Mouse Model Of Autoimmune Diabetes Research Articles

Dissecting the Roles of the Nuclear and Mitochondrial Genomes in a Mouse Model of Autoimmune Diabetes.

Mitochondria have been previously implicated in diabetes, but the specific genetic factors remain unclear. To better understand the contributions of mitochondrial genes in nuclear DNA (nDNA) versus mtDNA, we created mitochondrial-nuclear exchange (MNX) mice carrying nDNA from a diabetic strain and mtDNA from nondiabetic mice. Long-term tracking of MNX mice showed occasional large shifts in heteroplasmy consistent with an mtDNA bottleneck in the germ line. In addition, the MNX mice showed improved survival and delayed incidence of diabetes relative to the unaltered diabetic mice, which appeared to be linked to the activity of respiratory complex I.

Immunomodulation by subcutaneously injected methacrylic acid-based hydrogels and tolerogenic dendritic cells in a mouse model of autoimmune diabetes

Type 1 diabetes is an autoimmune disease associated with the destruction of insulin-producing β cells. Immunotherapies are being developed to mitigate autoimmune diabetes. One promising option is the delivery of tolerogenic dendritic cells (DCs) primed with specific β-cell-associated autoantigens. These DCs can combat autoreactive cells and promote expansion of β-cell-specific regulatory immune cells, including Tregs. Tolerogenic DCs are typically injected systemically (or near target lymph nodes) in suspension, precluding control over the microenvironment surrounding tolerogenic DC interactions with the host. In this study we show that degradable, synthetic methacrylic acid (MAA)-based hydrogels are an inherently immunomodulating delivery vehicle that enhances tolerogenic DC therapy in the context of autoimmune diabetes. MAA hydrogels were found to affect the local recruitment and activation state of macrophages, DCs, T cells and other cells. Delivering tolerogenic DCs in the MAA hydrogel improved the local host response (e.g., fewer cytotoxic T cells) and enhanced peripheral Treg expansion. Non obese diabetic (NOD) mice treated with tolerogenic DCs subcutaneously injected in MAA hydrogels showed a delay in onset of autoimmune diabetes compared to control vehicles. Our findings further demonstrate the usefulness of MAA-based hydrogels as platforms for regenerative medicine in the context of type 1 diabetes.

295-OR: Mechanisms of Antigen-Specific Immunotherapy following Tolerance Induction to a CD4 Hybrid Insulin Peptide Epitope and the Impact on CD8 T Cell Dysfunction

Type 1 diabetes is the culmination of T cell mediated autoimmunity resulting in the destruction of insulin-producing beta cells. Islet-derived hybrid insulin peptides (HIPs), recognized by autoreactive CD4 T cells, are likely dominant T cell epitopes in the NOD mouse model of autoimmune diabetes. The 2.5HIP is formed by the fusion of a fragment of insulin C-peptide with a natural cleavage product of chromogranin A, generating a neoepitope. When the 2.5HIP was delivered as an immunotherapy on tolerance inducing PLG-nanoparticles, islet grafts functioned longer in transplanted, diabetic NOD mice. Tolerance induction to this single neoepitope induced dysfunctional cytokine production in autoreactive CD4 and CD8 T cells specific for other known islet antigens. Here we used both prediabetic NOD mice and diabetic transplant recipients to examine mechanisms of peripheral tolerance induction to a HIP. Using the Tiger IL10-GFP reporter mouse, we observed a substantial increase in the proportion of (CD25+) Treg and (CD25-) Tr1 2.5HIP tetramer+ CD4 T cells that expressed IL10 in the islets, spleen, and draining lymph node of 2.5HIP nanoparticle treated mice. Autoreactive, IGRP tetramer+ CD8 T cells displayed a trafficking defect and accumulated in secondary lymphoid tissue with a concomitant reduction within islets. The proportion of cytolytic, CX3CR1+ IGRP tetramer+ CD8 T cells that infiltrated islets was also reduced. IL10 generated by both Treg and Tr1 2.5HIP tet+ T cells may directly suppress IGRP tetramer+ CD8 T cells or suppress an intermediate cell type such as a dendritic cell, causing CD8 T cell dysfunction. Examining how antigen-specific immunotherapies suppress multiple autoreactive T cells specificities may help inform future therapies without the need for immunosuppression. Disclosure J.E.Dilisio: None. R.L.Baker: None. K.M.Haskins: None. Funding National Institutes of Health (5T32DK120520-03, 2R01DK081166-11); JDRF (2-SRA-2020-907-S-B)

