Deciphering Adverse Drug Reactions:In VitroPriming and Characterization of Vancomycin-Specific T Cells From Healthy Donors Expressing HLA-A*32:01

Abstract

Drug rash with eosinophilia with systemic symptoms (DRESS) is a serious adverse event associated with use of the glycopeptide antibiotic vancomycin. Vancomycin-induced DRESS is associated with the expression of HLA-A*32:01, suggesting that the drug interacts with this HLA to activate CD8+ T-cells. The purpose of this study was to utilize PBMC from healthy donors to: (i) investigate whether expression of HLA-A*32:01 is critical for the priming naïve of T-cells with vancomycin and (ii) generate T-cell clones (TCC) to determine whether vancomycin exclusively activates CD8+ T-cells and to define cellular phenotype, pathways of drug presentation and cross-reactivity. Dendritic cells were cultured with naïve T-cells and vancomycin for 2 weeks. On day 14, cells were re-stimulated with vancomycin and T-cell proliferation was assessed by [3H]-thymidine incorporation. Vancomycin-specific TCC were generated by serial dilution and repetitive mitogen stimulation. Naïve T-cells from HLA-A*02:01 positive and negative donors were activated with vancomycin; however the strength of the induced response was significantly stronger in donors expressing HLA-A*32:01. Vancomycin-responsive CD4+ and CD8+ TCC from HLA-A*32:01+ donors expressed high levels of CXCR3 and CCR4, and secreted IFN-γ, IL-13 and cytolytic molecules. Activation of CD8+ TCC was HLA class I-restricted and dependent on a direct vancomycin HLA binding interaction with no requirement for processing. Several TCC displayed cross-reactivity with teicoplanin and daptomycin. To conclude, this study provides evidence that vancomycin primes naïve T-cells from healthy donors expressing HLA-A*32:01 through a direct pharmacological binding interaction. Cross-reactivity of CD8+ TCC with teicoplanin provides an explanation for the teicoplanin reactions observed in vancomycin hypersensitive patients.

Risk Assessment in Drug Hypersensitivity: Detecting Small Molecules Which Outsmart the Immune System

“…The outgrowth of drug reactive cells could be detected after 2–6 weeks by measuring cytokine secretion, upregulation of activation markers, or proliferation. Notably, similar effects could also be induced in cell cultures not expressing the risk allele, as the drug was bound to various HLA-proteins or TCR ( 46 , 47 , 49 ). Under these conditions, the outgrowth of cells seemed to require more time compared to cell cultures expressing the risk allele.…”

“…This stimulation occurs with both hapten-like and nonhapten drugs (p-i). It is cumbersome and requires optimal cell culture conditions for T cell expansion, such as the addition of IL-2 and feeder cells (45)(46)(47)(48)(49).…”

“…The feasibility of this approach using drug-naive individuals was originally assessed using cell cultures stimulated with sulfamethoxazole, flucloxacillin, abacavir, allopurinol, oxypurinol, and antiepileptics and relied mostly on known blood donors expressing the risk allele (45)(46)(47)(48)(49). The outgrowth of drug reactive cells could be detected after 2-6 weeks by measuring cytokine secretion, upregulation of activation markers, or proliferation.…”

“…A research group from Liverpool intensively investigated the cell culture approach using a panel of HLA typed donors ( 40 , 48 , 49 ). They concluded, that a negative cell culture could mean a true lack of stimulation, an absence of presenting HLA in the selected blood donors, or unsuccessful stimulations (due to the experimental conditions).…”

Characterization of Teicoplanin-Specific T-Cells from Drug Naïve Donors Expressing HLA-A*32:01

“…These data are concordant with previous mechanistic findings relating to T-cell responses to vancomycin for which it has been hypothesized glycopeptide compounds possess the capacity to displace and mimic native HLA peptides. 7 Proliferative T-cell cross-reactivity of teicoplanin-responsive TCCs generated from healthy volunteers to daptomycin highlights the complex patterns of reactivity encountered within clinical settings. The observed in vitro T-cell cross-reactivity may be explained by structural similarities between both teicoplanin and daptomycin, specifically the presence of a hydrophobic lipid chain.…”