Hybrid Insulin Peptides Research Articles

Recognition of Multiple Hybrid Insulin Peptides by a Single Highly Diabetogenic T-Cell Receptor.

The mechanisms underlying the major histocompatibility complex class II (MHCII) type 1 diabetes (T1D) association remain incompletely understood. We have previously shown that thymocytes expressing the highly diabetogenic, I-Ag7-restricted 4.1-T-cell receptor (TCR) are MHCII-promiscuous, and that, in MHCII-heterozygous mice, they sequentially undergo positive and negative selection/Treg deviation by recognizing pro- and anti-diabetogenic MHCII molecules on cortical thymic epithelial cells and medullary hematopoietic antigen-presenting cells (APCs), respectively. Here, we use a novel autoantigen discovery approach to define the antigenic specificity of this TCR in the context of I-Ag7. This was done by screening the ability of random epitope–GS linker–I-chain fusion pools to form agonistic peptide–MHCII complexes on the surface of I- chain-transgenic artificial APCs. Pool deconvolution, I-Ag7-binding register-fixing, TCR contact residue mapping, and alanine scanning mutagenesis resulted in the identification of a 4.1-TCR recognition motif XL(G/A)XEXE(D/E)X that was shared by seven agonistic hybrid insulin peptides (HIPs) resulting from the fusion of several different chromogranin A and/or insulin C fragments, including post-translationally modified variants. These data validate a novel, highly sensitive MHCII-restricted epitope discovery approach for orphan TCRs and suggest thymic selection of autoantigen-promiscuous TCRs as a mechanism for the murine T1D–I-Ag7-association.

T cell receptor recognition of hybrid insulin peptides bound to HLA-DQ8

HLA-DQ8, a genetic risk factor in type I diabetes (T1D), presents hybrid insulin peptides (HIPs) to autoreactive CD4+ T cells. The abundance of spliced peptides binding to HLA-DQ8 and how they are subsequently recognised by the autoreactive T cell repertoire is unknown. Here we report, the HIP (GQVELGGGNAVEVLK), derived from splicing of insulin and islet amyloid polypeptides, generates a preferred peptide-binding motif for HLA-DQ8. HLA-DQ8-HIP tetramer+ T cells from the peripheral blood of a T1D patient are characterised by repeated TRBV5 usage, which matches the TCR bias of CD4+ T cells reactive to the HIP peptide isolated from the pancreatic islets of a patient with T1D. The crystal structure of three TRBV5+ TCR-HLA-DQ8-HIP complexes shows that the TRBV5-encoded TCR β-chain forms a common landing pad on the HLA-DQ8 molecule. The N- and C-termini of the HIP is recognised predominantly by the TCR α-chain and TCR β-chain, respectively, in all three TCR ternary complexes. Accordingly, TRBV5 + TCR recognition of HIP peptides might occur via a ‘polarised’ mechanism, whereby each chain within the αβTCR heterodimer recognises distinct origins of the spliced peptide presented by HLA-DQ8.

109-OR: T-Cell Responses to Hybrid Insulin Peptides Precede Type 1 Diabetes Development