Suppression of IGRP-specific CD8 T cells following induction of tolerance to a Hybrid Insulin Peptide CD4 neoepitope

Abstract An autoimmune-mediated death of insulin-producing beta cells, orchestrated by effector CD4 and CD8 T cells that recognize islet antigens, results in Type 1 Diabetes. A dominant CD4 islet autoantigen in the NOD mouse model of autoimmune diabetes is a neoepitope termed the 2.5 Hybrid Insulin Peptide (2.5HIP). By delivering the 2.5HIP as an antigen-specific immunotherapy on tolerogenic PLG-nanoparticles, islet grafts in diabetic NOD mice survived longer and cytokine production in autoreactive CD4 and CD8 T cells was suppressed, including the well-studied IGRP tetramer+ (tet+) CD8 T cells. Using both diabetic transplant recipients and prediabetic NOD mice treated with 2.5HIP nanoparticles, we examined mechanisms of peripheral tolerance induction to a dominant CD4 neoepitope and the impact on the function of IGRP CD8 T cells. Following induction of tolerance to the 2.5HIP, there was an increase in dysfunctional surface marker expression (PD1+ TIM3+) on effector 2.5HIP tet+ CD4 T cells in the spleen of treated mice. Along with a decrease in effector function, we observed an increase in the fraction of both Treg and Tr1 2.5HIP tet+ T cells expressing IL10 in the islets, spleen, and draining lymph nodes of tolerized mice. Concurrently, IGRP tet+ CD8 T cells accumulated in the draining lymph nodes and were less able to traffic and infiltrate islets. Of the IGRP tet+ cells that entered islets, there were fewer cytolytic CX 3CR1 +effector cells in tolerized mice. The robust functional increase in both Treg and Tr1 2.5HIP tet+ T cells may provide a potential mechanism for the accumulation of IGRP tet+ CD8 T cells in the draining lymph node and explain their inefficient trafficking into, and function within, islets following tolerance induction. NIH - T32 5T32DK120520-03, R01 2R01DK081166-11) JDRF 2-SRA-2020-907-S-B

Compound A attenuates proinflammatory cytokine-induced endoplasmic reticulum stress in beta cells and displays beneficial therapeutic effects in a mouse model of autoimmune diabetes.

Type 1 diabetes (T1D) is characterized by an immune-mediated progressive destruction of the insulin-producing β-cells. Proinflammatory cytokines trigger endoplasmic reticulum (ER) stress and subsequent insulin secretory deficiency in cultured β-cells, mimicking the islet microenvironment in T1D. β-cells undergo physiologic ER stress due to the high rate of insulin production and secretion under stimulated conditions. Severe and uncompensated ER stress in β-cells is induced by several pathological mechanisms before onset and during T1D. We previously described that the small drug Compound A (CpdA), a selective glucocorticoid receptor (GR/NR3C1, nuclear receptor subfamily 3, group C, member 1) ligand with demonstrated inflammation-suppressive activity in vivo, is an effective modulator of effector T and dendritic cells and of macrophages, yet, in a GR-independent manner. Here, we focus on CpdA's therapeutic potential in T1D cellular and animal models. We demonstrate that CpdA improves the unfolded protein response (UPR) by attenuating ER stress and favoring the survival and function of β-cells exposed to an environment of proinflammatory cytokines. CpdA administration to NODscid mice adoptively transferred with diabetogenic splenocytes (from diabetic NOD mice) led to a delay of disease onset and reduction of diabetes incidence. Histological analysis of the pancreas showed a reduction in islet leukocyte infiltration (insulitis) and preservation of insulin expression in CpdA-treated normoglycemic mice in comparison with control group. These new findings together with our previous reports justify further studies on the administration of this small molecule as a novel therapeutic strategy with dual targets (effector immune and β-cells) during autoimmune diabetes.

