Inflamed Islets Research Articles

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)

Unique pathological changes in the pancreas of fulminant type 1 diabetes.

Distinct features of the pancreas of fulminant type 1 diabetes (FT1DM) include (1) enterovirus infection of the islets and exocrine acinar tissue. (2) Activated innate immune responses: MDA5 and RIG-I expression and TLR4 and TLR9 expression in the islets of FT1DM. (3) Combined activation of the STAT/JNK and NFkB pathways, resulting in Type I interferon (IFN) and proinflammatory cytokine (i.e., IFNγ) expression in islet beta cells and MHC class I hyper-expression. (4) Activation of dendritic cells followed by effector cell infiltration of CD8+ T cells and CD68+ macrophages, resulting in apoptosis and neurosis of islet cells and exocrine acinar cells. (5) Many chemo-attractants (i.e., CXCL10) and chemotactic activators (i.e., l-plastin) were induced by a viral infection. (6) Mutual stimulating effect of cytokines expressed in beta cells in autocrine and paracrine mechanisms may enhance beta-cell destruction through the STA1-caspase pathway. (7) Proteomics analysis using laser capture microdissection followed by mass spectrometry found 38 molecules in inflamed islets of FT1DM, which were not highlighted before. Our pathologically verified model of beta-cell destruction in FT1DM will contribute to anti-virus therapy of type 1 diabetes in the near future.

Persistent IL-2 Receptor Signaling by IL-2/CD25 Fusion Protein Controls Diabetes in NOD Mice by Multiple Mechanisms.

Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2-based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies.

Studies of insulin and proinsulin in pancreas and serum support the existence of aetiopathological endotypes of type 1 diabetes associated with age at diagnosis

Aims/hypothesisIt is unclear whether type 1 diabetes is a single disease or if endotypes exist. Our aim was to use a unique collection of pancreas samples recovered soon after disease onset to resolve this issue.MethodsImmunohistological analysis was used to determine the distribution of proinsulin and insulin in the islets of pancreas samples recovered soon after type 1 diabetes onset (<2 years) from young people diagnosed at age <7 years, 7–12 years and ≥13 years. The patterns were correlated with the insulitis profiles in the inflamed islets of the same groups of individuals. C-peptide levels and the proinsulin:C-peptide ratio were measured in the circulation of a cohort of living patients with longer duration of disease but who were diagnosed in these same age ranges.ResultsDistinct patterns of proinsulin localisation were seen in the islets of people with recent-onset type 1 diabetes, which differed markedly between children diagnosed at <7 years and those diagnosed at ≥13 years. Proinsulin processing was aberrant in most residual insulin-containing islets of the younger group but this was much less evident in the group ≥13 years (p < 0.0001). Among all individuals (including children in the middle [7–12 years] range) aberrant proinsulin processing correlated with the assigned immune cell profiles defined by analysis of the lymphocyte composition of islet infiltrates. C-peptide levels were much lower in individuals diagnosed at <7 years than in those diagnosed at ≥13 years (median <3 pmol/l, IQR <3 to <3 vs 34.5 pmol/l, IQR <3–151; p < 0.0001), while the median proinsulin:C-peptide ratio was increased in those with age of onset <7 years compared with people diagnosed aged ≥13 years (0.18, IQR 0.10–0.31) vs 0.01, IQR 0.009–0.10 pmol/l; p < 0.0001).Conclusions/interpretationAmong those with type 1 diabetes diagnosed under the age of 30 years, there are histologically distinct endotypes that correlate with age at diagnosis. Recognition of such differences should inform the design of future immunotherapeutic interventions designed to arrest disease progression.

CCL21 Expression in β-Cells Induces Antigen-Expressing Stromal Cell Networks in the Pancreas and Prevents Autoimmune Diabetes in Mice.

