title: Characterisation of the chemical basis of T-cell mediated β-lactam hypersensitivity reactions
Creators: name: Yaseen, Fiazia
Publisher: name: University of Liverpool Repository
Year: 2015

description: The idiosyncratic nature of drug hypersensitivity reactions poses a major clinical problem as they are difficult to predict and can be severe in nature. β-lactam antibiotics provide the cornerstone of treatment for pulmonary exacerbations in patients with cystic fibrosis (CF). Unfortunately, their use is complicated due to a high rate of delayed-type hypersensitivity reactions. This body of work was undertaken to investigate the chemical and cellular mechanisms of β-lactam-induced T-cell mediated hypersensitivity. Approximately 20% of patients with CF develop multiple drug hypersensitivity (MDH). Peripheral mononuclear blood cells (PBMCs) isolated from 4 patients, but not controls, were activated with three drugs (piperacillin, meropenem &amp; aztreonam). T-cell clones were isolated from the MDH patients and characterised for phenotype, function and cross reactivity. Piperacillin-, meropenem- and aztreonam-responsive T-cell clones (mainly CD4+) proliferated and released IFN-γ following drug stimulation. Clones were drug-specific; cross reactivity was not observed. All 3 drugs formed structurally distinct haptenic structures with specific lysine residues on HSA in a time- and concentration-dependent manner. β-lactam antibiotics are widely thought to activate immune cells through the covalent modification of proteins, but the link between conjugate formation and an immune response is still to be defined. T-cell clones from piperacillin hypersensitive patients were stimulated by piperacillin-pulsed antigen presenting cells (APCs) in a time dependent manner and T-cell responses were inhibited following inhibition of protein processing. Synthetic conjugates generated via two methods were investigated. Conjugates were similar in terms of sites of modification and relative level of binding at each site. Conjugate 1 did not stimulate T-cells while responses were observed with conjugate 2. The higher concentration of unbound piperacillin detected in conjugate 2 led to the hypothesis that HSA conjugates may facilitate the transfer of piperacillin to appropriate binding sites on MHC molecules to initiate a T-cell response. T-cell clones were subsequently generated from PBMC cultured with piperacillin conjugate 1. Several clones were stimulated with conjugate 1, but not the parent drug highlighting the existence of T-cells with specificity against different drug-derived antigens. Using a synthetic piperacillin-modified HSA peptide (PIP-K541 peptide) as a standard, a novel mass spectrometric method was developed to quantify the level of piperacillin protein binding needed to activate T-cells. Levels of PIP-K541 in patient plasma ranged from 2.5-6.5%. In vitro modification ranged from low (1%) coinciding with weak proliferative responses piperacillin-specific T-cell clones to high at around 4%. From these experiments it was possible to estimate that 2.8% modification of albumin is sufficient for effective T-cell activation. The β-lactam antibiotic flucloxacillin is associated with a high incidence of liver reactions. Genome-wide association studies identified HLA-B*57:01 as an important susceptibility factor. CD8+ T-cell clones from patients with liver injury were activated by flucloxacillin to proliferate and secrete cytokines in an HLA-B*57:01 restricted and dose-dependent manner. T-cell activation was detected with both soluble drug and drug-pulsed APC. In contrast, clones from volunteers were only activated with soluble drug and the response was not HLA-B*57:01 restricted. This work quantifies β-lactam-protein binding in patients and relates this to T-cell activation. Furthermore, the immunological studies show that T-cells from hypersensitive patients are activated via multiple pathways.