Characterisation of Drug-Specific T-Cell Responses in Hypersensitive Patients and Healthy Donors

Al-Attar, ZI (2016) Characterisation of Drug-Specific T-Cell Responses in Hypersensitive Patients and Healthy Donors. PhD thesis, University of Liverpool.

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Abstract

Drug hypersensitivity reactions represent a significant clinical problem and an impediment to drug development. It is currently impossible develop drugs with no immunological liability; furthermore, it is very difficult to predict which individuals will develop hypersensitivity when exposed to a therapeutic treatment regimen. This is because susceptibility is a function of the chemistry of the drug, the genetic background of the patient and environmental factors such as patient demographics, disease and concomitant medications. In this study, we focused on two drugs: piperacillin and telaprevir as examples of idiosyncratic hypersensitivity reactions. Regarding piperacillin, we characterized the piperacillin reactive T-cells response of piperacillin hypersensitive patients with cystic fibrosis in terms of proliferation, cytokine secretion in addition to TCR-Vβ and chemokine receptor expression. Piperacillin-responsive CD4+ clones expressing a diverse TCR-Vβs proliferated and secreted Th1/Th2 cytokines in a dose-dependent manner. Clones expressed chemokine receptors consistent with a mixed Th1/Th2 response. Piperacillin responsive T-cells displaying a similar phenotype were also generated from naïve healthy volunteers using a dendritic cell-T-cell priming technique. However, TCR-Vβ expression was more restricted; high expression of TCR-Vβ 9, 13.2, 18 and 4 was detected. Piperacillin is β-lactam antibiotic that is known to covalently modify lysine residues on proteins such as human serum albumin. Therefore, we characterized the absolute levels of piperacillin protein binding in patients and in vitro culture and whether piperacillin albumin adducts activate patient T-cells. With the aid of mass spectrometry, we identified the main lysine residues that piperacillin modifies and synthesized a piperacillin modified peptide incorporating amino acid residues of albumin to establish a standard curve for quantification of binding. The level of modified Lys541 ranged from 2.7-4.7% in patients. Analysis of incubation medium from piperacillin-responsive clones revealed that a similar level of piperacillin-modified Lys541 in albumin was required for the stimulation of T-cells. Antigen presenting cells cultured with piperacillin for 24h also activated the clones with 2.8% Lys541 modification at this time-point. Piperacillin-albumin conjugates that had levels and epitopes identical to those detected in patients were synthesized and purified, and were shown to stimulate T cells in an antigen processing dependent manner. Piperacillin-albumin conjugate clones expressed a restricted pattern of TCR-Vβ with high expression of TCR-Vβ 9 in many clones. Telaprevir is antiviral drug used for the management of hepatitis C. We succeeded in generating clones responsive to a telaprevir metabolite (VRT-127394) using PBMCs priming technique and these clones were found to be 100% cross reactive with telaprevir. These clones were found to be activated via the direct binding of the drug (metabolite) to MHC molecules on antigen presenting cells. Most clones were CD4 T-cells and they displayed a restricted pattern of TCR-Vβ expression. Together, our results define in immunological, chemical and quantitative terms the drug immune receptor interactions that can drive a T-cell response. For β-lactam antibiotics, the levels of modification that activated T-cells in vitro are equivalent to the ones formed in patients.

Item Type:	Thesis (PhD)
Divisions:	Faculty of Health and Life Sciences
Depositing User:	Symplectic Admin
Date Deposited:	17 Aug 2017 14:33
Last Modified:	19 Jan 2023 07:24
DOI:	10.17638/03004974
Supervisors:	Naisbitt, D Park, ORCID: 0000-0001-8384-824X
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3004974