Coxsackievirus infection induces direct pancreatic ß cell killing but poor antiviral CD8+ T cell responses.

Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß cell autoimmunity and type 1 diabetes. We investigated how CVB affects human ß cells and anti-CVB T cell responses. ß cells were efficiently infected by CVB in vitro, down-regulated human leukocyte antigen (HLA) class I, and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized a fraction of these peptides; only another subfraction was targeted by effector/memory T cells that expressed exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with ß cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Our in vitro and ex vivo data highlight limited CD8+ T cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and nonstructural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

Coxsackievirus infection induces direct pancreatic ß-cell killing but poor anti-viral CD8+ T-cell responses.

Coxsackievirus B (CVB) infection of pancreatic ß cells is associated with ß-cell autoimmunity. We investigated how CVB impacts human ß cells and anti-CVB T-cell responses. ß cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the ß-cell antigen GAD. Infected ß cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

The extent and magnitude of islet T cell infiltration as powerful tools to define the progression to type 1 diabetes.

AIMS/HYPOTHESIS: Insulitis is not present in all islets, and it is elusive in humans. Although earlier studies focused on islets that fulfilled certain criteria (e.g. ≥15 CD45+ cells or ≥6 CD3+ cells), there is a fundamental lack of understanding of the infiltration dynamics in terms of its magnitude (i.e. how much) and extent (i.e. where). Here, we aimed to perform an in-depth characterisation of T cell infiltration by investigating islets with moderate (1-5 CD3+ cells) and high (≥6 CD3+ cells) infiltration in individuals with and without type 1 diabetes. METHODS: Pancreatic tissue sections from 15 non-diabetic, eight double autoantibody-positive and ten type 1 diabetic (0-2 years of disease duration) organ donors were obtained from the Network for Pancreatic Organ Donors with Diabetes, and stained for insulin, glucagon, CD3 and CD8 by immunofluorescence. T cell infiltration was quantified in a total of 8661 islets using the software QuPath. The percentage of infiltrated islets and islet T cell density were calculated. To help standardise the analysis of T cell infiltration, we used cell density data to develop a new T cell density threshold capable of differentiating non-diabetic and type 1 diabetic donors. RESULTS: Our analysis revealed that 17.1% of islets in non-diabetic donors, 33% of islets in autoantibody-positive and 32.5% of islets in type 1 diabetic donors were infiltrated by 1 to 5 CD3+ cells. Islets infiltrated by ≥6 CD3+ cells were rare in non-diabetic donors (0.4%) but could be found in autoantibody-positive (4.5%) and type 1 diabetic donors (8.2%). CD8+ and CD8- populations followed similar patterns. Likewise, T cell density was significantly higher in the islets of autoantibody-positive donors (55.4 CD3+ cells/mm2) and type 1 diabetic donors (74.8 CD3+ cells/mm2) compared with non-diabetic individuals (17.3 CD3+ cells/mm2), which was accompanied by higher exocrine T cell density in type 1 diabetic individuals. Furthermore, we showed that the analysis of a minimum of 30 islets and the use of a reference mean value for T cell density of 30 CD3+ cells/mm2 (the 30-30 rule) can differentiate between non-diabetic and type 1 diabetic donors with high specificity and sensitivity. In addition, it can classify autoantibody-positive individuals as non-diabetic or type 1 diabetic-like. CONCLUSIONS/INTERPRETATION: Our data indicates that the proportion of infiltrated islets and T cell density change dramatically during the course of type 1 diabetes, and these changes can be already observed in double autoantibody-positive individuals. This suggests that, as disease progresses, T cell infiltration extends throughout the pancreas, reaching the islets and exocrine compartment. While it predominantly targets insulin-containing islets, large accumulations of cells are rare. Our study fulfils the need to further understand T cell infiltration, not only after diagnosis but also in individuals with diabetes-related autoantibodies. Furthermore, the development and application of new analytical tools based on T cell infiltration, like the 30-30 rule, will allow us to correlate islet infiltration with demographic and clinical variables with the aim of identifying individuals at the very early stages of the disease.

Means, Motive, and Opportunity: Do Non-Islet-Reactive Infiltrating T Cells Contribute to Autoimmunity in Type 1 Diabetes?