Tumors induce tolerance toward their antigens by producing the chemokine CCL21, leading to the formation of tertiary lymphoid organs (TLOs). Ins2-CCL21 transgenic, nonobese diabetic (NOD) mice express CCL21 in pancreatic β-cells and do not develop autoimmune diabetes. We investigated by which mechanisms CCL21 expression prevented diabetes. Ins2-CCL21 mice develop TLOs by 4 weeks of age, consisting of naive CD4+ T cells compartmentalized within networks of CD45-gp38+CD31- fibroblastic reticular cell (FRC)-like cells. Importantly, 12-week-old Ins2-CCL21 TLOs contained FRC-like cells with higher contractility, regulatory, and anti-inflammatory properties and enhanced expression of β-cell autoantigens compared with nontransgenic NOD TLOs found in inflamed islets. Consistently, transgenic mice harbored fewer autoreactive T cells and a higher proportion of regulatory T cells in the islets. Using adoptive transfer and islet transplantation models, we demonstrate that TLO formation in Ins2-CCL21 transgenic islets is critical for the regulation of autoimmunity, and although the effect is systemic, the induction is mediated locally likely by lymphocyte trafficking through TLOs. Overall, our findings suggest that CCL21 promotes TLOs that differ from inflammatory TLOs found in type 1 diabetic islets in that they resemble lymph nodes, contain FRC-like cells expressing β-cell autoantigens, and are able to induce systemic and antigen-specific tolerance leading to diabetes prevention.

Natural Killer Cell Re-Modeling of the Extra-Cellular Matrix by the Degradation of Hyaluronan: Potential Role in Type 1 Diabetes

Abstract Natural Killer (NK) cells have been shown to contribute to early insulitis during development of Type 1 Diabetes (T1D). We previously identified increased Hyaluronan (HA) deposition within inflamed pancreatic islets, indicative of remodeling of the extra cellular matrix (ECM) of which HA is a component. NK cells express CD44, a ligand for HA, and granzymes known to facilitate extravasation through the ECM. Thus, we asked whether NK cells may directly interact with the ECM, potentially contributing to HA modification in pancreatic islets. Isolated NK cells activated with IL-12, IL-15, and IL-18 up-regulated Has3, CD44, and Hyal1: genes related ECM re-modeling suggesting that NK cells may act upon the ECM. To test NK-ECM interactions, we used a co-culture system of stimulated NK cells and HA producing fibroblasts. We found down-regulation of Hyaluronidases Hyal1 and Hyal2 and up-regulation of HA Synthases Has1 and Has2. These results are consistent with NK cell modulation of ECM, that was also indicated by depletion of the fibroblast cell layer. Comparatively, CD4 and CD8 T cells did not show depletion of the cell layer and differed in the degree and composition of ECM related transcripts. Together, these data suggest that NK cells play a critical role in ECM re-modeling; this NK-ECM interaction may contribute to pancreatic inflammation in the setting of T1D.

CD11c+ Cells Are Gatekeepers for Lymphocyte Trafficking to Infiltrated Islets During Type 1 Diabetes.

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease that affects more than 19 million people with incidence increasing rapidly worldwide. For T cells to effectively drive T1D, they must first traffic to the islets and extravasate through the islet vasculature. Understanding the cues that lead to T cell entry into inflamed islets is important because diagnosed T1D patients already have established immune infiltration of their islets. Here we show that CD11c+ cells are a key mediator of T cell trafficking to infiltrated islets in non-obese diabetic (NOD) mice. Using intravital 2-photon islet imaging we show that T cell extravasation into the islets is an extended process, with T cells arresting in the islet vasculature in close proximity to perivascular CD11c+ cells. Antigen is not required for T cell trafficking to infiltrated islets, but T cell chemokine receptor signaling is necessary. Using RNAseq, we show that islet CD11c+ cells express over 20 different chemokines that bind chemokine receptors expressed on islet T cells. One highly expressed chemokine-receptor pair is CXCL16-CXCR6. However, NOD. CXCR6−/− mice progressed normally to T1D and CXCR6 deficient T cells trafficked normally to the islets. Even with CXCR3 and CXCR6 dual deficiency, T cells trafficked to infiltrated islets. These data reinforce that chemokine receptor signaling is highly redundant for T cell trafficking to inflamed islets. Importantly, depletion of CD11c+ cells strongly inhibited T cell trafficking to infiltrated islets of NOD mice. We suggest that targeted depletion of CD11c+ cells associated with the islet vasculature may yield a therapeutic target to inhibit T cell trafficking to inflamed islets to prevent progression of T1D.

