and activate T-cells that are thought to be the ultimate mediators of drug hypersensitivity reactions in skin and liver.