In human type 1 diabetes and animal models of the disease, a diverse assortment of immune cells infiltrates the pancreatic islets. CD8+ T cells are well represented within infiltrates and HLA multimer staining of pancreas sections provides clear evidence that islet epitope reactive T cells are present within autoimmune lesions. These bona fide effectors have been a key research focus because these cells represent an intellectually attractive culprit for ß cell destruction. However, T cell receptors are highly diverse in human insulitis. This suggests correspondingly broad antigen specificity, which includes a majority of T cells for which there is no evidence of islet-specific reactivity. The presence of "non-cognate" T cells in insulitis raises suspicion that their role could be beyond that of an innocent bystander. In this perspective, we consider the potential pathogenic contribution of non-islet-reactive T cells. Our intellectual framework will be that of a criminal investigation. Having arraigned islet-specific CD8+ T cells for the murder of pancreatic ß cells, we then turn our attention to the non-target immune cells present in human insulitis and consider the possible regulatory, benign, or effector roles that they may play in disease. Considering available evidence, we overview the case that can be made that non-islet-reactive infiltrating T cells should be suspected as co-conspirators or accessories to the crime and suggest some possible routes forward for reaching a better understanding of their role in disease.

Whole-Slide Image Analysis of Human Pancreas Samples to Elucidate the Immunopathogenesis of Type 1 Diabetes Using the QuPath Software.

Type 1 diabetes is a chronic disease of the pancreas characterized by the loss of insulin-producing beta cells. Access to human pancreas samples for research purposes has been historically limited, restricting pathological analyses to animal models. However, intrinsic differences between animals and humans have made clinical translation very challenging. Recently, human pancreas samples have become available through several biobanks worldwide, and this has opened numerous opportunities for scientific discovery. In addition, the use of new imaging technologies has unraveled many mysteries of the human pancreas not merely in the presence of disease, but also in physiological conditions. Nowadays, multiplex immunofluorescence protocols as well as sophisticated image analysis tools can be employed. Here, we described the use of QuPath-an open-source platform for image analysis-for the investigation of human pancreas samples. We demonstrate that QuPath can be adequately used to analyze whole-slide images with the aim of identifying the islets of Langerhans and define their cellular composition as well as other basic morphological characteristics. In addition, we show that QuPath can identify immune cell populations in the exocrine tissue and islets of Langerhans, accurately localizing and quantifying immune infiltrates in the pancreas. Therefore, we present a tool and analysis pipeline that allows for the accurate characterization of the human pancreas, enabling the study of the anatomical and physiological changes underlying pancreatic diseases such as type 1 diabetes. The standardization and implementation of these analysis tools is of critical importance to understand disease pathogenesis, and may be informative for the design of new therapies aimed at preserving beta cell function and halting the inflammation caused by the immune attack.

Potential Mimicry of Viral and Pancreatic ß Cell Antigens Through Non-Spliced and cis-Spliced Zwitter Epitope Candidates in Type 1 Diabetes.

Increasing evidence suggests that post-translational peptide splicing can play a role in the immune response under pathological conditions. This seems to be particularly relevant in Type 1 Diabetes (T1D) since post-translationally spliced epitopes derived from T1D-associated antigens have been identified among those peptides bound to Human Leucocyte Antigen (HLA) class I and II complexes. Their immunogenicity has been confirmed through CD4+ and CD8+ T cell-mediated responses in T1D patients. Spliced peptides theoretically have a large sequence variability. This might increase the frequency of viral-human zwitter peptides, i.e. peptides that share a complete sequence homology irrespective of whether they originate from human or viral antigens, thereby impinging upon the discrimination between self and non-self antigens by T cells. This might increase the risk of autoimmune responses triggered by viral infections. Since enteroviruses and other viral infections have historically been associated with T1D, we investigated whether cis-spliced peptides derived from selected viruses might be able to trigger CD8+ T cell-mediated autoimmunity. We computed in silico viral-human non-spliced and cis-spliced zwitter epitope candidates, and prioritized peptide candidates based on: (i) their binding affinity to HLA class I complexes, (ii) human pancreatic ß cell and medullary thymic epithelial cell (mTEC) antigens' mRNA expression, (iii) antigen association with T1D, and (iv) potential hotspot regions in those antigens. Neglecting potential T cell receptor (TCR) degeneracy, no viral-human zwitter non-spliced peptide was found to be an optimal candidate to trigger a virus-induced CD8+ T cell response against human pancreatic ß cells. Conversely, we identified some zwitter peptide candidates, which may be produced by proteasome-catalyzed peptide splicing, and might increase the likelihood of pancreatic ß cells recognition by virus-specific CD8+ T cell clones, therefore promoting ß cell destruction in the context of viral infections.

