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.

Keywords: HLA-B*57:01; drug hypersensitivity; flucloxacillin; hapten; immunogenicity; transgenic mice.

Figure 1

Flucloxacillin (FLX) does not significantly alter the B721-5701 cell proteome. Protein lysates were obtained from ten million B721-5701 cells treated with FLX at 150 μg/ml (low) or 453 μg/ml (high) for 5 days, or from untreated cultures (Unt), in duplicate. Samples were processed by LC-MS/MS and analyzed by Peaks 8.5 software. (A) Correlation among biological replicates. Protein abundance is represented in arbitrary units of intensity. (B, C) Increased (log₂ fold change ≥ 1) and decreased (log₂ fold change ≤ -1) abundance of proteins in the proteome of cells treated with low-FLX (B) or high-FLX concentration (C) compared to Unt, considering the average abundance of the two biological replicates per treatment. Percent values indicate divergence in abundance. Statistical differences were calculated by Spearman correlation. Proteome datasets are included in Table S1 .

Figure 2

Flucloxacillin (FLX) treatment induces diversity in the peptide repertoire of B721-5701 cells. Common and unique peptides eluted from human leukocyte antigen (HLA)/peptide complexes obtained from two independent cultures of untreated B721-5701 cells (Unt) and of cells treated with 150 μg/ml of FLX (FLX) for 5 days were combined for the analysis (A). Absolute counts (B) or frequency (C) of 8–15 amino acid peptides. Frequency of peptides with post-translational modification (PTM) within the peptide repertoire (D) or among modified peptides (E). Common and unique proteins represented by immunopeptidome-contained sequences (in Figure 2A ) (F). Protein distribution showing the number of peptides per protein (G). Data analysis was performed using Peaks 8.5 and X software. The datasets of peptides in the B721-5701 peptidomes are provided in Table S2A, B .

Figure 3

Flucloxacillin (FLX) treatment promotes a higher peptide sampling rate of proteins, independently of the length and abundance of the polypeptide. Abundance of human leukocyte antigen (HLA) peptides from 435 and 699 proteins in the proteome of B721-5701 cells untreated or FLX-treated, respectively, as a function of the protein amino acid length (A) or abundance (log₂) (B). Individual proteins and trendlines are represented in the graphs. (C) Protein fold over-presentation was calculated based on the ratio between observed (D) and expected (D’) peptide density, and represented as function of protein abundance. The dataset of the represented proteins is provided in Table S3 .

Figure 4

Flucloxacillin (FLX) treatment does not alter consensus peptide binding motif of HLA-B*57:01 peptides but favors peptides with tryptophan in P9/PΩ. (A) Unsupervised analysis of the sequence motif from 9-mer sequences in the immunopeptidome of B721-5701 cells, untreated (Unt) or treated with FLX for 5 days (FLX), using Gibbs Cluster 2.0 software (peptide sequence details provided in Table S2A-B). Peptides with PTMs were excluded from this analysis. (B) Ratio of the abundance of the common peptides in the FLX vs Unt immunopeptidome samples, including total and C-terminal tryptophan (W) peptides. Ratios are represented as FLX : Unt lower than two-fold, between two- and four-fold or higher than four-fold. Abundance values were calculated using Quant analysis of common peptide quantifiable IDs (<1% FDR). (C) Frequency of the different amino acid residues at P7 of 9-mers with predicted binding affinity <50 nM (by NetMHC 4.0 software).

Figure 5

Flucloxacillin modified peptides identified from drug treated HLA-B*57:01 expressing cells. (A) MS/MS spectra of parent ions with characteristic b and y ions, drug fragments (160.04, 295.03, 454.06/07) and diagnostic b+294 fragments identified: RTKK(FLX)VGIVGKY (FLX-RTKK_K4, top panel), KAAK(FLX)LKEKY (FLX-KAA_K4, middle panel), and FLX-modified TAAQITQRK(FLX)W (FLX-TAA_K9, bottom panel). (B) Two FLX-RTKK_K4 isoforms, with different retention time by RP-HPLC (peaks at minute 30.46 and 31.92) (top chromatogram) but identical mass (m/z=851.4191/2+, m/z=567.85/3+ (data not shown)) (mid and low LC-MS1 spectra). All peptides were identified using PEAKS analysis and verified manually.

Figure 6

Immunogenicity potential of flucloxacillin (FLX)-haptenated peptide is dependent on the peptide sequence, the position of the lysine to which drug is conjugated and the expression of human leukocyte antigen (HLA). (A, B) Tg/KO mice (n=3–9) were immunized by 2 s.c. doses of 100 μg of FLX-peptides together with 150 μg of HBV_128-140 CD4⁺ T helper peptide with IFA. Splenocytes were subsequently stimulated in vitro with 10 μg/ml peptides (as indicated) for 5 days. IFN-γ was measured in the supernatant of the cultures by ELISA. In vitro responses to the in vivo immunogen were blocked by anti-HLA B/C antibody. (C) Immunizations and cultures were set up similarly to that described in A but with peptide isoforms of the FLX-RTKK peptides. (D) IFN-γ intracellular staining of day 5 splenic cell cultures from animals treated with FLX-peptides and stimulated in vitro with the same peptide. Graphs show results gated in CD8⁺ or CD4⁺ T cell populations (one representative of two experiments). Statistical analysis by one-way ANOVA using the no peptide condition as baseline. p-values are only shown if <0.999.

Figure 7

In silico prediction of the interactions of parent and flucloxacillin (FLX)-RTKK peptides with HLA-B*57:01 cleft. KAF11 peptide residues (PDB ID: 2YPK) were replaced with those of the RTKK peptide. Docking the peptide into the HLA cleft was performed using FlexPepDock and addition of the drug to the appropriate lysine and energy minimization of the structure using OpenBable (v3.0.0) software. Structures were visualized in PyMOL (v2.3.4). Lateral (A–C) and top (D–F) views of the human leukocyte antigen (HLA) complex with the unmodified parent sequence of RTKK (A, D), FLX-RTKK_K4 (B, E) and FLX-RTKK_K10 (C, F). Peptide sequences are represented in blue spheres, drug is represented by red spheres and α1α2 domains of the HLA are depicted in gray color.