Diabetes In Non-obese Diabetic Mice Research Articles

8439 Targeting Interferon Gamma-Mediated CXCL16-CXCR6 Chemokine-Receptor Interactions As A Strategy To Prevent Immune Checkpoint Inhibitor Autoimmune Diabetes

Abstract Disclosure: S. Wang: None. N. Huang: None. J.G. Ortega: None. J. Lee: None. K. Kimbrell: None. J. Nguyen: None. J. Olay: None. E. McCarthy: None. E. Peluso: None. H. Chen: None. M.G. Lechner: None. Autoimmune diabetes mellitus is a rare but life-threatening side effect of immune checkpoint inhibitor (ICI) cancer therapy. Checkpoint inhibitors increase immune activation by blocking regulatory molecules on T cells, including programmed death protein (PD1). As a result of this heightened immune activation, patients can develop autoimmunity in healthy tissues, known as immune related adverse events (IRAEs). ICI-associated diabetes mellitus (ICI-T1DM) is an IRAE that results in autoimmune destruction of insulin-producing pancreatic beta-cells. Patients with ICI-T1DM require life-long insulin therapy and more than half present initially with diabetic ketoacidosis. Unfortunately, the mechanism of this IRAE is not fully understood. Anti-PD1 antibody treatment (10mg/kg/dose, twice weekly, i.p.) of male and female non-obese diabetic (NOD) mice leads to autoimmune infiltrates in multiple tissues, including accelerated insulitis and DM. Single cell RNA sequencing of islet infiltrating immune cells from ICI-treated mice previously showed a role for CD8+ T cells and cytokine interferon gamma (IFNγ) in the development of ICI-T1DM. Using CellChat to perform receptor-ligand interaction analysis of these data, we identified both CD8+ and CD4+ T cells as the primary source of IFNγ in immune infiltrates and islet myeloid cells as the primary target. Furthermore, CellChat analysis showed increased receptor-ligand interactions between T cell receptor CXCR6 and its chemokine CXCL16, as well as CXCR3 and chemokines CXCL9 and CXCL10. In vitro IFNγ stimulation of myeloid cells induced strong expression of CXCL16, 9, and 10, suggesting a potential mechanism by which effector T cells are recruited to islets during ICI therapy. Immunophenotyping of islet-infiltrating immune cells indeed showed increased IFNγ+CXCR6+ effector CD8+ cells in anti-PD1 treated mice compared to isotype-treated controls. Moreover, these IFNγ+CXCR6+CD8+ T cells stained positively for the NRP-V7 mimotope tetramer, a known antigen for CD8+ T cells in spontaneous autoimmune diabetes in NOD mice, suggesting this as an antigen-specific population contributing to the immunopathogenesis of ICI-T1DM. Finally, genetic deletion of CXCR6 in NOD mice delayed the onset of autoimmune diabetes during anti-PD1 treatment in female (p=0.0162) but not male mice (p=0.3656). In summary, these data further elucidate the cellular mechanisms by which IFNγ and chemokine signaling pathways drive the development of ICI-T1DM and identify the CXCR6-CXCL16 axis as a potential therapeutic target to prevent this IRAE in checkpoint inhibitor-treated cancer patients. Presentation: 6/2/2024

Probiotic treatment with viable α-galactosylceramide-producing Bacteroides fragilis reduces diabetes incidence in female nonobese diabetic mice.

We aimed to investigate whether alpha-galactosylceramide (α-GalCer)-producing Bacteroides fragilis could induce natural killer T (NKT) cells in nonobese diabetic (NOD)mice and reduce their diabetes incidence. Five-week-old female NOD mice were treated orally with B. fragilis, and islet pathology and diabetes onset were monitored. Immune responses were analyzed by flow cytometry and multiplex technology. Effects of ultraviolet (UV)-killed α-GalCer-producing B. fragilis and their culture medium on invariant NKT (iNKT) cells were tested ex vivo on murine splenocytes, and the immunosuppressive capacity of splenocytes from B. fragilis-treated NOD mice were tested by adoptive transfer to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. B. fragilis reduced the diabetes incidence from 69% to 33% and the percent of islets with insulitis from 40% to 7%, which doubled the serum insulin level compared with the vehicle-treated control mice. Furthermore, the early treatment reduced proinflammatory mediators in the serum, whereas the proportion of CD4+ NKT cell population was increased by 33%. B. fragilis growth media stimulated iNKT cells and anti-inflammatory M2 macrophages ex vivo in contrast to UV-killed bacteria, which had no effect, strongly indicating an α-GalCer-mediated effect. Adoptive transfer of splenocytes from B. fragilis-treated NOD mice induced a similar diabetes incidence as splenocytes from untreated NOD mice. B. fragilis induced iNKT cells and M2 macrophages and reduced type 1 diabetes in NOD mice. The protective effect seemed to be more centered on gut-pancreas interactions rather than a systemic immunosuppression. B. fragilis should be considered for probiotic use in individuals at risk of developing type 1 diabetes.

