Background: Vancomycin, a glycopeptide antibiotic routinely used to treat severe Gram-positive bacterial infections, is associated with the development of drug reaction with eosinophilia and systemic symptoms (DRESS) in individuals expressing HLA-A*32:01. Previous studies have identified the potential role of T-cells using HLA-A*32:01 positive healthy donor models. However, DRESS pathogenesis remains poorly defined and a deeper mechanistic understanding is now required to aid the diagnosis and prediction of vancomycin-induced DRESS. The present study aims to elucidate CD4+ and CD8+ T-cell involvement within the pathogenesis of vancomycin-induced DRESS following the isolation and functional study of cloned T-cells from hypersensitive patients. Methods: CD4+ and CD8+ vancomycin-responsive T-cell clones (TCCs) were generated by serial dilution from PBMC samples collected from suspected vancomycin-DRESS patients. Functionality of drug-responsive TCCs was assessed using T-cell proliferation ([ 3H]-thymidine). Cytokine analysis was performed using intracellular cytokine staining (ICS), ELISpot assay and LEGENDplex immunoassays. Results: Vancomycin-responsive TCCs expressing CD4+ and CD8+ phenotypes were successfully generated from suspected vancomycin-DRESS patients (n=3). CD45RO + memory T-cells were the primary activated population, with both CD4+ and CD8+ T-cells associated with the release of IFN-γ, IL-5, IL-13, granzyme B and perforin. Vancomycin-responsive CD4+ and CD8+ T-cells are activated by direct, pharmacological interactions, with antigen presentation possible through both HLA class I and HLA class II molecules. Conclusion: This study provides in vitro evidence for the dual role of antigen-specific CD4+ and CD8+ T-cells within the pathogenesis of vancomycin-induced DRESS. This has been demonstrated following the generation of cloned T-cells with strong vancomycin specificity from patients presenting with vancomycin-DRESS and positive for expression of HLA-A*32:01.