Type 1 diabetes (T1D) results from the T cell-mediated destruction of insulin-producing beta-cells within pancreatic islets. T cell responses to post-translationally modified hybrid insulin peptides (HIPs) have been implicated in T1D pathogenesis, and we hypothesized that CD4 T cell responses to HIPs precede clinical diabetes onset. Therefore, we measured longitudinal T cell responses to native and hybrid insulin peptides in a cohort of genetically at-risk individuals from the Diabetes AutoImmunity Study in the Young (DAISY). Using cytokine ELISPOT assays, both pro-inflammatory (IFN-γ) and anti-inflammatory (IL-10) cytokine responses to HIPs were more robust than to native peptides. Three of the 38 autoantibody negative individuals (med. age 18.7 yrs) developed islet autoimmunity during 2 years of follow-up, and the C-peptide-islet amyloid polypeptide-2 (C:IAPP-2), C-peptide-neuropeptide Y (C:NPY), and C-peptide-insulin A chain (C:A chain) HIPs induced more frequent pro-inflammatory T cell responses compared to those that did not develop autoantibodies. Five of the 25 autoantibody positive individuals (med. age 18.7 yrs) progressed to clinical T1D during the study, and the C:IAPP-2 HIP more frequently induced a pro-inflammatory response compared to those that did not progress (p=0.037). Additionally, length of time from autoantibody seroconversion correlated with proinflammatory T cell responses to the insulin B:9-23 (B22E) HIP (p=0.007) and the native B:9-23 peptide (p=0.022). T cell responses to the B22E HIP also strongly correlated with insulin autoantibody levels (p=0.0001). Furthermore, responses to the C:IAPP-2 HIP correlated with worsening measurements of blood glucose (A1c %, p=0.046; glucose area under the curve, p=0.054). Monitoring T cell responses to post-translationally modified antigens prior to T1D onset holds promise for determining which individuals are likely to progress to clinical disease and may benefit from immune intervention to prevent T1D onset. Disclosure A. M. Mitchell: None. M. Rewers: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; RTI, Research Support; Self; Janssen Research & Development, LLC, JDRF. A. W. Michels: Stock/Shareholder; Self; IM Therapeutics. A. Alkanani: None. K. Mcdaniel: None. L. Pyle: None. K. Waugh: None. A. Steck: None. M. Nakayama: None. L. Yu: None. P. Gottlieb: Advisory Panel; Self; Janssen Research & Development, LLC, Tolerion, Inc., Viacyte, Inc., Other Relationship; Self; ImmunoMolecular Therapeutics, Inc., Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Mercia Pharma Inc., National Institute of Diabetes and Digestive and Kidney Diseases, Precigen, Inc., Tolerion, Inc. Funding National Institutes of Health (DK108868, DK110845, DK032083, DK032493, DK116073); JDRF (3-PDF-2020-943-A-N); Children’s Diabetes Foundation; Colorado Clinical and Translational Science Institute (TR002535)

108-OR: Islet-Derived Preproinsulin T Cell Receptor Sequences Are Present throughout the Stages of Type 1 Diabetes Development

Type 1 diabetes (T1D) results from the T cell-mediated destruction of insulin-producing pancreatic β-cells as evidenced by CD8 and CD4 T cells being present within the inflamed islets of recent-onset T1D organ donors. We isolated, sequenced the T cell receptor (TCR) from ∼3,500 T cells in the residual islets of T1D organ donors (n=9), and determined antigen specificity to 41 preproinsulin-reactive T cells from these islet sequences. Among the T1D organ donor islet sequences, 32% are shared (identical Vβ, Jβ, CDR3β) within the peripheral blood of new-onset T1D patients that had sequencing done from whole blood DNA (n=162, 34x106 TCR sequences, Adaptive Biotechnologies). Similar numbers of sequences are shared between both CD4 and CD8 T cells. Furthermore, 53 unique sequences were disease-specific, as they were identified in <1% of TCR sequences from controls (n=786, 275x106 sequences), whereas they were detected in 54% of new-onset T1D patients (p<0.001). These disease-specific and preproinsulin-reactive TCR sequences are good candidates for biomarkers in T1D. Therefore, we next sequenced the TCR beta chains throughout the disease course of five participants in the Diabetes AutoImmunity Study in the Young (DAISY) at 4 time points (early in life, prior to islet autoantibody seroconversion, post-autoantibody seroconversion, and at T1D onset). 10% of these insulin-reactive TCR sequences, including those to hybrid insulin peptides, are present throughout the disease course. Interestingly, insulin-reactive CD4 TCRs were predominant early in life, while higher frequencies of both insulin-reactive CD8 and CD4 were present at clinical T1D diagnosis. With continued TCR sequencing from longitudinal patient samples across the stages of T1D and identifying antigen specificity to more islet-derived T cells outside of preproinsulin, TCR sequences from genomic DNA have the potential to be a powerful disease biomarker during the progression to clinical type 1 diabetes. Disclosure E. E. Baschal: None. A. W. Michels: Stock/Shareholder; Self; IM Therapeutics. A. M. Mitchell: None. K. Mcdaniel: None. A. Alkanani: None. T. Armstrong: None. L. Landry: None. K. Waugh: None. M. Rewers: Advisory Panel; Self; Provention Bio, Inc., Consultant; Self; RTI, Research Support; Self; Janssen Research & Development, LLC, JDRF. M. Nakayama: None. Funding National Institutes of Health (DK108868, DK110845, DK032083, DK099317, DK032493)

Characterization of Human CD4 T Cells Specific for a C-Peptide/C-Peptide Hybrid Insulin Peptide.

