Autoantibody and T cell responses to oxidative post-translationally modified insulin neoantigenic peptides in type 1 diabetes

• Published in: Diabetologia Vol. 66; no. 1; pp. 132 - 146
• Main Authors: Strollo, Rocky, Vinci, Chiara, Man, Y. K. Stella, Bruzzaniti, Sara, Piemonte, Erica, Alhamar, Ghadeer, Briganti, Silvia Irina, Malandrucco, Ilaria, Tramontana, Flavia, Fanali, Chiara, Garnett, James, Buccafusca, Roberto, Guyer, Perrin, Mamula, Mark, James, Eddie A., Pozzilli, Paolo, Ludvigsson, Johnny, Winyard, Paul G., Galgani, Mario, Nissim, Ahuva
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.01.2023; Springer Nature B.V
• Subjects: Antibodies; Antibody response; Antigenic determinants; Autoantibodies; CD4 antigen; CD8 antigen; CD8-Positive T-Lymphocytes; Cell activation; Chromatography, Liquid; Diabetes; Diabetes mellitus (insulin dependent); Diabetes Mellitus, Type 1; Enzyme-linked immunosorbent assay; Epitopes; Human Physiology; Humans; Hydroxyl radicals; Immunogenicity; Immunopathogenesis; Insulin; Internal Medicine; Lymphocytes; Lymphocytes T; Medicine; Medicine & Public Health; Metabolic Diseases; Neoantigens; Oxidants; Oxidative stress; Peptides; Post-translation; Tandem Mass Spectrometry; Autoimmunity; Oxidative post-translational modifications; Neoepitope; Immune response; Post-translational modifications; Insulin autoantibodies; Insulin; Neoantigen; Insulin neoepitope peptide
• ISSN: 0012-186X; 1432-0428; 1432-0428
• DOI: 10.1007/s00125-022-05812-4

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. Graphical abstract