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. 2025 Aug;68(8):1721-1734.
doi: 10.1007/s00125-025-06458-8. Epub 2025 Jun 9.

T cell and autoantibody recognition of nucleus-associated islet autoantigens in individuals with type 1 diabetes

Perrin Guyer  1 , Kapitolina Seminova  1 , Marija Lugar  2 , Anthony Manganaro  3 , Erandi E Velarde de la Cruz  3 , Rachel Hartley  1 , Megan E Smithmyer  1 , Cate Speake  1 , Ezio Bonifacio  2 , Sally C Kent  3 , Eddie A James  4
Affiliations

T cell and autoantibody recognition of nucleus-associated islet autoantigens in individuals with type 1 diabetes

Perrin Guyer et al. Diabetologia. 2025 Aug.

Abstract

Aims/hypothesis: There is a progressive loss of self-tolerance in type 1 diabetes, manifested by the appearance of various autoantibodies. Array-based screening identified antibodies that recognise nucleus-associated proteins in individuals with type 1 diabetes, but the role of these antigens in the disease is poorly understood. Antibodies against MutL homologue 1 (MLH1) and nucleoporin 50 (NUP50) are enriched in DR4-positive and DR3-positive individuals, respectively. Therefore, we sought to investigate CD4+ T cell recognition of these antigens and to assess whether cellular and humoral recognition of these autoantigens are linked.

Methods: We used a systematic discovery process to identify CD4+ T cell epitopes within MLH1 and NUP50. We synthesised peptides derived from these antigens and then measured their ability to bind to recombinant DRB1*04:01 or DRB1*03:01 protein, our two HLA class II types of interest. We assessed peptide immunogenicity by expanding peripheral blood T cells in vitro and visualising peptide-specific T cells using HLA class II tetramers. We then performed direct tetramer staining of samples from individuals with type 1 diabetes and HLA-matched control individuals to enumerate MLH1- or NUP50-reactive CD4+ T cells and characterise their cell surface phenotype. Responses were also characterised using islet-derived T cells from pancreatic organ donors with type 1 diabetes using cytokine release as a readout. Antibody responses against both antigens were measured in matched serum samples using a previously published ELISA assay.

Results: Our discovery process revealed three novel DRB1*03:01-restricted NUP50 epitopes and four novel DRB1*04:01-restricted MLH1 epitopes that are present within the peripheral blood of individuals with type 1 diabetes and among pancreatic islet infiltrates. T cells specific for these epitopes were significantly more frequent in individuals with diabetes than in HLA haplotype-matched control individuals (p=0.0012 and 0.030 for NUP50 and MLH1, respectively). Variable levels of antibody responses were observed: elevated levels of MLH1 and NUP50 antibodies were present in individuals with type 1 diabetes, especially those with the HLA-DR types with previously reported associations, but high titres did not always directly correlate with elevated T cell frequency.

Conclusions/interpretation: The observation that T cell and antibody responses can target nucleus-associated self-antigens confirms and extends previously published studies. Disease-associated recognition of a class of proteins that are not exclusively expressed in pancreatic islets implies a systemic autoimmune component to the disease process. Linked antibody recognition does not appear to be a general phenomenon, suggesting a subtle relationship between humoral and cellular responses to these self-antigens.

Keywords: Autoantibody; CD4+ T cell; HLA tetramer; Human immunology; Nuclear antigen; Type 1 diabetes.

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Conflict of interest statement

Acknowledgements: We thank T.-S. Nguyen (BRI Clinical Core) for organising and assisting with shipment of archived samples and V. Green and C. Cousens-Jacobs (BRI) for administrative support. This manuscript used data acquired from the Human Pancreas Analysis Program (HPAP-RRID:SCR_016202) Database ( https://hpap.pmacs.upenn.edu/ ), a Human Islet Research Network (RRID:SCR_014393) consortium (UC4-DK-112217, U01-DK-123594, UC4-DK-112232 and U01-DK-123716). This research was performed with the support of the Network for Pancreatic Organ Donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project supported by Breakthrough T1D and The Leona M. & Harry B. Helmsley Charitable Trust (Grant 3-SRA-2023-1417-S-B). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPOs) partnering with nPOD to provide research resources are listed at https://npod.org/for-partners/npod-partners/ . Data availability: Data are available on request from the corresponding author. Funding: Funding for this research was provided by Breakthrough T1D (formerly known as JDRF) Grant 2-SRA-2018-551-S-B to EAJ. SCK was funded by NIH-NIDDK UC4DK116284 and The George F. and Sybil H. Fuller Term Chair in Diabetes. Authors’ relationships and activities: The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their work. Contribution statement: EAJ conceptualised and designed the study. KS and PG acquired peptide binding assay and T cell assay data and performed data analysis. ML and EB designed and executed LIPS assays to measure autoantibodies directed against NUP50 and MLH1, and analysed and interpreted the resulting data. AM and EEVC made substantial contributions to the analysis or interpretation of data and SCK made substantial contributions to the design of the work, or the acquisition, analysis, and interpretation of data, drafting the article, reviewing it critically for important intellectual content. RH, MES and CS were responsible for participant selection and collected and interpreted clinical data. EAJ wrote the manuscript with assistance and approval from all co-authors. EAJ obtained funding and was responsible for the entire project. EAJ is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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