Investigation of immunogenetic risk factors for carbamazepine-induced hypersensitivity reactions

T-cell mediated hypersensitivity reactions (HSRs) to carbamazepine (CBZ), a commonly used anti-epileptic drug, occur only in a small proportion of patients, but can often be severe in nature. As the underlying pathomechanisms are not fully understood, it has proven difficult to predict who may be at risk of developing CBZ-induced HSRs. Recently, specific human leukocyte antigen (HLA) alleles have been identified as susceptibility factors for CBZ hypersensitivity in diverse populations, indicating that HLA molecules may be functionally involved in CBZ-induced T-cell activation. HLA-A*31:01 represents the latest example and has been implicated in CBZ-induced HSRs in Caucasian patients. Thus, the aim of this work was to explore the molecular interactions of CBZ with HLA-A*31:01 and drug-specific T-cells. The HLA restriction pattern of CBZ-reactive T-cells from a patient expressing HLA-A*31:01 was investigated. It was shown that CD8+ T-cells were activated in a HLA-A*31:01 dependent way. Further, HLA-DRB1*04:04 was found to be responsible for the stimulation of CD4+ T-cells, suggesting a common HLA haplotype may be involved in mediating T-cell responses to CBZ in Europeans. Next, in vitro priming of drug-naïve T-cells from HLA-A*31:01+ healthy volunteers against CBZ was attempted. Weak responses to CBZ could be detected in some but not all volunteers, indicating factors additional to HLA- A*31:01 are required to induce a primary stimulation of T-cells to CBZ. Besides, the removal of T-regulatory cells and use of dendritic cells as antigen- presenting cells seemed to generally improve priming conditions. In order to investigate whether CBZ affected the peptide-binding specificity of HLA-A*31:01, in silico and in vitro analysis were performed. In silico modelling provided a possible binding site for CBZ within the HLA peptide-binding cleft. A peptide elution study provided a preliminary indication that binding of CBZ to HLA-A*31:01 may alter the peptide repertoire presented by the allele, which could potentially result in T-cell activation. Most recently, it has been suggested that the T-cell receptor (TCR) may represent an additional predisposing factor for CBZ-induced HSRs. Accordingly, a protocol for the analysis of the TCR Vβ repertoire of drug-reactive T-cells by flow cytometry as well as CDR3 spectratyping was set up using T-cells from healthy donors primed against the model antigen SMX-NO. Both methods showed that antigen stimulation resulted in skewing of common TCR Vβ subtypes among the donors. Combined, the optimised methods will allow assessment of whether specific TCR clonotypes may be implicated in HLA- A*31:01 associated HSRs to CBZ. In summary, the data presented in this thesis provide initial evidence that CBZ is able to interact directly, through a non-covalent binding mechanism, with HLA-A*31:01 causing T-cell activation. However, it cannot be excluded that the stimulation of T-cells in vivo requires the formation of a hapten complex. Further work is needed to define other factors that are involved in predisposing an individual to CBZ hypersensitivity