Hybrid Insulin Peptides (HIPs), which consist of insulin fragments fused to other peptides from β-cell secretory granule proteins, are CD4 T cell autoantigens in type 1 diabetes (T1D). We have studied HIPs and HIP-reactive CD4 T cells extensively in the context of the non-obese diabetic (NOD) mouse model of autoimmune diabetes and have shown that CD4 T cells specific for HIPs are major contributors to disease pathogenesis. Additionally, in the human context, HIP-reactive CD4 T cells can be found in the islets and peripheral blood of T1D patients. Here, we performed an in-depth characterization of the CD4 T cell response to a C-peptide/C-peptide HIP (HIP11) in human T1D. We identified the TCR expressed by the previously-reported HIP11-reactive CD4 T cell clone E2, which was isolated from the peripheral blood of a T1D patient, and determined that it recognizes HIP11 in the context of HLA-DQ2. We also identified a HIP11-specific TCR directly in the islets of a T1D donor and demonstrated that this TCR recognizes a different minimal epitope of HIP11 presented by HLA-DQ8. We generated and tested an HLA-DQ2 tetramer loaded with HIP11 that will enable direct ex vivo interrogation of CD4 T cell responses to HIP11 in human patients and control subjects. Using mass spectrometric analysis, we confirmed that HIP11 is present in human islets. This work represents an important step in characterizing the role of CD4 T cell responses to HIPs in human T1D.

Contribution of a Hybrid Insulin Peptide-reactive T cell to Diabetes progression in the NOD mouse.

Abstract Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the immune destruction of pancreatic β cells. The search for natural equivalents to the mimetopes that can stimulate diabetogenic T cell clones from the Non-Obese Diabetic (NOD) mouse model of T1D led to the realization that hybrid peptides (HP) formed by the joining of β cell granule protein-derived peptide fragments are highly stimulatory to some clones. These hybrid peptides were later identified in vivo and proposed as a new class of post-translationally modified autoantigens mediating this disease. Recent data suggest high concentrations of these proteins in β cell lysosomes during granule turnover can lead to the hybridization of peptide fragments which may then be presented on MHC molecules. In the thymus, medullary thymic epithelial cells (mTECs) mediate T cell tolerance through promiscuous gene expression, but may not mediate HP-reactive T cell tolerance. We hypothesize that HP-reactive T cells preferentially escape the thymus and contribute significantly to T1D progression. We attempted to enhance T cell central tolerance to 2.5HIP, a hybrid insulin peptide formed from Proinsulin and Chromogranin A, the only two antigens known to be critical for T1D progression in the NOD mice, to better understand its impact on T1D incidence. Using an antibody-peptide conjugation system, we targeted 2.5HIP to a subset of thymic resident DCs to enhance presentation of 2.5HIP in early neonatal thymus. In neonatal BDC2.5 TCR-transgenic mice we found that a single dose of antibody-2.5HIP enhanced antigen-specific thymocyte apoptosis and regulatory T cell development. Importantly, a single dose in wild-type neonates resulted in a significant delay of diabetes onset.

Identifying New Hybrid Insulin Peptides (HIPs) in Type 1 Diabetes.

In 2016 Delong et al. discovered a new type of neoepitope formed by the fusion of two unrelated peptide fragments. Remarkably these neoepitopes, called hybrid insulin peptides, or HIPs, are recognized by pathogenic CD4+ T cells in the NOD mouse and human pancreatic islet-infiltrating T cells in people with type 1 diabetes. Current data implicates CD4+ T-cell responses to HIPs in the immune pathogenesis of human T1D. Because of their role in the immune pathogenesis of human T1D it is important to identify new HIPs that are recognized by CD4+ T cells in people at risk of, or with, T1D. A detailed knowledge of T1D-associated HIPs will allow HIPs to be used in assays to monitor changes in T cell mediated beta-cell autoimmunity. They will also provide new targets for antigen-specific therapies for T1D. However, because HIPs are formed by the fusion of two unrelated peptides there are an enormous number of potential HIPs which makes it technically challenging to identify them. Here we review the discovery of HIPs, how they form and discuss approaches to identifying new HIPs relevant to the immune pathogenesis of human type 1 diabetes.

