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Title: The role of T-helper 17 and T-helper 22 lymphocytes in beta-lactam hypersensitivity
Author: Sullivan, Andrew
ISNI:       0000 0004 6059 8542
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2016
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Beta-lactam antibiotics are used to treat recurrent opportunistic infections that occur in patients with cystic fibrosis. However, the incidence of reported hypersensitivity in these patients is greatly higher than the healthy population. Due to the delayed nature of beta-lactam hypersensitivity, a T-cell mediated immune response is implicated. Type IV drug hypersensitivity is a major clinical concern, with a cutaneous aetiology driven through antigen specific T cells. Pro- and anti-inflammatory cytokines can be detected in reactions to β-lactam drugs in responsive T cells from hypersensitive patients. Classical Th1/Th2 phenotypes are currently used to classify the reactions, though these do not accurately characterise the function of the immune cells. In addition, this classification does not reference the newer Th subsets such as Th9, Th17 and Th22. To characterise the nature of the T-cell response observed in piperacillin hypersensitive patients with cystic fibrosis, T-cell clones from both blood and inflamed skin of hypersensitive patients were generated, then characterised in terms of phenotype and function. Additional investigations into PBMC responses were conducted. Naive T cells from healthy donors were also primed to piperacillin, as well as attempted regulation of the cytokine response through modulation of a selected nuclear receptor involved in the progression of a Th22 mediated response. Drug-specific clones were generated from both blood (n=570, 84% CD4) and skin (n=96, 83% CD4) samples obtained from patients hypersensitive to piperacillin. All clones secreted high levels of IFN-γ and IL-13. Interleukin-22, perforin and granzyme B were secreted in over 50% of clones, with none from either blood or skin showing any detectable level of IL-17A. Naive T cells primed to piperacillin via autologous dendritic cells showed proliferative responses (p=0.001, SI > 2). Clones generated from primed T-cells showed similar patterns of cytokine secretion when compared alongside clones generated from hypersensitive patients. Significant differences in chemokine receptor expressions were observed between blood-derived piperacillin-specific clones, skin-derived piperacillin-specific clones and skin-derived non piperacillin-specific clones. CLA, CXCR6 and CCR1 expression was higher on piperacillin-specific skin derived clones when compared to non-piperacillin specific skin derived clones (p=0.01). CCR2, CCR4, CXCR1 and E-cadherin were higher on skin specific clones when compared to blood specific clones (p=0.01). Piperacillin specific clones isolated from blood and skin of hypersensitive patients, as well as healthy donor PBMC migrated in the presence of chemokines specific to their respective cell surface receptors, with migration to CCR4 and CCR10 most prevalent. In addition, modulation of the aryl hydrocarbon receptor showed that an abrogation of the cytokine response was observed in cells treated with an AhR inhibitor. This abrogation was only observed in the secretion of interleukin-22, a key cytokine in a Th22 response. Currently, T-cell mediated hypersensitivity involving drug binding to the HLA molecule has only been shown for HLA class I molecules; no data has provided evidence for HLA class II interactions with drugs being able to activate CD4+ T-cells. To investigate whether HLA class II molecules binding to drugs could activate T-cells, an investigation into the COX-2 specific NSAID lumiracoxib was performed. Lumiracoxib was withdrawn from use after incidences of liver toxicity were reported in 2008. Utilising a naive T-cell dendritic cell co-culture assay, attempts were made to generate drug-specific responses from lymphocytes to either lumiracoxib or its major/minor metabolites. No positive responses were generated, with all assays performed showing no activation of drug-specific T-cells following re-challenge with the drugs. In conclusion, the data generated over the course of this thesis has shown that there is a subset of piperacillin-specific T-cells in hypersensitive patients with cystic fibrosis that secrete IL-22, IFN-γ, perforin and granzyme B in response to antigen challenge. No cells secreted IL17, suggesting a strong Th22 phenotype rather than Th17. In addition, The AhR signalling pathway is also heavily implicated in the development of hypersensitivity, giving further evidence for the role of both IL-22 and Th22 lymphocytes in beta-lactam hypersensitivity.
Supervisor: Naisbitt, D. J. ; Park, B. K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral