Abacavir is an antiretroviral drug used in the treatment of HIV-1 infection. abacavir hypersensitivity syndrome (AHS) is a CD8+ T cell-mediated drug hypersensitivity reaction strongly associated with human leukocyte antigen (HLA)-B*57:01. AHS clinically manifests with systemic symptoms such as fever, gastrointestinal disturbances and rash, which becomes more severe with continued use. The molecular mechanism of AHS involves the occupation of the peptide-binding cleft of HLA-B*57:01 by abacavir, which alters the repertoire of self-peptides presented to T cells. The broad range of novel epitopes induced by the drug results in a polyclonal response by CD8+ T cells. However, abacavir/peptide/HLA complexes that stimulate specific CD8+ T cell receptors (TCRs) have yet to be identified.
Here, we describe the identification epitopes of two abacavir-specific TCRs, which differ in their requirement for the transporter associated with antigen processing (TAP). HLA-B*57:01 peptide ligands were identified from drug-treated HLA-B*57:01+ cell lines (TAP-sufficient C1R.B*57:01 and 1106 KERTr, and TAP-deficient T2.B*57:01). The immunopeptidome workflow involved immunoaffinity purification of HLA molecules, fractionation of peptides and HLA molecules by RP-HPLC and peptide identification by tandem mass spectrometry. Selected peptides were screened with SKW3 reporter cells transduced with TCRs derived from abacavir-stimulated T cells of HLA-B*57:01+ donors, using a CD69 upregulation assay. The screening was performed with 39 peptide ligands of HLA-B*57:01 (identified in abacavir-treated C1R.B*57:01) and 10 signal peptides (identified in the overlap of abacavir-treated C1R.B*57:01, 1106 KERTr and T2.B*57:01 cell lines). The stimulation via the first TCR was TAP-dependent, and the epitope mapped to a widely expressed protein, Ribophorin II. The stimulation of the second TCR was TAP-independent and the epitope mapped to the signal peptide of HLA-DPB1.
These findings highlight different pathways and sources of epitopes for abacavir-specific TCRs, which likely contribute to the complexity of AHS immunopathology. Additionally, this study demonstrates that the discovery of drug-induced peptides by mass spectrometry can be combined with functional assays to identify drug-specific T cell epitopes. Current work strives to characterise the molecular interactions within the abacavir/peptide/HLA/TCR complexes that could be targeted to abrogate these life-threatening immune responses.