Costimulation blockade in combination with IL-2 permits regulatory T cell sparing immunomodulation that inhibits autoimmunity

Blockade of CD28 costimulation with CTLA-4-Ig/Abatacept is used to dampen effector T cell responses in autoimmune and transplantation settings. However, a significant drawback of this approach is impaired regulatory T cell homeostasis that requires CD28 signaling. Therefore, strategies that restrict the effects of costimulation blockade to effector T cells would be advantageous. Here we probe the relative roles of CD28 and IL-2 in maintaining Treg. We find provision of IL-2 counteracts the regulatory T cell loss induced by costimulation blockade while minimally affecting the conventional T cell compartment. These data suggest that combining costimulation blockade with IL-2 treatment may selectively impair effector T cell responses while maintaining regulatory T cells. Using a mouse model of autoimmune diabetes, we show combined therapy supports regulatory T cell homeostasis and protects from disease. These findings are recapitulated in humanised mice using clinically relevant reagents and provide an exemplar for rational use of a second immunotherapy to offset known limitations of the first.

1443-P: Real-Time In Vivo Analysis of Beta-Cell Autophagy in Autoimmune Diabetes

Type 1 Diabetes (T1D) is an autoimmune disease caused by a combination of genetic and environmental factors. The onset of disease is associated with autoimmune attack against pancreatic beta-cells leading to a reduction of their number. While a role for the beta-cell in these events is widely hypothesized, a complete understanding of disease pathogenesis is still lacking. Our group recently found that beta-cell autophagy is defective in the context of human T1D as well as in the non-obese diabetic (NOD) mouse model of autoimmune diabetes. Importantly, our data suggested that defective autophagy is present prior to the onset of diabetes but stopped short of definitively demonstrating pre-existing defects. Therefore, here we evaluated autophagic flux in the pancreatic beta-cells of live mice using a fluorescent biosensor coupled to intravital microscopy. A custom beta-cell-selective autophagy biosensor (AAV8-INS-mCherry-EGFP-LC3B) was intraperitoneally (IP) injected into 7-week-old mice of three strains: wild type C57Bl/6J, NOD and NOD scid gamma (NSG) mice. Three weeks after biosensor injections, mice were placed under inhaled isoflurane anesthesia, then externalized pancreata were imaged using a LEICA SP8 DIVE two-photon microscope mounted with a 40x/1.1NA water objective. To evaluate autophagic flux, we assessed fluorescent signal over time in response to IP infusion of either saline or chloroquine, an autophagy inhibitor. Autophagic flux was assessed by quantifying the rate of change of the ph-sensitive GFP and the ph-insensitive RFP, as well as signal colocalization. In wild type mice, we observed clear evidence of autophagic flux over time that was altered in the presence of chloroquine. However, in contrast, in pre-diabetic NOD mice, islet autophagic flux did not respond effectively to chloroquine treatment. Collectively, these data support the conclusion that autophagy defects precede hyperglycemia and suggest a potential role for these defects in beta cell demise during T1D pathogenesis. Disclosure O.Melnyk: None. C.Muralidharan: None. M.M.Martinez irizarry: None. J.Crowder: None. A.K.Linnemann: Consultant; Janssen Research & Development, LLC. Funding National Institutes of Health (R01DK124380) . Startup funds to AKL from the Herman B Wells Center for Pediatric Research at Indiana University School of Medicine

Induction of tolerance to a CD4 T cell hybrid insulin peptide epitope prolongs islet graft survival and suppresses autoreactive CD8 T cells

Abstract Autoreactive T cells are thought to drive autoimmune diabetes by recognizing beta cell-derived peptide antigens. We previously discovered that hybrid insulin peptides (HIPs) are potent neoantigen peptide ligands for a subset of autoreactive CD4 T cells in both the NOD mouse model and human type 1 diabetes patients. Inducing tolerance to prominent T cell autoantigens through antigen-specific immunotherapy could prevent disease onset or recurrence after islet transplantation without the need for broad immunosuppression. Here we show that tolerogenic nanoparticle (NP) delivery of the 2.5HIP, a dominant CD4 T cell HIP epitope in the NOD mouse, can prolong islet graft survival in transplanted diabetic NOD mice. Tolerance induction to the 2.5HIP not only suppressed 2.5HIP tetramer+ CD4 T cells but also autoreactive CD4 and CD8 T cells specific for different islet antigens. 2.5HIP NP treatment induced a dysfunctional state in graft-infiltrating T cells characterized by increased expression of anergic markers on CD4 T cells and reduced inflammatory cytokine production in 2.5HIP tetramer+ CD4 T cells as well as IGRP (islet specific glucose-6-phosphatase catalytic subunit-related protein) tetramer+ CD8 T cells. In conclusion, we demonstrate that antigen-specific immunotherapy aimed at inducing tolerance to a single CD4 T cell HIP epitope can prolong islet graft survival and suppress autoreactive CD8 T cells in the NOD mouse model of autoimmune diabetes. Supported by grants from NIH (R01 DK122566, R01 DK081166, T32 DK120520) and JDRF (2-SRA-2018-566-S-B)

