Definition of antigenic determinants in drug hypersensitive patients: an integrated clinical, chemical and cellular approach to quantify and characterize the drug signals presented to T-Lymphocytes

Ogese, Monday ORCID: 0000-0002-1873-4032
Definition of antigenic determinants in drug hypersensitive patients: an integrated clinical, chemical and cellular approach to quantify and characterize the drug signals presented to T-Lymphocytes. Doctor of Philosophy thesis, University of Liverpool.

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Idiosyncratic drug hypersensitivity remains a major challenge as it causes high morbidity and mortality. This is complicated by the multiple risk factors implicated and the inability to predict these reactions during the early stages of drug development. Thus, this study attempted to delineate the molecular pathomechanism(s) involved in sulfamethoxazole (SMX) hypersensitivity. The reactive metabolite, nitroso-SMX (SMX.NO) generated through the hepatic bioactivation of SMX has long been hypothesised as a major trigger of these reactions. SMX hypersensitivity has been used as a paradigm to study the role of drug metabolism in the activation of T-cells as the synthetic nitroso metabolite is available for functional studies. Metabolism of SMX in hepatic tissue has been extensively studied. CYP2C9 and Myeloperoxidase (MPO) are implicated in the formation of SMX.NO. However, it is unclear whether the SMX.NO generated in the liver migrates to the skin; the primary target in SMX hypersensitivity. It is possible that localised SMX metabolism by immune cells resident in the skin are implicated in the observed reactions. ELISA data revealed SMX metabolism in EBV-transformed B-cells used as antigen presenting cells (APCs). SMX-metabolism was significantly inhibited by methimazole. Furthermore, Western blotting and RT-PCR analyses suggested the presence of low concentrations of MPO in EBV-transformed B-cells. Interestingly, RT-PCR revealed mRNA expression of flavine containing monooxygenases (FMO1-5), TPO and LPO but the protein levels of these enzymes were not detected in immune cells. Subsequent experiments involved the generation and LC-MS/MS characterization of SMX.NO-modified MPO adducts. Although SMX.NO formed both the sulphinamide and N-hydroxysulfinamide adducts, drug specific T-cell clones failed to proliferate in response to drug-modified peptides. Since SMX.NO binds to multiple cellular proteins, it is assumed that peptides derived from the modified protein interact with a number of diverse HLA molecules to activate T-cells. However, the HLA molecules that interact with SMX.NO-modified peptides have not been defined. This study therefore examined the HLA molecules that present SMX.NO (derived peptides) to T-cells. T-cell clones (TCCs) were generated from 5 hypersensitive patients with cystic fibrosis. Fast growing TCCs from 2 SMX hypersensitive patients were used for HLA restriction studies. Drug-specific proliferative response, cytokine secretion and cytolytic markers were measured using [3H]-thymidine incorporation and ELIspot assays. Anti-human class I and class II (DR, DP, and DQ) antibodies were used to determine HLA restriction of drug-specific T-cell activation. APCs expressing similar or different HLAs were used to define the alleles involved in the presentation of SMX.NO-derived antigens to T-cells. A total of 1578 clones were tested for SMX.NO reactivity. Seventy-seven CD4+ clones were activated to proliferate and secrete IFN-ϒ, IL-5, IL-13 and granzyme-B by SMX.NO. Only one TCC was CD8+No cross reactivity with SMX was observed. The SMX.NO-specific response of clones was blocked with antibodies against MHC class II and HLA-DQ. Clones from 2 patients (Patient 1: HLA-DQB1*05:01:01G/ DQB1*06:03:01G; Patient 2: HLA-DQB1*02:01:01G/DQB1*02:01:01G) were used to define the DQ alleles involved in the presentation of SMX.NO derived antigens. SMX.NO-specific responses were detected with heterologous APCs expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. Activation of PD-1 on T-cells is thought to inhibit antigen-specific T-cell priming and regulate T-cell differentiation. Thus, this study sought to measure the drug-specific activation of naïve T-cells after perturbation of PD-L1/PD-1 binding and investigate whether PD-1 signalling influences the differentiation of T-cells. Naive T-cells were co-cultured with monocyte-derived dendritic cells in the presence of SMX.NO for a period of 8 days (±PD-1/2 block) and T-cell priming investigated using readouts for proliferation and cytokine secretion. Priming of naïve T-cells against SMX.NO was found to be more effective when PD-L1 signalling was blocked. Drug-specific TCCs generated through priming and from hypersensitive patients were found to secrete IFN-γ, IL-5 and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL22 secreting clones expressed the skin homing receptors CCR4, CCR10 and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the antigen-specific proliferative response or the secretion of cytokines/cytolytic molecules. In conclusion, this study used a variety of in vitro assays to investigate the multiple factors involved in the pathomechanism of SMX hypersensitivity. A clear understanding of mechanisms of drug hypersensitivity will provide insights that aid drug design and reduce the frequency of such reactions.

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2014-04 (completed)
Subjects: ?? RM ??
Divisions: Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 05 Aug 2014 10:35
Last Modified: 16 Dec 2022 04:42
DOI: 10.17638/00018733