Poster Presentation 27th Annual Lorne Proteomics Symposium 2022

Use of functional T-cell assays and immunopeptidomics to decipher the molecular mechanism underpinning penicillin allergy (#118)

Shawn Goh 1 , Hovey Lu 1 , Kirti Pandey 1 , Kate Scull 1 , Robert Puy 2 , Robyn O'Hehir 2 , Nicole Mifsud 1 , Patricia Illing 1 , Anthony Purcell 1
  1. Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia
  2. Department of Allergy, Immunology and Respiratory Medicine, Monash University, Central Clinical School, The Alfred Hospital, Melbourne, VIC, Australia

Penicillin allergy affects 1 in 10 patients administered with this class of antibiotic1. A genetic risk factor associated with penicillin hypersensitivity reactions is the expression of HLA-A*02:012,3. Given evidence that T-cells are involved in these immune-mediated reactions, the mechanism underpinning HLA-mediated T-cell recognition of penicillins is of key interest. Penicillins can form neoantigens, through covalent modification of proteins and peptides via the beta-lactam chemical warhead (haptenation). These neoantigens are postulated to trigger T-cell activation when presented on the surface of cells by HLA molecules.

We report the identification of a dominant Benzylpenicillin (BP)-specific T-cell receptor (TCR) cloned from drug-specific T-cells isolated from a penicillin-allergic patient. This TCR recognises a BP-modified peptide presented by HLA-A*02:01. The BP-TCR demonstrated reactivity consistent with the recognition of a covalently modified peptide-HLA*02:01 complex. Further investigation demonstrated that the immunogenic peptide traverses the HLA class-I antigen processing pathway, and that T-cell recognition can be blocked using an anti-penicillin antibody suggesting that the ligand includes a solvent-exposed drug adduct.

We therefore sought to identify BP-modified neoepitopes by isolating peptides bound to HLA-A*02:01 after drug treatment using mass spectrometry4,5. This workflow led to the discovery of multiple drug-modified peptides presented by HLA-A*02:01. To shortlist potential immunogenic ligands, we screened HLA-A*02:01 bound peptide fractions for recognition by a BP-TCR expressing reporter T-cell line. We demonstrated that BP-TCR was activated by peptides contained in two fractions. These fractions are being further analysed by mass spectrometry for peptide identification and immunological studies.

Our work highlights 1) the use of mass spectrometry to discover novel BP-induced HLA-A*02:01 ligands and 2) the complementary use of biochemical techniques and functional T-cell assays, for epitope discovery. Identification of the BP-modified immunogenic ligands will allow us to decipher key interactions between the TCR and drug-peptide-HLA complex, providing insights into the nature of penicillin hypersensitivity.

  1. Macy, E. (2014). Penicillin and beta-lactam allergy: epidemiology and diagnosis. Current allergy and asthma reports, 14(11), 476.
  2. Romano, A., Di Fonso, M., Venuti, A., De Santis, A., Romito, A., Gasbarrini, G. B., & Manna, R. (1998). Delayed hypersensitivity to aminopenicillins is related to major histocompatibility complex genes. Annals of Allergy, Asthma & Immunology, 80(5), 433-437.
  3. Lucena, M. I., Molokhia, M., Shen, Y., Urban, T. J., Aithal, G. P., Andrade, R. J., ... & Eudragene, D. (2011). Susceptibility to amoxicillin-clavulanate-induced liver injury is influenced by multiple HLA class I and II alleles. Gastroenterology, 141(1), 338-347.
  4. Purcell, A. W., Ramarathinam, S. H., & Ternette, N. (2019). Mass spectrometry–based identification of MHC-bound peptides for immunopeptidomics. Nature protocols, 14(6), 1687-1707.
  5. Pandey, K., Ramarathinam, S. H., & Purcell, A. W. (2021). Isolation of HLA Bound Peptides by Immunoaffinity Capture and Identification by Mass Spectrometry. Current Protocols, 1(3), e92.