Identification of spliced peptides in pancreatic islets uncovers errors leading to false assignments

Proteasomal spliced peptides (PSPs) have been identified in the class I major histocompatibility complex (MHC) peptidomes of several tumors and have emerged as novel neoantigens that can stimulate highly specific T cells. Much debate has surrounded the percentage of PSPs in the immunopeptidome; reported numbers have ranged from <1-5% to 12-45%. Recently, our laboratory demonstrated in nonobese diabetic (NOD) mice that hybrid insulin peptides (HIPs), a special class of spliced peptides, are formed during insulin granule degradation in crinosomes of the pancreatic β cells and that modified peptides comprised a significant source of false positive HIP assignments. Herein, this study is extended to crinosomes isolated from other mouse strains and to two recent MHC class I studies, to see if modified peptides explained discrepancies in reported percentages of PSPs. This analysis revealed that both MHC-I peptidomes contained many spectra erroneously assigned as PSPs. While many false positive PSPs did arise from modified peptides, others arose from probable data processing errors. Thus, the reported numbers of PSPs in the literature are likely elevated due to errors associated with data processing and analysis.

Induction of Antigen-Specific Tolerance in Autoimmune Diabetes with Nanoparticles Containing Hybrid Insulin Peptides

Autoreactive T cells are thought to orchestrate the onset and progression of autoimmune diabetes. Key cognate antigens of these diabetogenic T cells include hybrid insulin peptides, formed by the fusion of insulin fragments to cleavage products of other β-cell granule proteins. Here we review initial work exploring tolerance induction to a hybrid insulin peptide using a biodegradable, nanoparticle delivery system in non-obese diabetic (NOD) mice. The immune phenotype(s) and possible mechanism(s) behind antigen-specific tolerance induction were dissected with a disease transfer model using transgenic autoreactive mouse T cells. Treatment of NOD mice with peptide-coupled nanoparticles appeared to have a dual function in preventing diabetes onset, inducing anergy in effector T cells and enhancing the activity of regulatory T cells. Importantly, the ratio of these two cell types in the pancreas was pushed toward tolerance. Antigen-specific tolerance induction to hybrid insulin peptides has the translational potential to preserve islet β-cells in new-onset or at-risk patients and prevent recurrent autoimmunity in transplant patients.

Non-Genetically Encoded Epitopes Are Relevant Targets in Autoimmune Diabetes.

Islet antigen reactive T cells play a key role in promoting beta cell destruction in type 1 diabetes (T1D). Self-reactive T cells are typically deleted through negative selection in the thymus or deviated to a regulatory phenotype. Nevertheless, those processes are imperfect such that even healthy individuals have a reservoir of potentially autoreactive T cells. What remains less clear is how tolerance is lost to insulin and other beta cell specific antigens. Islet autoantibodies, the best predictor of disease risk, are known to recognize classical antigens such as proinsulin, GAD65, IA-2, and ZnT8. These antibodies are thought to be supported by the expansion of autoreactive CD4+ T cells that recognize these same antigenic targets. However, recent studies have identified new classes of non-genetically encoded epitopes that may reflect crucial gaps in central and peripheral tolerance. Notably, some of these specificities, including epitopes from enzymatically post-translationally modified antigens and hybrid insulin peptides, are present at relatively high frequencies in the peripheral blood of patients with T1D. We conclude that CD4+ T cells that recognize non-genetically encoded epitopes are likely to make an important contribution to the progression of islet autoimmunity in T1D. We further propose that these classes of neo-epitopes should be considered as possible targets for strategies to induce antigen specific tolerance.