Glucagon-receptor-antagonism-mediated β-cell regeneration as an effective anti-diabetic therapy.

Type 1 diabetes mellitus (T1D) is a chronic disease with potentially severe complications, and β-cell deficiency underlies this disease. Despite active research, no therapy to date has been able to induce β-cell regeneration in humans. Here, we discover the β-cell regenerative effects of glucagon receptor antibody (anti-GcgR). Treatment with anti-GcgR in mouse models of β-cell deficiency leads to reversal of hyperglycemia, increase in plasma insulin levels, and restoration of β-cell mass. We demonstrate that both β-cell proliferation and α- to β-cell transdifferentiation contribute to anti-GcgR-induced β-cell regeneration. Interestingly, anti-GcgR-induced α-cell hyperplasia can be uncoupled from β-cell regeneration after antibody clearance from the body. Importantly, we are able to show that anti-GcgR-induced β-cell regeneration is also observed in non-human primates. Furthermore, anti-GcgR and anti-CD3 combination therapy reverses diabetes and increases β-cell mass in a mouse model of autoimmune diabetes.

D-mannose administration improves autoimmune hepatitis by upregulating regulatory T cells

A recent study revealed that d-mannose suppressed immunopathology in mouse models of autoimmune diabetes and airway inflammation and increased the proportion of regulatory T cells (Tregs) in mice. We investigated the effect of d-mannose on liver injury in murine autoimmune hepatitis (AIH) models induced by concanavalin A (ConA) and α-galactosylceramide (GalCer). Mouse models of AIH were created by intraperitoneal injection of GalCer or intravenous injection of ConA. Drinking water was supplemented with d-mannose and biochemically and pathologically examined over time. The administration of d-mannose to AIH model mice significantly reduced liver injury and reduced inflammatory cytokine expression. In addition, Tregs among splenocytes and intrahepatic lymphocytes were significantly increased by the administration of d-mannose. These results indicate that treatment with d-mannose reduced the inflammatory response in the liver and suppressed liver damage by increasing Tregs.

Dynamic Number and Function of IL-10-Producing Regulatory B Cells in the Immune Microenvironment at Distinct Stages of Type 1 Diabetes.

The critical role of IL-10-producing B cells (B10 cells) with a unique CD1dhiCD5+ phenotype in suppressing autoimmune responses and relieving inflammation has been demonstrated in several models of autoimmune diseases. However, the regulatory role of B10 cells in T cell-mediated autoimmune responses during the natural history of type 1 diabetes is unclear. In this study, we used the NOD mouse model of autoimmune diabetes to clarify the changes and potential mechanisms of B10 cells for disease. Compared with B10 cells present in the 4-wk-old normoglycemic NOD mice, the frequency of B10 cells was increased in the insulitis and diabetic NOD mice, with the highest proportion in the insulitis NOD mice. The changes in the relative number of B10 cells were most pronounced in the pancreas-draining lymph nodes. The pathogenic T cells, including Th1 and Th17 cells, remarkably increased. The assays in vitro showed that B10 cells in the NOD mice did not inhibit the proliferation of CD4+CD25- T cells. They also had no regulatory effect on IFN-γ and IL-4 secretion or on Foxp3 expression of T cells. B10 cells suppressed T cell-mediated autoimmune responses via an IL-10-dependent pathway. In contrast, B10 cells in the NOD mice exhibited a significant reduction in IL-10 production. In summary, a defect in the number and function of B10 cells may participate in the development and progression of type 1 diabetes.

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.

Negative regulation of FOXP3 expression by c-Rel O-GlcNAcylation.

