Proinsulin C-peptide is a major source of HLA-DQ8 restricted HIPs recognized by human Islet-Infiltrating CD4+ T cells

Bhattacharjee, P.; Pakusch, M.; Lacorcia, M.; Tresoldi, E.; Rubin, A. F.; Foster, A.; King, L. S.; Chiu, C.; Kay, T.; Karas, J. A.; Cameron, F. J.; Mannering, S. I.

doi:10.1101/2024.05.09.593303

bioRxiv · 10.1101/2024.05.09.593303

Proinsulin C-peptide is a major source of HLA-DQ8 restricted HIPs recognized by human Islet-Infiltrating CD4+ T cells

Bhattacharjee, P.·Pakusch, M.·Lacorcia, M.·Tresoldi, E.·Rubin, A. F.·Foster, A.·King, L. S.·Chiu, C.·Kay, T.·Karas, J. A.·Cameron, F. J.·Mannering, S. I.

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Abstract

Type 1 diabetes (T1D) is an autoimmune disease that develops when T cells destroy the pancreatic insulin-producing beta cells that reside in the pancreatic islets. Immune cells, including T cells infiltrate the islets and gradually destroy the beta cells. Human islet-infiltrating CD4+ T cells recognize peptide epitopes derived from proinsulin, particularly C-peptide. Hybrid Insulin peptides (HIPs) are neoepitopes formed by the fusion of two peptides derived from beta-cell granule proteins and are known to be the targets of pathogenic CD4+ T cells in the NOD mouse and human islet-infiltrating CD4+ T cells. Proinsulin is widely recognized as a central antigen in T1D, but its role in forming HIPs is unclear. We developed a method to functionally screen TCRs derived from human islet-infiltrating CD4+ T cells and applied this to the identification of new proinsulin-derived HIPs. We generated a library of 4,488 candidate HIPs formed by fusion of proinsulin fragments and predicted to bind to HLA-DQ8. This library was screened against 109 islet-infiltrating CD4+ T-cell TCRs isolated from four organ donors who had T1D. We identified 13 unique HIPs recognized by 9 different TCRs from two organ donors. HIP specific T-cell avatars responded specifically to a peptide extract from human islets. These new HIPs predominantly stimulated CD4+ T-cell proliferation in PBMCs from people with T1D in contrast to HLA-matched controls. This is the first unbiased functional, islet-infiltrating T-cell based, screen to identify proinsulin derived HIPs. It has revealed many new HIPs and a central role of proinsulin C-peptide in their formation. SUMMARYType 1 diabetes is an autoimmune disease caused by T cells destroying the pancreatic insulin-producing beta cells. The antigens/epitopes seen by disease promoting CD4+ T cells are poorly understood. Hybrid insulin peptides (HIPs) are a new class of CD4+ antigen recognized by pathogenic NOD mouse CD4+ T cells. In humans very few HIPs recognized by human islet-infiltrating CD4+ T cells are known. We show that proinsulin HIPs are recognized by human islet-infiltrating CD4+ T cells from T1D donors and describe 13 new HIPs formed by fusion of proinsulin peptides. This work shows that proinsulin, particularly C-peptide, is a major contributor to the pool HIPs recognized by human islet-infiltrating CD4+ T cells and are therefore central to autoimmunity in T1D.

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Bhattacharjee, P., Pakusch, M., Lacorcia, M., Tresoldi, E., Rubin, A. F., Foster, A., King, L. S., Chiu, C., Kay, T., Karas, J. A., Cameron, F. J., Mannering, S. I.. 2024-05-10. Proinsulin C-peptide is a major source of HLA-DQ8 restricted HIPs recognized by human Islet-Infiltrating CD4+ T cells. https://doi.org/10.1101/2024.05.09.593303

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