T-cell responses to hybrid insulin peptides prior to type 1 diabetes development

T-cell responses to posttranslationally modified self-antigens are associated with many autoimmune disorders. In type 1 diabetes, hybrid insulin peptides (HIPs) are implicated in the T-cell-mediated destruction of insulin-producing β-cells within pancreatic islets. The natural history of the disease is such that it allows for the study of T-cell reactivity prior to the onset of clinical symptoms. We hypothesized that CD4 T-cell responses to posttranslationally modified islet peptides precedes diabetes onset. In a cohort of genetically at-risk individuals, we measured longitudinal T-cell responses to native insulin and hybrid insulin peptides. Both proinflammatory (interferon-γ) and antiinflammatory (interluekin-10) cytokine responses to HIPs were more robust than those to native peptides, and the ratio of such responses oscillated between pro- and antiinflammatory over time. However, individuals who developed islet autoantibodies or progressed to clinical type 1 diabetes had predominantly inflammatory T-cell responses to HIPs. Additionally, several HIP T-cell responses correlated to worsening measurements of blood glucose, highlighting the relevance of T-cell responses to posttranslationally modified peptides prior to autoimmune disease development.

Soluble Antigen Arrays Efficiently Deliver Peptides and Arrest Spontaneous Autoimmune Diabetes.

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address the limited clinical efficacy of ASIT. Soluble antigen arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using either hydrolysable linkers (hSAgAs) or stable click chemistry linkers (cSAgAs). They were evaluated for the ability to block spontaneous development of disease in a nonobese diabetic mouse model of type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgAs) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers, such as CD73, interleukin-10, programmed death-1, and KLRG-1. Anaphylaxis caused by free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely nonoverlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to free peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.

Proinsulin-specific T-cell responses correlate with estimated c-peptide and predict partial remission duration in type 1 diabetes.

ObjectiveType 1 diabetes (T1D) is an autoimmune disorder in which autoreactive T cells destroy insulin‐producing β‐cells. Interventions that preserve β‐cell function represent a fundamental therapeutic goal in T1D and biomarkers that predict and monitor β‐cell function, and changes in islet autoantigenic signatures are needed. As proinsulin and neoantigens derived from proinsulin peptides (hybrid insulin peptides, HIPs) are important T1D autoantigens, we analysed peripheral blood CD4+ T‐cell autoantigen‐specific proliferative responses and their relationship to estimated β‐cell function.MethodsWe recruited 72 people with and 42 without T1D, including 17 pre‐diabetic islet antibody‐positive and 9 antibody‐negative first‐degree relatives and 16 unrelated healthy controls with T1D‐risk HLA types. We estimated C‐peptide level at 3‐month intervals for 2 years post‐diagnosis and measured CD4+ T‐cell proliferation to proinsulin epitopes and HIPs using an optimised bioassay.ResultsWe show that CD4+ T‐cell proliferation to any islet peptide and to multiple epitopes were significantly more frequent in pre‐diabetic islet antibody‐positive siblings and participants with T1D ≤ 3 months of duration, than in participants with T1D > 3 months or healthy controls. Among participants with T1D and first‐degree relatives, CD4+ T‐cell proliferation occurred most frequently in response to proinsulin33‐63 (full‐length C‐peptide). Proinsulin33‐63‐specific responses were associated with HLA‐DR3‐DQ2 and/or HLA‐DR4/DQ8. In children with T1D, proinsulin33‐63‐specific T‐cell proliferation positively associated with concurrent estimated C‐peptide and predicted survival in honeymoon.ConclusionCD4+ T‐cell proliferative responses to proinsulin‐containing autoantigens are common before and immediately after diagnosis of T1D but decline thereafter. Proinsulin33‐63‐specific CD4+ T‐cell response is a novel marker of estimated residual endogenous β‐cell function and predicts a better 2‐year disease outcome.