Neoepitopes in Type 1 Diabetes: Etiological Insights, Biomarkers and Therapeutic Targets.

The mechanisms underlying type 1 diabetes (T1D) pathogenesis remain largely unknown. While autoantibodies to pancreatic beta-cell antigens are often the first biological response and thereby a useful biomarker for identifying individuals in early stages of T1D, their role in T1D pathogenesis is not well understood. Recognition of these antigenic targets by autoreactive T-cells plays a pathological role in T1D development. Recently, several beta-cell neoantigens have been described, indicating that both neoantigens and known T1D antigens escape central or peripheral tolerance. Several questions regarding the mechanisms by which tolerance is broken in T1D remain unanswered. Further delineating the timing and nature of antigenic responses could allow their use as biomarkers to improve staging, as targets for therapeutic intervention, and lead to a better understanding of the mechanisms leading to loss of tolerance. Multiple factors that contribute to cellular stress may result in the generation of beta-cell derived neoepitopes and contribute to autoimmunity. Understanding the cellular mechanisms that induce beta-cells to produce neoantigens has direct implications on development of therapies to intercept T1D disease progression. In this perspective, we will discuss evidence for the role of neoantigens in the pathogenesis of T1D, including antigenic responses and cellular mechanisms. We will additionally discuss the pathways leading to neoepitope formation and the cross talk between the immune system and the beta-cells in this regard. Ultimately, delineating the timing of neoepitope generation in T1D pathogenesis will determine their role as biomarkers as well as therapeutic targets.

Altered ß-Cell Prohormone Processing and Secretion in Type 1 Diabetes.

Analysis of data from clinical cohorts, and more recently from human pancreatic tissue, indicates that reduced prohormone processing is an early and persistent finding in type 1 diabetes. In this article, we review the current state of knowledge regarding alterations in islet prohormone expression and processing in type 1 diabetes and consider the clinical impact of these findings. Lingering questions, including pathologic etiologies and consequences of altered prohormone expression and secretion in type 1 diabetes, and the natural history of circulating prohormone production in health and disease, are considered. Finally, key next steps required to move forward in this area are outlined, including longitudinal testing of relevant clinical populations, studies that probe the genetics of altered prohormone processing, the need for combined functional and histologic testing of human pancreatic tissues, continued interrogation of the intersection between prohormone processing and autoimmunity, and optimal approaches for analysis. Successful resolution of these questions may offer the potential to use altered prohormone processing as a biomarker to inform therapeutic strategies aimed at personalized intervention during the natural history of type 1 diabetes and as a pathogenic anchor for identification of potential disease-specific endotypes.

Islet expression of type I interferon response sensors is associated with immune infiltration and viral infection in type 1 diabetes.

Previous results indicate the presence of an interferon (IFN) signature in type 1 diabetes (T1D), capable of inducing chronic inflammation and compromising b cell function. Here, we determined the expression of the IFN response markers MxA, PKR, and HLA-I in the islets of autoantibody-positive and T1D donors. We found that these markers can be coexpressed in the same islet, are more abundant in insulin-containing islets, are highly expressed in islets with insulitis, and their expression levels are correlated with the presence of the enteroviral protein VP1. The expression of these markers was associated with down-regulation of multiple genes in the insulin secretion pathway. The coexistence of an IFN response and a microbial stress response is likely to prime islets for immune destruction. This study highlights the importance of therapeutic interventions aimed at eliminating potentially persistent infections and diminishing inflammation in individuals with T1D.

One in Ten CD8+ Cells in the Pancreas of Living Individuals With Recent-Onset Type 1 Diabetes Recognizes the Preproinsulin Epitope PPI15-24.

