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 β 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 β 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.

Carbonyl Post-Translational Modification Associated with Early Onset Type 1 Diabetes Autoimmunity

Inflammation and oxidative stress in pancreatic islets amplify the appearance of various post-translational modifications (PTMs) to self-proteins. Herein, we identified a select group of carbonylated islet proteins arising before the onset of hyperglycemia in non-obese diabetic (NOD) mice. Of particular interest, we identified carbonyl modification of the prolyl-4-hydroxylase beta subunit (P4Hb) that is responsible for proinsulin folding and trafficking as an autoantigen in both human and murine type 1 diabetes. We found the carbonylated-P4Hb is amplified in stressed islets coincident with decreased glucose-stimulated insulin secretion and altered proinsulin to insulin ratios. Moreover, circulating autoantibodies against P4Hb were detected in prediabetic NOD mice and in early human type 1 diabetes prior to the onset of anti-insulin autoimmunity. Our studies provide mechanistic insight into the pathways of proinsulin metabolism and those creating autoantigenic forms of insulin in type 1 diabetes.

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 and characterisation of tertiary lymphoid organs in human type 1 diabetes

Aims/hypothesis We and others previously reported the presence of tertiary lymphoid organs (TLOs) in the pancreas of NOD mice, where they play a role in the development of type 1 diabetes. Our aims here are to investigate whether TLOs are present in the pancreas of individuals with type 1 diabetes and to characterise their distinctive features, in comparison with TLOs present in NOD mouse pancreases, in order to interpret their functional significance. Methods Using immunofluorescence confocal microscopy, we examined the extracellular matrix (ECM) and cellular constituents of pancreatic TLOs from individuals with ongoing islet autoimmunity in three distinct clinical settings of type 1 diabetes: at risk of diabetes; at/after diagnosis; and in the transplanted pancreas with recurrent diabetes. Comparisons were made with TLOs from 14-week-old NOD mice, which contain islets exhibiting mild to heavy leucocyte infiltration. We determined the frequency of the TLOs in human type 1diabetes with insulitis and investigated the presence of TLOs in relation to age of onset, disease duration and disease severity. Results TLOs were identified in preclinical and clinical settings of human type 1 diabetes. The main characteristics of these TLOs, including the cellular and ECM composition of reticular fibres (RFs), the presence of high endothelial venules and immune cell subtypes detected, were similar to those observed for TLOs from NOD mouse pancreases. Among 21 donors with clinical type 1 diabetes who exhibited insulitis, 12 had TLOs and had developed disease at younger age compared with those lacking TLOs. Compartmentalised TLOs with distinct T cell and B cell zones were detected in donors with short disease duration. Overall, TLOs were mainly associated with insulin-containing islets and their frequency decreased with increasing severity of beta cell loss. Parallel studies in NOD mice further revealed some differences in so far as regulatory T cells were essentially absent from human pancreatic TLOs and CCL21 was not associated with RFs. Conclusions/interpretation We demonstrate a novel feature of pancreas pathology in type 1 diabetes. TLOs represent a potential site of autoreactive effector T cell generation in islet autoimmunity and our data from mouse and human tissues suggest that they disappear once the destructive process has run its course. Thus, TLOs may be important for type 1 diabetes progression. Graphical abstract

Footprints of Immune Cells in the Pancreas in Type 1 Diabetes; to “B” or Not to “B”: Is That Still the Question?

Significant progress has been made in understanding the phenotypes of circulating immune cell sub-populations in human type 1 diabetes but much less is known about the equivalent populations that infiltrate the islets to cause beta-cell loss. In particular, considerable uncertainties remain about the phenotype and role of B-lymphocytes in the pancreas. This gap in understanding reflects both the difficulty in accessing the gland to study islet inflammation during disease progression and the fact that the number and proportion of islet-associated B-lymphocytes varies significantly according to the disease endotype. In very young children (especially those <7 years at onset) pancreatic islets are infiltrated by both CD8+ T- and CD20+ B-lymphocytes in roughly equal proportions but it is widely held that the CD8+ T-lymphocytes are responsible for driving beta-cell toxicity. By contrast, the role played by B-lymphocytes remains enigmatic. This is compounded by the fact that, in older children and teenagers (those ≥13 years at diagnosis) the proportion of B-lymphocytes found in association with inflamed islets is much reduced by comparison with those who are younger at diagnosis (reflecting two endotypes of disease) whereas CD8+ T-lymphocytes form the predominant population in both groups. In the present paper, we review the current state of understanding and develop a proposal to stimulate further discussion of the roles played by islet-associated B-lymphocytes in human type 1 diabetes. We cite evidence indicating that sites of direct contact can be found between CD8+ and CD20+-lymphocytes in and around inflamed islets and propose that such interactions may be important in determining the efficiency of beta cell killing.

New Insights Into the Role of Autoreactive CD8 T Cells and Cytokines in Human Type 1 Diabetes

Since the establishment of the network for pancreatic organ donors with diabetes (nPOD), we have gained unprecedented insight into the pathology of human type 1 diabetes. Many of the pre-existing “dogmas”, mostly derived from studies of animal models and sometimes limited human samples, have to be revised now. For example, we have learned that autoreactive CD8 T cells are present even in healthy individuals within the exocrine pancreas. Furthermore, their “attraction” to islets probably relies on beta-cell intrinsic events, such as the over-expression of MHC class I and resulting presentation of autoantigens such as (prepro)insulin. In addition, we are discovering other signs of beta-cell dysfunction, possibly at least in part due to stress, such as the over-expression of certain cytokines. This review summarizes the latest developments focusing on cytokines and autoreactive CD8 T cells in human type 1 diabetes pathogenesis.

