Alterations in the HLA-B*57:01 Immunopeptidome by Flucloxacillin and Immunogenicity of Drug-Haptenated Peptides

Abstract

Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a β-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.

Immunopharmacogenomics: Mechanisms of HLA‐Associated Drug Reactions

“…Landsteiner and Jacobs proposed the hapten hypothesis, a process by which chemically reactive compounds covalently bind self‐proteins to form hapten‐carrier complexes and undergo intracellular processing to drug hapten‐modified peptide(s) that subsequently elicit T‐cell response ( Figure a , i ). The beta‐lactam antibiotic flucloxacillin is a key example of a hapten‐forming drug, described to bind protein via lysine residues with risk HLA‐B*57:01–presented flucloxacillin‐modified peptides recently annotated as derived from self‐protein 3 . However, the hapten hypothesis is not suitable for chemically inert drugs such as sulfamethoxazole, which may similarly stimulate patient T cells in cell cultures without capacity for antigen uptake and processing.…”

“…The betalactam antibiotic flucloxacillin is a key example of a haptenforming drug, described to bind protein via lysine residues with risk HLA-B*57:01-presented flucloxacillin-modified peptides recently annotated as derived from self-protein. 3 However, the hapten hypothesis is not suitable for chemically inert drugs such as sulfamethoxazole, which may similarly stimulate patient T cells in cell cultures without capacity for antigen uptake and processing. Thus, the PI concept was established with direct, labile, and noncovalent interaction between drug antigen and HLA or TCR driving rapid T-cell activation (Figure 2a,ii,iii).…”

In Vitro Monitoring of Human T Cell Responses to Skin Sensitizing Chemicals—A Systematic Review

“…This has been shown for MHC I-restricted CD8+ T cells specific for the model chemical 2,4,6-trinitrobenzenesulphonic acid (TNBS) or the β-lactam antibiotic flucloxacillin. Murine responses seem to focus on a lysine modification at peptide position 4 (red-grey striped) [ 44 , 45 ]. ( B ) Some drugs associated with hypersensitivity reactions bind non-covalently, which is called pharmacological interaction (p-i) [ 46 , 47 ].…”

“…Patient-derived T cells mainly recognize a covalently modified peptide [ 65 , 66 ]. In mice, hypersensitivity could be induced with a peptide modified at a p4 lysine residue [ 45 ].…”

“…Flucloxacillin activity dependent on high drug concentrations was independent of proteasomal processing and immediate, indicating direct binding to the TCR-pMHC interface [ 67 ]. A third mechanism for flucloxacillin T cell epitope formation was recently shown, which involves the binding in the peptide-anchoring pockets of HLA-B*57:01 and the presentation of an altered peptide repertoire ( Figure 1 B) [ 45 ]. In summary, the flucloxacillin case demonstrates the importance of patient analysis to determine the in vivo relevance of different epitope formation mechanisms.…”