Abacavir induces loading of novel self-peptides into HLA-B*57: 01: an autoimmune model for HLA-associated drug hypersensitivity

Abstract

Background: Abacavir drug hypersensitivity in HIV-treated patients is associated with HLA-B57:01 expression. To understand the immunochemistry of abacavir drug reactions, we investigated the effects of abacavir on HLA-B57:01 epitope-binding in vitro and the quality and quantity of self-peptides presented by HLA-B57:01 from abacavir-treated cells.

Design and methods: An HLA-B57:01-specific epitope-binding assay was developed to test for effects of abacavir, didanosine or flucloxacillin on self-peptide binding. To examine whether abacavir alters the peptide repertoire in HLA-B57:01, a B-cell line secreting soluble human leucocyte antigen (sHLA) was cultured in the presence or absence of abacavir, peptides were eluted from purified human leucocyte antigen (HLA), and the peptide epitopes comparatively mapped by mass spectroscopy to identify drug-unique peptides.

Results: Abacavir, but not didansosine or flucloxacillin, enhanced binding of the FITC-labeled self-peptide LF9 to HLA-B57:01 in a dose-dependent manner. Endogenous peptides isolated from abacavir-treated HLA-B57:01 B cells showed amino acid sequence differences compared with peptides from untreated cells. Novel drug-induced peptides lacked typical carboxyl (C) terminal amino acids characteristic of the HLA-B57:01 peptide motif and instead contained predominantly isoleucine or leucine residues. Drug-induced peptides bind to soluble HLA-B57:01 with high affinity that was not altered by abacavir addition.

Conclusion: Our results support a model of drug-induced autoimmunity in which abacavir alters the quantity and quality of self-peptide loading into HLA-B57:01. Drug-induced loading of novel self-peptides into HLA, possibly by abacavir either altering the binding cleft or modifying the peptide-loading complex, generates an array of neo-antigen peptides that drive polyclonal T-cell autoimmune responses and multiorgan systemic toxicity.

Fig. 1. Fluorescence polarization-based HLA-B*57:01 competition assay

Soluble HLA B*57:01 was derived from 721.221 B-cell transfectants and affinity purified before use in binding assays. The tracer is the self-peptide LF9 (LSSPVTKSF) from IgKappa substituted with a FITC-lysine residue at P6 that is not involved with direct HLA binding based on examination of the crystal structure [14]. Serial dilutions of nonlabeled LF9 competitor were incubated with a constant concentration of heat activated sHLA-B*57:01, excess β2m and the FITC-labeled tracer peptides until equilibrium was reached by monitoring changes in fluorescent polarization (FP) on an Analyst AD plate reader (Molecular Devices Sunnyvale, California, USA). The IC₅₀ concentration of unlabeled competitor that produces FITC-labeled peptide binding half way between the upper and lower plateau is indicated.

Fig. 2. (a) Effect of abacavir sulfate, didanosine and flucloxacillin sodium on LF9FITC binding to sHLA-B*57:01

Structures for drugs are shown in (b) abacavir, (c) flucloxacillin and (d) didanosine. To monitor drug effects on peptide binding, abacavir, didanosine or flucloxacillin were added over a wide concentration range and fluorescent polarization (FP) measured at equilibrium. The increase in FP represents an increase in epitope binding to HLA.

Fig. 3. Examples of MS–MS fragmentation spectrums from abacavir-unique peptides from fraction 70

(a) Peptide molecular weight (MW) 990.5868 HSLPALIQI. (b) Abacavir-unique peptide MW 1002.6061 STIRLLTSL.

Fig. 4. Competitive binding evaluation of HLA-B*57:01 drug-induced peptides

(a) STIRLLTSL and (b) HSLPALIQI with and without abacavir. IC₅₀ values for two DI peptides were determined applying serial dilutions of nonlabeled HSL and STIR competitor, respectively, in the presence and absence of 200 μmol/l of abacavir. Results showed the expected increase in reference LF9FITC peptide binding signal with abacavir. No significant difference in inhibition between experiments in both cases could be observed. DI, drug-induced