O-GlcNAcylation is a reversible post-translational protein modification that regulates fundamental cellular processes including immune responses and autoimmunity. Previously, we showed that hyperglycemia increases O-GlcNAcylation of the transcription factor, nuclear factor kappaB c-Rel at serine residue 350 and enhances the transcription of the c-Rel-dependent proautoimmune cytokines interleukin-2, interferon gamma and granulocyte macrophage colony stimulating factor in T cells. c-Rel also plays a critical role in the transcriptional regulation of forkhead box P3 (FOXP3)-the master transcription factor that governs development and function of Treg cells. Here we show that the regulatory effect of c-Rel O-GlcNAcylation is gene-dependent, and in contrast to its role in enhancing the expression of proautoimmune cytokines, it suppresses the expression of FOXP3. Hyperglycemia-induced O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; streptozotocin-induced diabetes and spontaneous diabetes in nonobese diabetic mice. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases c-Rel binding at the FOXP3 promoter and negatively regulates FOXP3 expression. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating immunosuppressive FOXP3 expression and proautoimmune gene expression in autoimmune diabetes with potential therapeutic implications.

Regulatory T Cell-Derived TRAIL Is Not Required for Peripheral Tolerance.

TRAIL (Tnfsf10/TRAIL/CD253/Apo2L) is an important immune molecule that mediates apoptosis. TRAIL can play key roles in regulating cell death in the tumor and autoimmune microenvironments. However, dissecting TRAIL function remains difficult because of the lack of optimal models. We have now generated a conditional knockout (Tnfsf10L/L) for cell type–specific analysis of TRAIL function on C57BL/6, BALB/c, and NOD backgrounds. Previous studies have suggested a role for TRAIL in regulatory T cell (Treg)–mediated suppression. We generated mice with a Treg-restricted Tnfsf10 deletion and surprisingly found no impact on tumor growth in C57BL/6 and BALB/c tumor models. Furthermore, we found no difference in the suppressive capacity of Tnfsf10-deficient Tregs and no change in function or proliferation of T cells in tumors. We also assessed the role of TRAIL on Tregs in two autoimmune mouse models: the NOD mouse model of autoimmune diabetes and the myelin oligodendrocyte glycoprotein (MOG) C57BL/6 model of experimental autoimmune encephalomyelitis. We found that deletion of Tnfsf10 on Tregs had no effect on disease progression in either model. We conclude that Tregs do not appear to be dependent on TRAIL exclusively as a mechanism of suppression in both the tumor and autoimmune microenvironments, although it remains possible that TRAIL may contribute in combination with other mechanisms and/or in different disease settings. Our Tnfsf10 conditional knockout mouse should prove to be a useful tool for the dissection of TRAIL function on different cell populations in multiple mouse models of human disease.

Innate immune stimulation of whole blood reveals IFN-1 hyper-responsiveness in type 1 diabetes.

Self-antigen-specific T cell responses drive type 1 diabetes pathogenesis, but alterations in innate immune responses are also critical and not as well understood. Innate immunity in human type 1 diabetes has primarily been assessed via gene-expression analysis of unstimulated peripheral blood mononuclear cells, without the immune activation that could amplify disease-associated signals. Increased responsiveness in each of the two main innate immune pathways, driven by either type 1 IFN (IFN-1) or IL-1, have been detected in type 1 diabetes, but the dominant innate pathway is still unclear. This study aimed to determine the key innate pathway in type 1 diabetes and assess the whole blood immune stimulation assay as a tool to investigate this. The TruCulture whole blood ex vivo stimulation assay, paired with gene expression and cytokine measurements, was used to characterise changes in the stimulated innate immune response in type 1 diabetes. We applied specific cytokine-induced signatures to our data, pre-defined from the same assays measured in a separate cohort of healthy individuals. In addition, NOD mice were stimulated with CpG and monocyte gene expression was measured. Monocytes from NOD mice showed lower baseline vs diabetes-resistant B6.g7 mice, but higher induced IFN-1-associated gene expression. In human participants, ex vivo whole blood stimulation revealed higher induced IFN-1 responses in type 1 diabetes, as compared with healthy control participants. In contrast, neither the IL-1-induced gene signature nor response to the adaptive immune stimulant Staphylococcal enterotoxin B were significantly altered in type 1 diabetes samples vs healthy control participants. Targeted gene-expression analysis showed that this enhanced IFN response was specific to IFN-1, as IFN-γ-driven responses were not significantly different. Our study identifies increased responsiveness to IFN-1 as a feature of both the NOD mouse model of autoimmune diabetes and human established type 1 diabetes. A stimulated IFN-1 gene signature may be a potential biomarker for type 1 diabetes and used to evaluate the effects of therapies targeting this pathway. Mouse gene expression data are found in the gene expression omnibus (GEO) repository, accession GSE146452 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146452 ). Nanostring count data from the human experiments were deposited in the GEO repository, accession GSE146338 ( www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE146338 ). Data files and R code for all analyses are available at https://github.com/rodriguesk/T1D_truculture_diabetologia . Graphical abstract.