In type 1 diabetes (T1D), a lifelong autoimmune disease, T cells infiltrate the islets and the exocrine pancreas in high numbers. CD8+ T cells are the main cell type found in the insulitic lesion, and CD8+ T cells reactive against ß-cell antigens have been detected in peripheral blood and in the pancreas of patients with short- or long-term disease. In the Diabetes Virus Detection (DiViD) study, researchers collected pancreatic tissue, by pancreatic tail resection, from living patients with recent-onset T1D. These tissues have been extensively studied by the scientific community, but the autoreactive nature of the T-cell infiltrate has remained unexplored. Our objective was to determine the number and localization of these cells in pancreas samples obtained through the DiViD study. Here, we demonstrate the presence of high frequencies of CD8+ T cells reactive against a highly relevant epitope derived from the preproinsulin signal peptide in pancreatic tissue samples from these donors. We also show the heterogeneity of islet distribution and CD8+ T-cell infiltration. Our findings contribute to the current limited existing knowledge of T-cell reactivity in the pancreas of donors with recent-onset T1D and indicate that antigen-specific therapies directed toward preproinsulin could have high clinical impact.

The healthy exocrine pancreas contains preproinsulin-specific CD8 T cells that attack islets in type 1 diabetes.

Preproinsulin (PPI) is presumably a crucial islet autoantigen found in patients with type 1 diabetes (T1D) but is also recognized by CD8+ T cells from healthy individuals. We quantified PPI-specific CD8+ T cells within different areas of the human pancreas from nondiabetic controls, autoantibody-positive donors, and donors with T1D to investigate their role in diabetes development. This spatial cellular quantitation revealed unusually high frequencies of autoreactive CD8+ T cells supporting the hypothesis that PPI is indeed a key autoantigen. To our surprise, PPI-specific CD8+ T cells were already abundantly present in the nondiabetic pancreas, thus questioning the dogma that T1D is caused by defective thymic deletion or systemic immune dysregulation. During T1D development, these cells accumulated in and around islets, indicating that an islet-specific trigger such as up-regulation of major histocompatibility complex class I might be essential to unmask beta cells to the immune system.

Human herpesvirus-6 is present at higher levels in the pancreatic tissues of donors with type 1 diabetes.

Human herpesvirus-6 (HHV-6) is a ubiquitous pathogen associated with nervous and endocrine autoimmune disorders. The aim of this study was to investigate the presence of HHV-6 in pancreatic tissue sections from non-diabetic, auto-antibody positive (AAB+), and donors with type 1 diabetes (T1D) and explore whether there is any association between HHV-6 and MHC class I hyperexpression and CD8 T cell infiltration. HHV-6 DNA was detected by PCR and its protein was examined by indirect immunofluorescence assay followed by imaging using high-resolution confocal microscopy. Viral DNA (U67) was found in most pancreata of non-diabetic (3 out of 4), AAB+ (3 out of 5) and T1D donors (6 out of 7). Interestingly, HHV-6 glycoprotein B (gB) was more expressed in islets and exocrine pancreas of donors with T1D. However, gB expression was not directly associated with other pathologies. Out of 20 islets with high gB expression, only 3 islets (15%) showed MHC class I hyperexpression. Furthermore, no correlation was found between gB expression and CD8 T cell infiltration on a per-islet basis in any of the groups. Our observations indicate that HHV-6 DNA and protein are present in the pancreas of non-diabetic subjects but gB expression is higher in the pancreas of donors with T1D. The possible role of HHV-6 as a contributory factor for T1D should therefore be further investigated.

Pancreas Pathology During the Natural History of Type 1 Diabetes.

PURPOSE OF REVIEW: We provide an overview of pancreas pathology in type 1 diabetes (T1D) in the context of its clinical stages. RECENT FINDINGS: Recent studies of pancreata from organ donors with T1D and non-diabetic donors expressing T1D-associated autoantibodies reveal pathological changes/disease mechanisms beyond the well-known loss of ß cells and lymphocytic infiltrates of the islets (insulitis), including ß-cell stress, dysfunction, and viral infections. Pancreas pathology evolves through disease stages, is asynchronous, and demonstrates a chronic disease that remains active years after diagnosis. Critically, ß-cell loss is not complete at onset, although young age is associated with increased severity. The recognition of multiple pathogenic alterations and the chronic nature of disease mechanisms during and after the development of T1D inform improved clinical trial design and reveal additional targets for therapeutic manipulation, in the context of an expanded time window for intervention.

Enteroviral Infections as a Trigger for Type 1 Diabetes.