Investigating the Role of CD137 Ligand in the Pathogenesis of Type 1 Diabetes

Pathogenesis of type 1 diabetes (T1D) involves various interactions between genetic and environmental factors. The gene encoding the co-stimulatory molecule CD137 is located within the Idd9.3 T1D susceptibility locus and contributes to diabetes progression in NOD mice. We have previously shown that CD137 expression in T cells has dual functions: CD4+CD137+ T cells negatively regulate T1D development while CD8+CD137+ T cells showed potent diabetogenecity. The protective function of CD137 in CD4+ T cells is likely due to the significant amounts of soluble CD137 (CD137) produced by Foxp3+ Tregs. The interaction between CD137 and its ligand (CD137L) induces two signaling pathways, forward one driven by CD137 and the reverse signaling mediated by CD137L, both of which modulate T cell function. Here, we study the impact of CD137L deficiency on T1D to gain further insight into disease pathogenesis. We successfully generated a mouse strain with the NOD background and knockout of the gene encoding CD137L (Tnfsf9) using CRISPR/Cas9 technology. Relative to wild type NOD, Tnfsf9-/- mice showed significant delay in T1D development, less islet-infiltrating autoreactive CD8 T cells, reduced high-avidity IGRP autoreactive CD8 T cells in the spleen and pancreatic lymph node, and less inflamed islets. Interestingly, we detect significant increase in serum levels of sCD137. Furthermore, we could not detect differences in CD137+ Treg populations between NOD and Tnfsf9-/- mice. In bone marrow transfer experiments, CD137L deficiency in either hosts or donors was able to suppress T1D development. Tnfsf9-/- and wild type T cells showed similar capacity to induce T1D in NOD.Rag1-/- recipients. We are working on identifying the cellular source of increased sCD137 and the mechanism behind this elevation. We direct our efforts to understand how CD137-CD137L interaction can modulate T1D pathogenesis and how to translate the data from NOD mouse to understand the pathogenesis of human diabetes. Disclosure B. Foda: None. M.H. Forsberg: None. A.E. Ciecko: None. K.W. Mueller: None. A. Geurts: None. Y. Chen: None.

Revealing the specificity of regulatory T cells in murine autoimmune diabetes

Regulatory T cells (Tregs) control organ-specific autoimmunity in a tissue antigen-specific manner, yet little is known about their specificity in a natural repertoire. In this study, we used the nonobese diabetic (NOD) mouse model of autoimmune diabetes to investigate the antigen specificity of Tregs present in the inflamed tissue, the islets of Langerhans. Compared with Tregs present in spleen and lymph node, Tregs in the islets showed evidence of antigen stimulation that correlated with higher proliferation and expression of activation markers CD103, ICOS, and TIGIT. T cell receptor (TCR) repertoire profiling demonstrated that islet Treg clonotypes are expanded in the islets, suggesting localized antigen-driven expansion in inflamed islets. To determine their specificity, we captured TCRαβ pairs from islet Tregs using single-cell TCR sequencing and found direct evidence that some of these TCRs were specific for islet-derived antigens including insulin B:9-23 and proinsulin. Consistently, insulin B:9-23 tetramers readily detected insulin-specific Tregs in the islets of NOD mice. Lastly, islet Tregs from prediabetic NOD mice were effective at preventing diabetes in Treg-deficient NOD.CD28-/- recipients. These results provide a glimpse into the specificities of Tregs in a natural repertoire that are crucial for opposing the progression of autoimmune diabetes.

Deciphering the Pathogenesis of Human Type 1 Diabetes (T1D) by Interrogating T Cells from the "Scene of the Crime".