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)

1668-P: Novel Mechanism of Negative Regulation of FOXP3 Expression in Autoimmune Diabetes

The NF-kappaB protein c-Rel is essential for the expression of forkhead box P3 (FOXP3), the master transcription factor that governs development and function of T regulatory cells. We found that O-GlcNAcylation of c-Rel at serine residue 350 suppresses the expression of FOXP3. Hyperglycemia-induced c-Rel O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; the streptozotocin (STZ)-induced diabetic mouse model and in the non-obese diabetic (NOD) mouse model. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases the DNA binding ability of c-Rel at the FOXP3 promoter. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism regulating immunosuppressive FOXP3 expression in autoimmune diabetes with potential therapeutic implications. Disclosure P. Ramakrishnan: None. T.J. de Jesus: None. J. Centore: None. Funding National Institutes of Health (R01AI116730, R21AI144264)

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.

Immunomodulation Followed by Antigen-Specific Treg Infusion Controls Islet Autoimmunity.

Optimal immune-based therapies for type 1 diabetes (T1D) should restore self-tolerance without inducing chronic immunosuppression. CD4+Foxp3+ regulatory T cells (Tregs) are a key cell population capable of facilitating durable immune tolerance. However, clinical trials with expanded Tregs in T1D and solid-organ transplant recipients are limited by poor Treg engraftment without host manipulation. We showed that Treg engraftment and therapeutic benefit in nonautoimmune models required ablative host conditioning. Here, we evaluated Treg engraftment and therapeutic efficacy in the nonobese diabetic (NOD) mouse model of autoimmune diabetes using nonablative, combinatorial regimens involving the anti-CD3 (αCD3), cyclophosphamide (CyP), and IAC (IL-2/JES6-1) antibody complex. We demonstrate that αCD3 alone induced substantial T-cell depletion, impacting both conventional T cells (Tconv) and Tregs, subsequently followed by more rapid rebound of Tregs Despite robust depletion of host Tconv and host Tregs, donor Tregs failed to engraft even with interleukin-2 (IL-2) support. A single dose of CyP after αCD3 depleted rebounding host Tregs and resulted in a 43-fold increase in donor Treg engraftment, yet polyclonal donor Tregs failed to reverse diabetes. However, infusion of autoantigen-specific Tregs after αCD3 alone resulted in robust Treg engraftment within the islets and induced remission in all mice. This novel combinatorial therapy promotes engraftment of autoantigen-specific donor Tregs and controls islet autoimmunity without long-term immunosuppression.

Pancreatic nerve electrostimulation inhibits recent-onset autoimmune diabetes.

Vagus nerve stimulation can ameliorate autoimmune diseases such as rheumatoid arthritis by modulation of the immune system. Its efficacy for the treatment of type 1 diabetes has not been explored, in part because the nerves projecting to the pancreatic lymph nodes (pLNs) in mice are unmapped. Here, we map the nerve projecting to the pancreas and pLNs in mice and use a minimally invasive surgical procedure to implant micro-cuff electrodes onto the nerve. Pancreatic nerve electrical stimulation (PNES) resulted in β-adrenergic receptor-mediated-accumulation of B and T cells in pLNs and reduced production of pro-inflammatory cytokines following lipopolysaccharide stimulation. Autoreactive T cells showed reduced proliferation in pLNs of mice receiving PNES as compared to sham controls. In a spontaneous mouse model of autoimmune diabetes, PNES inhibited disease progression in diabetic mice.