CHARACTERISATION OF DRUG-SPECIFIC T-CELLS TOWARDS A MECHANISTIC INSIGHT OF DRUG HYPERSENSITIVITY REACTIONS

Drug hypersensitivity reactions are rare but remain a serious problem in clinical practice. Many reactions to a number of drugs are associated with expression of individual HLA class I markers. Carbamazepine (CBZ) is an anticonvulsant which causes severe cutaneous adverse reactions associated with expression of HLA-A*31:01, HLA-B*15:02, HLA-B*15:21, and HLA-B*57:01. Although, in vitro analyses has shown that the CBZ-mediated adverse reaction is associated with activation of drug-specific CD4+ and CD8+ T-cells, the association between drug HLA class I and class II between and T-cell activation has not been fully established. In this thesis, the CBZ-mediated response was evaluated using peripheral blood mononuclear cells from CBZ-naïve healthy donors and CBZ hypersensitive patients from the University of Liverpool, UK, and Prince of Songkla University, Thailand. The in vitro drug response of each participant was evaluated by lymphocyte transformation test and ELISpot and further characterised by T-cell cloning. A total of 76 CBZ-specific CD4+ T-cells were generated by T-cell cloning in 2/5 healthy donors from the University of Liverpool cohort and 4/6 CBZ hypersensitive patients from both cohorts. The generated T-cell clones were polyclonal in terms of CBZ-10,11-epoxide cross-reactivity pattern and T-cell receptor (TCR) Vβ phenotype. Cytokine secretion analysis revealed that the generated CD4+ T-cells secreted IFN-γ and granulysin in response to CBZ. A detailed characterisation showed that the CD4+ CBZ-mediated response was restricted to HLA-DR, particularly HLA-DRB1*07:01. A database review substantiated that this marker was previously reported to be associated with CBZ-induced Stevens-Johnson syndrome and toxic epidermal necrolysis. The HLA-DR allele is also often linked with HLA-B*57:01 as a haplotype. These findings suggested that this HLA class II marker may co-contribute with the HLA class I marker as a haplotype via selective presentation of CBZ to CD4+ T-cells. TCRs that interact with HLAs were characterised by TCR sequencing analyses of drug-specific T-cells. T-cell clones that respond specifically to CBZ, sulfamethoxazole, nitroso-sulfamethoxazole, dapsone, and nitroso-dapsone were included in the sequencing analysis. A remarkable heterogeneity was observed among drug-specific T-cells, notably even among clones that respond to the same drug. No clear relationship between TCR residue expression and T-cell responsiveness (cytokine release, cross-reactivity) was identified. The β-lactamase inhibitors tazobactam and clavulanic acid often cause hypersensitivity when administrated alongside β-lactam antibiotics. Unlike β-lactam hypersensitivity, the cross-reactivity pattern of T-cells responsive to β-lactamase inhibitors is not well understood. Herein, the cross-reactivity of tazobactam- and clavulanate-responsive T-cells was characterised. Clavulanate-specific T-cells showed a total cross-reactivity against tazobactam and the reverse was also true. Mass spectrometric analysis revealed that tazobactam and clavulanate form similar protein adducts, thereby potentially presenting similar modified peptides to T-cells. Collectively, this work showed that there are highly heterogeneous molecular components that underlie drug hypersensitivity reactions. Expression of specific HLA, TCR, and antigenic drug molecule alongside yet to be determined environmental factors all determine whether drug exposure will result in a T-cell response and hypersensitivity