Autoimmune-mediated destruction of insulin-producing β-cells within the pancreas results in type 1 diabetes (T1D), which is not yet preventable or curable. Previously, our understanding of the β-cell specific T cell repertoire was based on studies of autoreactive T cell responses in the peripheral blood of patients at risk for, or with, T1D; more recently, investigations have included immunohistochemical analysis of some T cell specificities in the pancreas from organ donors with T1D. Now, we are able to examine live, islet-infiltrating T cells from donors with T1D. Analysis of the T cell repertoire isolated directly from the pancreatic islets of donors with T1D revealed pro-inflammatory T cells with targets of known autoantigens, including proinsulin and glutamic acid decarboxylase, as well as modified autoantigens. We have assayed the islet-infiltrating T cell repertoire for autoreactivity and function directly from the inflamed islets of T1D organ donors. Design of durable treatments for prevention of or therapy for T1D requires understanding this repertoire.

Increased Effector Memory Insulin-Specific CD4+ T Cells Correlate With Insulin Autoantibodies in Patients With Recent-Onset Type 1 Diabetes.

Type 1 diabetes (T1D) results from T cell–mediated destruction of insulin-producing β-cells. Insulin represents a key self-antigen in disease pathogenesis, as recent studies identified proinsulin-responding T cells from inflamed pancreatic islets of organ donors with recent-onset T1D. These cells respond to an insulin B-chain (InsB) epitope presented by the HLA-DQ8 molecule associated with high T1D risk. Understanding insulin-specific T-cell frequency and phenotype in peripheral blood is now critical. We constructed fluorescent InsB10–23:DQ8 tetramers, stained peripheral blood lymphocytes directly ex vivo, and show DQ8+ patients with T1D have increased tetramer+ CD4+ T cells compared with HLA-matched control subjects without diabetes. Patients with a shorter disease duration had higher frequencies of insulin-reactive CD4+ T cells, with most of these cells being antigen experienced. We also demonstrate that the number of insulin tetramer+ effector memory cells is directly correlated with insulin antibody titers, suggesting insulin-specific T- and B-cell interactions. Notably, one of four control subjects with tetramer+ cells was a first-degree relative who had insulin-specific cells with an effector memory phenotype, potentially representing an early marker of T-cell autoimmunity. Our results suggest that studying InsB10–23:DQ8 reactive T-cell frequency and phenotype may provide a biomarker of disease activity in patients with T1D and those at risk.

Cutting Edge: Origins, Recruitment, and Regulation of CD11c+ Cells in Inflamed Islets of Autoimmune Diabetes Mice.

In NOD mice, CD11c+ cells increase greatly with islet inflammation and contribute to autoimmune destruction of pancreatic β cells. In this study, we investigated their origin and mechanism of recruitment. CD11c+ cells in inflamed islets resembled classical dendritic cells based on their transcriptional profile. However, the majority of these cells were not from the Zbtb46-dependent dendritic-cell lineage. Instead, monocyte precursors could give rise to CD11c+ cells in inflamed islets. Chemokines Ccl5 and Ccl8 were persistently elevated in inflamed islets and the influx of CD11c+ cells was partially dependent on their receptor Ccr5. Treatment with islet Ag-specific regulatory T cells led to a marked decrease of Ccl5 and Ccl8, and a reduction of monocyte recruitment. These results implicate a monocytic origin of CD11c+ cells in inflamed islets and suggest that therapeutic regulatory T cells directly or indirectly regulate their influx by altering the chemotactic milieu in the islets.

Identification of Donor Origin and Condition of Transplanted Islets in Situ in the Liver of a Type 1 Diabetic Recipient