Adaptive Immunity and Pathogenesis of Diabetes: Insights Provided by the α4–Integrin Deficient NOD Mouse

Background: The spontaneously diabetic “non-obese diabetic” (NOD) mouse is a faithful model of human type-1 diabetes (T1D). Methods: Given the pivotal role of α4 integrin (CD49d) in other autoimmune diseases, we generated NOD mice with α4-deficient hematopoiesis (NOD.α4-/-) to study the role of α4 integrin in T1D. Results: NOD.α4-/- mice developed islet-specific T-cells and antibodies, albeit quantitatively less than α4+ counterparts. Nevertheless, NOD.α4-/- mice were completely and life-long protected from diabetes and insulitis. Moreover, transplantation with isogeneic α4-/- bone marrow prevented progression to T1D of pre-diabetic NOD.α4+ mice despite significant pre-existing islet cell injury. Transfer of α4+/CD3+, but not α4+/CD4+ splenocytes from diabetic to NOD.α4-/- mice induced diabetes with short latency. Despite an only modest contribution of adoptively transferred α4+/CD3+ cells to peripheral blood, pancreas-infiltrating T-cells were exclusively graft derived, i.e., α4+. Microbiota of diabetes-resistant NOD.α4-/- and pre-diabetic NOD.α4+ mice were identical. Co- housed diabetic NOD.α4+ mice showed the characteristic diabetic dysbiosis, implying causality of diabetes for dysbiosis. Incidentally, NOD.α4-/- mice were protected from autoimmune sialitis. Conclusion: α4 is a potential target for primary or secondary prevention of T1D.

Pancreatic Beta Cell Autophagy is Impaired in Type 1 Diabetes

Aims/hypothesisPancreatic beta cells are highly metabolic secretory cells that are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species (ROS) during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce ROS to protect against apoptosis both in vitro and in vivo. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesized that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development.MethodsMouse pancreata were collected from chloroquine injected and non-injected NOR, nondiabetic NOD, and diabetic NOD mice. Age and BMI-matched pancreas tissue sections from human organ donors (n=34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). To assess autophagic flux, we injected the mice with chloroquine to inhibit the final stages of autophagy. We analyzed tissues for markers of autophagy via immunofluorescence analysis. Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), LC3A/B (autophagosome marker), Lamp1 (lysosome marker), and p62 (autophagy adaptor protein and marker for autophagic flux). Images were collected on a scanning laser confocal microscope then analyzed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes (formerly called lipofuscin bodies, residual bodies or tertiary lysosomes) were analyzed in electron micrographs of pancreatic tissue sections from human organ donors (nPOD; n=12) deposited in www.nanotomy.org/OA/nPOD. Energy Dispersive X-ray (EDX) analysis was also performed on these tissues to analyze distribution of elements such as nitrogen, phosphorus, and osmium in secondary lysosomes and telolysosomes of nondiabetic and autoantibody positive donor tissues (n=5).ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p=0.0077) and increased p62 in islets of both nondiabetic and diabetic NOD mice (p<0.0001 in both cases) when compared to NOR mice. There was also a significant reduction in autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to both nondiabetic NOD mice (p=0.0004) and NOR mice (p=0.0003). Chloroquine infusions elicited accumulation of autophagosomes in the islets of NOR (p=0.0029) and nondiabetic NOD mice (p<0.0001), but not in the islets of diabetic NOD mice. Chloroquine also stimulated an accumulation of the autophagy adaptor protein p62 in the islets of NOR mice (p<0.001), however this was not observed in NOD mice (regardless of diabetes status). In the human pancreata, we observed significantly reduced autophagosome:lysosome colocalization (p=0.0002) in the residual beta cells of donors with type 1 diabetes compared to nondiabetic controls. We also observed reduced colocalization of proinsulin with lysosomes in the residual beta cells of donors with type 1 diabetes compared to both nondiabetic (p<0.0001) and autoantibody positive donors (p<0.0001). Electron microscopy based analysis of human pancreas sections also revealed accumulation of telolysosomes in beta cells of autoantibody positive donors (p=0.0084), the majority of which had an nitrogen dense ring outside a phospholipid core.Conclusions/interpretationCollectively, we provide evidence of impairment in the final degradation stages of islet macroautophagy and crinophagy in human type 1 diabetes. We also document an accumulation of telolysosomes with nitrogen accumulation at their periphery in the beta cells of autoantibody positive donors. This demonstrates clear differences in the lysosome contents of autoantibody positive donors that may be associated with lysosome dysfunction prior to clinical hyperglycemia. We observe similar impairments in macroautophagy in the diabetic NOD mouse, a model of type 1 diabetes, suggesting that this mouse model can be appropriately used to study the pathogenesis of autophagy/crinophagy loss and how it relates to disease initiation and progression. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development.Research in contextWhat is already known about this subject?Autophagy confers a cytoprotective role in the beta cell.Autophagy is reduced in type 2 diabetes.Autophagy in the context of type 1 diabetes is unexplored.What is the key question?Is autophagy reduced during the pathogenesis of human type 1 diabetes?What are the new findings?We provide evidence of reduced autophagy and crinophagy in human type 1 diabetes.These data are supported by observations of reduced autophagy in a mouse model of autoimmune diabetes.How might this impact on clinical practice in the foreseeable future?This study provides evidence that autophagy is impaired in human type 1 diabetes. Prior studies have shown that loss of autophagy in the islet is associated with increased beta cell apoptosis, therefore we propose that therapeutic targeting of autophagy pathways may reduce beta cell death in type 1 diabetes development.