Simposio 14: Factores ambientales desencadenantes

Simposio 14: ¿Cuándo comienza la diabetes mellitus tipo 1?Factores ambientales desencadenantesEn los últimos años la diabetes mellitus tipo 1 (DM1) registra un incremento de la incidencia a una tasa de 3-4% anual, principalmente en niños menores de 5 años. La mayor contribución genética está representada por el HLA (50% de la contribución), mientras que la otra mitad está causada por más de 50 polimorfismos en genes no HLA. Aunque 70% de los casos porta alelos HLA de riesgo, sólo el 3-7% de los niños con esos alelos desarrolla DM1. Esto implica que son necesarios factores adicionales para desencadenar y desarrollar el proceso de enfermedad en sujetos predispuestos. En las últimas décadas, se ha observado un fuerte aumento en la presión contributiva del ambiente en el desarrollo de la DM1. La identificación y confirmación de los determinantes ambientales sigue resultando un desafío. Es probable que los factores del estilo de vida que aumentan las demandas de insulina, a través de producir un estrés en el retículo endoplásmico, promueva la aparición de alteraciones post-traduccionales de las proteínas endógenas de las células B, generando la producción de neoantígenos, péptidos híbridos de insulina que inician y/o sostienen la autoinmunidad.

247-OR: ADA Presidents’ Select Abstract: Detection of T-Cells Reactive to Hybrid Insulin Peptides in Subjects at Risk for Type 1 Diabetes

Our lab recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors and more recently, in PBMC from T1D patients. Using an IFN-gamma ELISPOT assay to test T cell reactivity in the peripheral blood of T1D patients or control subjects to a panel of 16 HIPs, we demonstrated that 70% of the responding patients showed reactivity to multiple HIPs. To determine whether the presence of HIP-reactive T cells might serve as a biomarker of disease activity in humans, we are following longitudinally a cohort of subjects positive for multiple islet autoantibodies (Ab+), individuals that are at high risk for developing T1D. Using IFN-gamma ELISPOT and CFSE dilution assays, our data indicate that HIP-reactive T cells with an inflammatory phenotype can be detected before disease onset. In one Ab+ individual that showed HIP reactivity, we used single-cell RNA sequencing to study the phenotype and TCR usage of HIP-responding T cells in 3 longitudinal samples collected 6 months apart. We observed that T cells with a regulatory phenotype (FoxP3+, CTLA-4+, LAG3+) can be observed when a proliferative response - but not inflammatory - is detected. In this ongoing study, we are reporting for the first time that HIP-reactive T cells can be observed in the peripheral blood of subjects at risk for T1D, and our data may indicate that the balance of inflammatory vs. regulatory signals of HIP-reactive T cells could be potential indicators of disease activity. Disclosure R.L. Baker: None. T. Delong: None. M. Rewers: None. P. Gottlieb: Advisory Panel; Self; Janssen Scientific Affairs, LLC., Tolerion, Inc., Viacyte, Inc. Research Support; Self; Caladrius Biosciences, Inc., Immune Tolerance Network, Janssen Pharmaceuticals, Inc., JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Mercia/Nova Laboratories, National Institute of Diabetes and Digestive and Kidney Diseases, Novo Nordisk Inc. Stock/Shareholder; Self; IM Therapeutics. K.M. Haskins: None. Funding American Diabetes Association (1-15-ACE-14 to T.D.); JDRF (2-SRA-2017-511-S-B); National Institutes of Health (R21AI133059-01A1)

Mapping of Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus (T1DM), an autoimmune disorder, is becoming widespread with approximately 97,700 children in India and 490,000 children worldwide affected. There are various etiological factors contributing to the expansion of its incidence on different geographical locations. Hence, the articles published in reputed journals were studies and data were collected for analyzing the etiology and prevention of T1DM. It has been observed that hybrid insulin peptides act as key antigens for the autoreactive T cells and cause the loss of self-tolerance in humans. The association of coxsackievirus B has been observed with the onset of T1DM. Accurate identification of the trigger can lead to the development of appropriate preventive measures. It can become a base for advance studies to prevent T1DM in humans. This review will highlight the causes and some preventive actions which can be considered to eliminate T1DM.