Transplantation of islet allografts into type 1 diabetic recipients usually requires multiple pancreas donors to achieve insulin independence. This adds to the challenges of immunological monitoring of islet transplantation currently relying on surrogate immune markers in peripheral blood. We investigated donor origin and infiltration of islets transplanted in the liver of a T1D patient who died of hemorrhagic stroke 4 months after successful transplantation with two intraportal islet grafts combining six donors. Immunohistological staining for donor HLA using a unique panel of human monoclonal HLA-specific alloantibodies was performed on liver cryosections after validation on cryopreserved kidney, liver, and pancreas and compared with auto- and alloreactive T-cell immunity in peripheral blood. HLA-specific staining intensity and signal-to-noise ratio varied between tissues from very strong on kidney glomeruli, less in liver, kidney tubuli, and endocrine pancreas to least in exocrine pancreas, complicating the staining of inflamed islets in an HLA-disparate liver. Nonetheless, five islets from different liver lobes could be attributed to donors 1, 2, and 5 by staining patterns with multiple HLA types. All islets showed infiltration with CD8+ cytotoxic T cells that was mirrored by progressive alloreactive responses in peripheral blood mononuclear cells (PBMCs) to donors 1, 2, and 5 after transplantation. Stably low rates of peripheral islet autoreactive T-cell responses after islet infusion fit with a complete HLA mismatch between grafts and recipient and exclude the possibility that the islet-infiltrating CD8 T cells were autoreactive. HLA-specific immunohistochemistry can identify donor origin in situ and differentiate graft dysfunction and immunological destruction.

Islet-Derived CD4 T Cells Targeting Proinsulin in Human Autoimmune Diabetes.

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19-35, and two clones from separate donors responded to insulin B-chain amino acids 9-23 (B:9-23), which are known to be a critical self-antigen-driving disease progress in animal models of autoimmune diabetes. These B:9-23-specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9-23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment.

Restoring Regulatory T Cells in Type 1 Diabetes.

Genetic and cellular studies of type 1 diabetes in patients and in the nonobese diabetic mouse model of type 1 diabetes point to an imbalance between effector T cells and regulatory T cells (Tregs) as a driver of the disease. The imbalance may arise as a consequence of genetically encoded defects in thymic deletion of islet antigen-specific T cells, induction of islet antigen-specific thymic Tregs, unfavorable tissue environment for peripheral Treg induction, and failure of islet antigen-specific Tregs to survive in the inflamed islets secondary to insufficient IL-2 signals. These understandings are the foundation for rationalized design of new therapeutic interventions to restore the balance by selectively targeting effector T cells and boosting Tregs.

Ubiquitin D Regulates IRE1α/c-Jun N-terminal Kinase (JNK) Protein-dependent Apoptosis in Pancreatic Beta Cells

Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown of UBD in human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1α in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells.

Homeostasis and function of Tregs at the site of chronic inflammation (LYM2P.728)

Abstract This study is aimed at characterizing Treg dynamics at the site of chronic inflammation in the islets of NOD mice. Tregs expressing CD103 were present primarily in the inflamed islets and low in the pancreatic LN and spleen. Both CD103+ and CD103- Tregs from inflamed NOD islets could prevent diabetes when transferred to Treg-deficient CD28KO mice. CD103 was selectively induced and maintained in inflamed islets after transfer. However, fewer CD103+ Tregs were recovered after transfer compared with CD103- Tregs, suggesting that CD103+ Tregs have a survival defect. Indeed, CD103+ Tregs had lower Bcl2 expression despite higher CD25 expression and higher responsiveness to IL-2. CD103+ islet Tregs have reduced TCRb repertoire diversity and higher BrdU incorporation, indicating they clonally expand in the islets. Lastly, in NOD mice expressing a TCR signaling reporter, the Nur77-GFP transgene, CD103+ islet Tregs were found to be uniformly Nur77hi suggesting they were more recently activated by antigens. TCRb sequences used by high frequency clones of CD103+ Tregs could be found in Nur77hiCD103- but not Nur77loCD103- Tregs, consistent with sequential TCR activation and CD103 expression. Together, our results demonstrate that chronically inflamed islets in NOD mice contain functional Tregs, but fail to maintain the highly antigen-specific activated subset. Maintenance of this subset may have important implications for the treatment of chronic inflammatory disease.

