Rocky Strollo1, Chiara Vinci2, Y K Stella Man2, Sara Bruzzaniti3,4, Erica Piemonte5, Ghadeer Alhamar6, Silvia Irina Briganti6, Ilaria Malandrucco7, Flavia Tramontana6, Chiara Fanali1, James Garnett8,9, Roberto Buccafusca9, Perrin Guyer10, Mark Mamula11, Eddie A James10, Paolo Pozzilli6, Johnny Ludvigsson12, Paul G Winyard13, Mario Galgani3,5, Ahuva Nissim14. 1. Department of Science and Technology for Humans and the Environment, Università Campus Bio-Medico di Roma, Rome, Italy. 2. Biochemical Pharmacology, William Harvey Research Institute, Queen Mary University of London, London, UK. 3. Institute for Experimental Endocrinology and Oncology 'G. Salvatore', Consiglio Nazionale delle Ricerche, Naples, Italy. 4. Department of Biology, Università degli Studi di Napoli 'Federico II', Naples, Italy. 5. Department of Molecular Medicine and Medical Biotechnology, Università degli Studi di Napoli 'Federico II', Naples, Italy. 6. Department of Medicine, Unit of Endocrinology & Diabetes, Università Campus Bio-Medico di Roma, Rome, Italy. 7. The UOSD of Endocrinology and Metabolic Diseases, Azienda Sanitaria Locale (ASL) Frosinone, Frosinone, Italy. 8. Centre for Host-Microbiome Interactions, Dental Institute, King's College London, London, UK. 9. School of Biological and Chemical Sciences, Queen Mary University of London, London, UK. 10. Program for Translational Immunology, Benaroya Research Institute, Seattle, WA, USA. 11. Department of Medicine, Yale University School of Medicine, New Haven, CT, USA. 12. Division of Pediatrics, Department of Biomedical and Clinical Sciences, Crown Princess Victoria Children's Hospital, Linköping University, Linköping, Sweden. 13. Institute of Biomedical and Clinical Science, University of Exeter Medical School, St Luke's Campus, Exeter, UK. 14. Biochemical Pharmacology, William Harvey Research Institute, Queen Mary University of London, London, UK. a.nissim@qmul.ac.uk.
Abstract
AIMS/HYPOTHESIS: Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. METHODS: oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. RESULTS: We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12-21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11-30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21-30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies. CONCLUSIONS/ INTERPRETATION: Our findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.
AIMS/HYPOTHESIS: Antibodies specific to oxidative post-translational modifications (oxPTM) of insulin (oxPTM-INS) are present in most individuals with type 1 diabetes, even before the clinical onset. However, the antigenic determinants of such response are still unknown. In this study, we investigated the antibody response to oxPTM-INS neoepitope peptides (oxPTM-INSPs) and evaluated their ability to stimulate humoral and T cell responses in type 1 diabetes. We also assessed the concordance between antibody and T cell responses to the oxPTM-INS neoantigenic peptides. METHODS: oxPTM-INS was generated by exposing insulin to various reactive oxidants. The insulin fragments resulting from oxPTM were fractionated by size-exclusion chromatography further to ELISA and LC-MS/MS analysis to identify the oxidised peptide neoepitopes. Immunogenic peptide candidates were produced and then modified in house or designed to incorporate in silico-oxidised amino acids during synthesis. Autoantibodies to the oxPTM-INSPs were tested by ELISA using sera from 63 participants with new-onset type 1 diabetes and 30 control participants. An additional 18 fresh blood samples from participants with recently diagnosed type 1 diabetes, five with established disease, and from 11 control participants were used to evaluate, in parallel, CD4+ and CD8+ T cell activation by oxPTM-INSPs. RESULTS: We observed antibody and T cell responses to three out of six LC-MS/MS-identified insulin peptide candidates: A:12-21 (SLYQLENYCN, native insulin peptide 3 [Nt-INSP-3]), B:11-30 (LVEALYLVCGERGFFYTPKT, Nt-INSP-4) and B:21-30 (ERGFFYTPKT, Nt-INSP-6). For Nt-INSP-4 and Nt-INSP-6, serum antibody binding was stronger in type 1 diabetes compared with healthy control participants (p≤0.02), with oxidised forms of ERGFFYTPKT, oxPTM-INSP-6 conferring the highest antibody binding (83% binders to peptide modified in house by hydroxyl radical [●OH] and >88% to in silico-oxidised peptide; p≤0.001 vs control participants). Nt-INSP-4 induced the strongest T cell stimulation in type 1 diabetes compared with control participants for both CD4+ (p<0.001) and CD8+ (p=0.049). CD4+ response to oxPTM-INSP-6 was also commoner in type 1 diabetes than in control participants (66.7% vs 27.3%; p=0.039). Among individuals with type 1 diabetes, the CD4+ response to oxPTM-INSP-6 was more frequent than to Nt-INSP-6 (66.7% vs 27.8%; p=0.045). Overall, 44.4% of patients showed a concordant autoimmune response to oxPTM-INSP involving simultaneously CD4+ and CD8+ T cells and autoantibodies. CONCLUSIONS/ INTERPRETATION: Our findings support the concept that oxidative stress, and neoantigenic epitopes of insulin, may be involved in the immunopathogenesis of type 1 diabetes.
Authors: Aaron W Michels; Laurie G Landry; Kristen A McDaniel; Liping Yu; Martha Campbell-Thompson; William W Kwok; Kenneth L Jones; Peter A Gottlieb; John W Kappler; Qizhi Tang; Bart O Roep; Mark A Atkinson; Clayton E Mathews; Maki Nakayama Journal: Diabetes Date: 2016-12-05 Impact factor: 9.461
Authors: Jenny Aurielle B Babon; Megan E DeNicola; David M Blodgett; Inne Crèvecoeur; Thomas S Buttrick; René Maehr; Rita Bottino; Ali Naji; John Kaddis; Wassim Elyaman; Eddie A James; Rachana Haliyur; Marcela Brissova; Lut Overbergh; Chantal Mathieu; Thomas Delong; Kathryn Haskins; Alberto Pugliese; Martha Campbell-Thompson; Clayton Mathews; Mark A Atkinson; Alvin C Powers; David M Harlan; Sally C Kent Journal: Nat Med Date: 2016-10-31 Impact factor: 53.440
Authors: Anette G Ziegler; Marian Rewers; Olli Simell; Tuula Simell; Johanna Lempainen; Andrea Steck; Christiane Winkler; Jorma Ilonen; Riitta Veijola; Mikael Knip; Ezio Bonifacio; George S Eisenbarth Journal: JAMA Date: 2013-06-19 Impact factor: 56.272
Authors: Ken T Coppieters; Francesco Dotta; Natalie Amirian; Peter D Campbell; Thomas W H Kay; Mark A Atkinson; Bart O Roep; Matthias G von Herrath Journal: J Exp Med Date: 2012-01-02 Impact factor: 14.307
Authors: Antti-Pekka Laine; Hanna Holmberg; Anita Nilsson; Eva Ortqvist; Minna Kiviniemi; Outi Vaarala; Hans K Akerblom; Olli Simell; Mikael Knip; Johnny Ludvigsson; Sten-A Ivarsson; Karin Larsson; Ake Lernmark; Jorma Ilonen Journal: Dis Markers Date: 2007 Impact factor: 3.434