PURPOSE OF REVIEW: To provide an overview of studies that have detected enteroviruses (EV) in samples from people with type 1 diabetes (T1D), the techniques they have used, and which challenges they have encountered. RECENT FINDINGS: Recent studies have detected EVs in serum, blood, stools, nasal swabs, and pancreas of people with T1D before or around clinical onset of disease, indicating that an association between EV infections and T1D exists. However, definitive evidence for its role as disease triggers is lacking. Recent access to human samples is starting to provide the necessary tools to define their role in disease pathogenesis. Emerging evidence suggests that chronic infections take place in the pancreas of diabetic donors. However, the development of sensitive techniques able to detect low amounts of viral protein and RNA still constitute a major challenge for the field. New evidence at the protein, RNA, and host immune response level suggests a role for EV infections in the development of autoimmunity. In the upcoming years, new technologies, collaborative efforts, and therapeutic interventions are likely to find a definitive answer for their role in disease pathogenesis.

Loss of IDO1 Expression From Human Pancreatic ß-Cells Precedes Their Destruction During the Development of Type 1 Diabetes.

Indoleamine 2,3 dioxygenase-1 (IDO1) is a powerful immunoregulatory enzyme that is deficient in patients with type 1 diabetes (T1D). In this study, we present the first systematic evaluation of IDO1 expression and localization in human pancreatic tissue. Although IDO1 was constitutively expressed in ß-cells from donors without diabetes, less IDO1 was expressed in insulin-containing islets from double autoantibody-positive donors and patients with recent-onset T1D, although it was virtually absent in insulin-deficient islets from donors with T1D. Scatter plot analysis suggested that IDO1 decay occurred in individuals with multiple autoantibodies, prior to ß-cell demise. IDO1 impairment might therefore contribute to ß-cell demise and could potentially emerge as a promising therapeutic target.

Recall T cell responses to bluetongue virus produce a narrowing of the T cell repertoire.

In most viral infections, recall T cell responses are critical for protection. The magnitude of these secondary responses can also affect the CD8 and CD4 epitope repertoire diversity. Bluetongue virus (BTV) infection in sheep elicits a T cell response that contributes to viremia control and could be relevant for cross-protection between BTV serotypes. Here, we characterized CD4+ and CD8+ T cell responses during primary and recall responses. During primary immune responses, both CD4+ and CD8+ T cell populations expanded by 14 days post-infection (dpi). CD4+ T cell populations showed a lower peak of expansion and prolonged contraction phase compared to CD8+ T cell populations. Recall responses to BTV challenge led to BTV-specific expansion and activation of CD8+ but not of CD4+ T cells. The evolution of the BTV-specific TCR repertoire was also characterized in response to VP7 peptide stimulation. Striking differences in repertoire development were noted over the time-course of infection. During primary responses, a broader repertoire was induced for MHC-I and MHC-II epitopes. However, during memory responses, a narrowed repertoire was activated towards a dominant motif in VP7 comprising amino acids 139-291. Monocytes were also examined, and expanded during acute infection resolution. In addition, pro-inflammatory cytokine levels increased after BTV inoculation and persisted throughout the experiment, indicative of a prolonged inflammatory state during BTV infections. These findings could have implications for vaccine design as the narrowing memory T cell repertoire induced after BTV re-infection could lead to the development of protective immunodominant TCR repertoires that differs between individual sheep.

Alpha cells, the main source of IL-1ß in human pancreas.

Interleukin-1ß (IL-1ß) is known to trigger beta cell dysfunction in vitro and could potentially play a role during the pathogenesis of type 1 diabetes and type 2 diabetes. However, several clinical trials attempting to block IL-1ß function have had minimal success. We therefore re-investigated local expression of IL-1ß in human diabetic and non-diabetic pancreata. We obtained pancreatic tissue sections from the Network for Pancreatic Organ Donors with Diabetes (nPOD) including non-diabetic (n = 9), non-diabetic auto-antibody positive (AAb+, n = 5), type 1 diabetes (n = 6), and type 2 diabetes (n = 6) donors. Islets were systematically investigated for the presence of IL-1ß mRNA by in situ hybridization and IL-1ß protein by indirect immunofluorescence. We found that intra-islet IL-1ß was produced at comparable level in both non-diabetic and diabetic donors. Interestingly, the main source for IL-1ß was alpha cells but not beta cells. Our findings call into question the role of IL-1ß in the diabetic pancreas as it has been proposed in previous literature. Additionally, our results regarding the localization of IL-1ß should lead to further investigation into the role of IL-1ß in the physiology of pancreatic alpha cells.