Proinflammatory role of tissue-resident fibroblastic reticular cells (CAM1P.146)

Abstract Fibroblastic reticular cells (FRC) play an important role in maintaining immune homeostasis and orchestrating immune responses by producing chemokines and cytokines that promote immune cell migration, organization, function, and survival in secondary lymphoid organs. However, little is known about their function in the development and maintenance of inflammation in the tissue. Here we show that FRC-like cells can be found in inflamed islets of Langerhans in type 1 diabetes-prone NOD mice. No FRC are found in non-inflamed islets and crucially their number increases with severity of islet infiltration. Islet FRC share many characteristics of lymph node FRC, including CCL19 and IL-7 expression. However, in contrast to lymph node FRC, islet FRC are a major source of IFNγ-induced CXCL9 in inflamed islets, indicating their more proinflammatory phenotype. In addition, co-culture of islet FRC with activated T cells results in increased expression of proinflammatory molecules including granzymes, perforin, and IFNγ by T cells. Finally, LTβR-Ig treatment leads to dramatic reduction of islet FRC, which is associated with a significant loss of CD4 and CD8 T cells among islet infiltrates as early as 3 days after treatment. These data indicates FRC-like cells in inflamed tissue actively contribute to the local inflammatory responses and may act as a viable therapeutic target for the treatment of type 1 diabetes.

Enterovirus infection and type 1 diabetes: closing in on a link?

The famous French writer Victor Hugo (1802–1885) once said: “Perseverance, secret of all triumphs!” (1). Due to the divergence seen in monozygotic twin cohorts, we know that both genetic and environmental factors must contribute to the development of human type 1 diabetes (T1D) (2,3). For many decades, we have searched for environmental triggers, but it has remained difficult to define the key players. In addition to viral infections, nutritional and hygiene conditions as well as sunlight exposure and geographical locations have all been investigated but often with inconclusive results (4). Now, we might be getting closer to identifying a key culprit, which could be enteroviral infections. In this issue of Diabetes, Krogvold et al. (5) document the presence of a low-grade enteroviral infection in pancreata from recently diagnosed patients. The importance of this study resides on the uniqueness of the six specimens examined, which were collected via partial pancreatectomy from volunteers with very recently diagnosed diabetes (3–9 weeks) as part of the Diabetes Virus Detection study (DiViD) (6). Interestingly, hyperexpression of major histocompatibility complex (MHC-I) molecules and the presence of the enteroviral capsid protein 1 (VP1) were detected in the islets from all the patients. Additional confirmation was obtained by sequencing techniques in four of the six cases. These findings indicate that infections with enteroviruses could, at least in some cases, be a precipitating key factor for the onset of T1D. The natural history tells us that T1D is an inflammatory disease affecting specifically the pancreatic islets of Langerhans, where autoantibodies to various islet antigens in many cases precede clinical diagnosis and autoreactive CD8 T cells can be documented in inflamed islets (7). Viruses have been traditionally implicated in the etiopathogenesis of the disease with no definitive proof so far. Enteroviruses are one of the main candidates because …

Therapeutic regulatory T cells subvert effector T cell function in inflamed islets to halt autoimmune diabetes.

Therapeutic regulatory T cells (Tregs) can reverse pre-established autoimmune pathology. In this study, using a mouse model of autoimmune diabetes, we aimed to determine the means by which therapeutic Tregs control islet inflammation. Islet Ag-specific Tregs infiltrated inflamed islets soon after infusion into prediabetic mice, which was quickly followed by a selective reduction of mRNA associated with effector T cells in the islets. This change was partially due to decreased CD8(+) T cell accumulation in the tissue. CD8(+) T cells that remained in the islets after Treg treatment were able to engage dendritic cells in a manner similar to that found in untreated mice, consistent with the retention of an activated phenotype by islet dendritic cells shortly after Treg treatment. Nonetheless, Treg treatment abrogated IFN-γ production by intraislet CD8(+) and CD4(+) T cells at the protein level with minimal effect on IFN-γ mRNA. Sustained expression of IFN-γ protein by effector T cells was dependent on common γ-chain cytokine activation of the mTOR pathway, which was suppressed in islet CD8(+) T cells in vivo after Treg treatment. These multifaceted mechanisms underlie the efficacy of therapeutic Treg subversion of effector T cell functions at the site of inflammation to restore normal tissue homeostasis.