Detection of T cells Reactive to Hybrid Insulin Peptides in Subjects at Risk for Type 1 Diabetes

Abstract Our lab recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors and more recently, in PBMC from T1D patients. Using an IFN-g ELISPOT assay to test T cell reactivity in the peripheral blood of T1D patients or control subjects to a panel of 16 HIPs, we demonstrated that 70% of the responding patients showed reactivity to multiple HIPs. To determine whether the presence of HIP-reactive T cells might serve as a biomarker of disease activity in humans, we are following longitudinally a cohort of subjects positive for multiple islet autoantibodies (Ab+), individuals that are at high risk for developing T1D. Using IFN-g ELISPOT and CFSE dilution assays, our data indicate that HIP-reactive T cells with an inflammatory phenotype can be detected before disease onset. In one Ab+ individual that showed HIP reactivity, we used single-cell RNA sequencing to study the phenotype and TCR usage of HIP-responding T cells. We observed that T cells with a regulatory phenotype (FoxP3+, CTLA-4+, LAG3+) can be observed when a proliferative (but not inflammatory) response is detected. In this ongoing study, we are reporting for the first time that HIP-reactive T cells can be observed in the peripheral blood of subjects at risk for T1D, suggesting that the balance of inflammatory vs. regulatory signals of HIP-reactive T cells could be potential indicators of disease activity.

Analysis of the nonobese diabetic mouse islet MHC-II peptidome reveals posttranslationally modified autoantigens

Abstract Autoimmune diabetes results from the destruction of insulin-producing beta cells, a process initiated by CD4 T cells recognizing MHC-II bound beta cell-derived peptides. We identified MHC-II bound peptides in the islets and throughout the peripheral lymphatic system in order to better understand diabetes initiation and progression. Toward this end, we performed unbiased nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis to obtain sequence information for MHC-II peptides isolated from islets, pancreatic lymph nodes and spleens of nonobese diabetic (NOD) mice. Peptides derived from beta cell proteins were further examined for their relative I-Ag7 binding strength and CD4 T cell autoreactivity. For a discussion of the major immunogenic peptides derived from the B chain of insulin and from C peptide, see the abstract of Wan et al. Herein we discuss the finding of a deamidated C peptide fragment and our search for fused peptides. We identified the deamidated insulin-1 C peptide fragment Ins1C:51–61, LQTLALEVARE. Evidence in the literature supports this C-terminal deamidation as being spontaneous rather than enzyme-mediated. Deamidation improved I-Ag7 binding compared to the wild type peptide and increased its immunogenicity, demonstrating the biological importance of this modification. The homologous peptide from insulin-2 was not identified, indicating a possible sequence bias for spontaneous deamidation. We identified only one fused peptide bound to I-Ag7, an insulin C peptide-islet amyloid polypeptide hybrid insulin peptide (HIP) that matches the previously reported HIP6.9. We also identified several free fused peptides in crinosomes and posit this as the probable site of their formation.

Hybrid Insulin Peptides Are Recognized by Human T Cells in the Context of DRB1*04:01.

T cells isolated from the pancreatic infiltrates of nonobese diabetic mice have been shown to recognize epitopes formed by the covalent cross-linking of proinsulin and secretory granule peptides. Formation of such hybrid insulin peptides (HIPs) was confirmed through mass spectrometry, and responses to HIPs were observed among the islet-infiltrating T cells of pancreatic organ donors and in the peripheral blood of individuals with type 1 diabetes (T1D). However, questions remain about the prevalence of HIP-specific T cells in humans, the sequences they recognize, and their role in disease. We identified six novel HIPs that are recognized in the context of DRB1*04:01, discovered by using a library of theoretical HIP sequences derived from insulin fragments covalently linked to one another or to fragments of secretory granule proteins or other islet-derived proteins. We demonstrate that T cells that recognize these HIPs are detectable in the peripheral blood of subjects with T1D and exhibit an effector memory phenotype. HIP-reactive T-cell clones produced Th1-associated cytokines and proliferated in response to human islet preparations. These results support the relevance of HIPs in human disease, further establishing a novel posttranslational modification that may contribute to the loss of peripheral tolerance in T1D.

Cluster of Differentiation 4 T Cells and Neoantigens in Autoimmune Diabetes

Our investigations of antigens for pathogenic T cells in autoimmune diabetes led to the discovery of hybrid insulin peptides as T cell epitopes. T cells reactive to hybrid insulin peptides can be found at high frequency in the nonobese diabetic mouse model of type 1 diabetes and are also present in human patients. Hybrid insulin peptides can also be administered to mice in a tolerogenic form, thereby suppressing the autoimmune response.