Biology subjects

Mannering, S. I.

Publications and source records attributed to Mannering, S. I..

4 recordsLinked to original sources

bioRxiv2025

BASTA, a simple whole blood assay for measuring beta-cell antigen specific CD4+ T-cell responses in type 1 diabetes

Type 1 diabetes (T1D) is an autoimmune disease where T-cells mediate the destruction of the insulin-producing {beta}-cells found within the Islets of Langerhans in the pancreas. Currently, autoantibodies to {beta}-cell antigens are the only tests available to detect {beta}-cell autoimmunity. T-cell responses to {beta}-cell antigens, which are known to cause T1D, can only be measured in research settings due to the complexity of assays and the large blood volumes required. Here we describe the {beta}-cell antigen specific T-cell assay - BASTA. BASTA is a simple whole blood assay that can detect human CD4+ T-cell responses to {beta}-cell antigens by measuring antigen-stimulated IL-2 production. BASTA is both more sensitive and specific than the CFSE-based Proliferation Assay. We used BASTA to identify the regions of preproinsulin which stimulated T-cell responses specifically in blood from people with T1D. BASTA can be done with as little as 2-3 mL of blood. We found that effector memory CD4+ T cells are the primary producers of IL-2 in response to preproinsulin peptides. We then evaluated responses to individual and pooled preproinsulin peptides in a cross-sectional study of pediatric subjects: without T1D, without T1D but with a first-degree relative with T1D, or diagnosed with T1D. In contrast to other preproinsulin peptides, full-length C-peptide (PI33-63) showed excellent specificity for T1D (AUC = 0.86). We suggest that BASTA will be a useful tool for monitoring changes in {beta}-cell specific CD4+ T-cell responses both in research and clinical settings. One Sentence Summary{beta}-cell antigen specific CD4+ T-cell responses in children with T1D can be measured using a simple whole blood assay.

immunology↗

bioRxiv2024

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

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.

immunology↗

bioRxiv2024

Structure and dynamics of the autoantigen GAD65 in complex with the human autoimmune polyendocrine syndrome type 2-associated autoantibody b96.11

The enzyme glutamate decarboxylase (GAD) produces the neurotransmitter GABA, using pyridoxal-5-phosphate. GAD exists as two isoforms, GAD65 and GAD67. Only GAD65 acts as a major autoantigen, with its autoantibodies frequently found in type 1 diabetes and other autoimmune diseases. Here we characterize the structure and dynamics of GAD65 and its interaction with the autoimmune polyendocrine syndrome type 2-associated autoantibody b96.11. Combining hydrogen-deuterium exchange mass spectrometry (HDX), X-ray crystallography, cryo-electron microscopy and computational approaches, we dissect the conformational dynamics of the inactive apo- and the active holo-forms of GAD65, as well as the structure of the GAD65-autoantibody complex. HDX reveals the time-resolved, local dynamics that accompany autoinactivation, with the catalytic loop playing a key role in promoting collective dynamics at the interface between CTD and PLP domains. In the GAD65-b96.11 complex, heavy chain CDRs dominate the interaction, with the relatively long CDRH3 at the interface centre and uniquely bridging the GAD65 dimer via extensive electrostatic interactions with the 260PEVKEK265 motif. The autoantibody bridges structural elements on GAD65 that contribute to conformational change in GAD65, thus connecting the unique and intrinsic conformational flexibility that governs the autoinactivation mechanism of the enzyme to its autoantigenicity. The intrinsic dynamics, rather than sequence differences within epitopes, appear to be responsible for the contrasting autoantigenicities of GAD65 and GAD67. Our data thus reveal insights into the structural and dynamic differences between GAD65 and GAD67 that dictate their contrasting autoantibody reactivities, provide a new structural rationalisation for the nature of the autoimmune response to GAD65, and may have broader implications for antigenicity in general.

biochemistry↗

bioRxiv2019

Replacing murine insulin 1 with human insulin protects NOD mice from diabetes

Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (INS) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.

immunology↗