Reg2 treatment is protective but the induced Reg2 autoantibody is destructive to the islets in NOD mice

Regenerating family protein 2 (Reg2) is a trophic factor which stimulates β-cell replication and resists islet destruction. However, Reg2 also serves as an islet autoantigen, which makes it complicated to judge the effectiveness in treating diabetes. How Reg2 treatment behaves in non-obese diabetic (NOD) mice is to be investigated. NOD mice were treated with recombinant Reg2 protein, Complete Freund’s adjuvant (CFA) + PBS and CFA+Reg2 vaccinations, CFA+PBS- and CFA+Reg2-immunized antisera, and single chain variable fragment (scFv)-Reg2 and mIgG2a-Reg2 antibodies. Glycemic level, bodyweight, serum Reg2 antibody titer, glucose tolerance, and insulin secretion were determined. Islet morphological characteristics, insulitis, cell apoptosis, islet cell components, and T cell infiltration were analyzed by histological examinations. The autoantigenicity of constructed Reg2C and Reg2X fragments was determined in healthy BALB/c mice, and the bioactivity in stimulating cell proliferation and survival was assessed in insulinoma MIN6 cells. Reg2 administration alleviated diabetes in NOD mice with improved glucose tolerance and insulin secretion but elevated serum Reg2 autoantibodies. Histomorphometry showed reduced inflammatory area, TUNEL signal and CD8 + T cell infiltration, and increased β-cell proportion in support of the islet-protective effect of Reg2 treatment. CFA+PBS and CFA+Reg2 immunizations prevented diabetic onset and alleviated insulitis while injections of the antisera offered mild protections. Antibody treatments accelerated diabetic onset without increasing the overall incidence. Reg2C fragment depletes antigenicity, but reserves protective activity in streptozotocin (STZ)-treated MIN6 cells. In conclusion, Reg2 treatment alleviates type 1 diabetes (T1D) by preserving islet β-cells, but induces Reg2 autoantibody production which poses a potential risk of accelerating diabetic progression.

Irisin delays the onset of type 1 diabetes in NOD mice by enhancing intestinal barrier

Type 1 diabetes (T1D), a complex autoimmune disease, is intricately linked to the gut's epithelial barrier function. Emerging evidence emphasizes the role of irisin, an exercise-related hormone, in preserving intestinal integrity. This study investigates whether irisin could delay T1D onset by enhancing the colon intestinal barrier. Impaired colon intestinal barriers were observed in newly diagnosed T1D patients and non-obese diabetic (NOD) mice, worsening with age and accompanied by islet inflammation. Using an LPS-induced colonic inflammation model, a dose-dependent impact of LPS on colon cells irisin expression, secretion, and barrier function was revealed. Exogenous irisin demonstrated remarkable effects, mitigating islet insulitis, enhancing energy expenditure, and alleviating autoimmune symptoms by reducing colon intestinal permeability. Single-cell RNA sequencing (scRNA-seq) highlighted irisin's positive impact on colon epithelial cell clusters, effectively restoring the intestinal barrier. Irisin also selectively modulated bacterial composition, averting potential bacterial translocation. Mechanistically, irisin enhanced colon intestinal barrier tight junction proteins through the AMPK/PI3K/AKT pathway, with FAM120A playing a crucial role. Irisin upregulated MUC3 expression, a protector against damage and inflammation. Harnessing irisin's exercise-mimicking properties suggests therapeutic potential in clinical settings for preventing T1D progression, offering valuable insights into fortifying the colon's intestinal barrier and managing autoimmune conditions associated with T1DM.

TIGIT acts as an immune checkpoint upon inhibition of PD1 signaling in autoimmune diabetes.

Chronic activation of self-reactive T cells with beta cell antigens results in the upregulation of immune checkpoint molecules that keep self-reactive T cells under control and delay beta cell destruction in autoimmune diabetes. Inhibiting PD1/PD-L1 signaling results in autoimmune diabetes in mice and humans with pre-existing autoimmunity against beta cells. However, it is not known if other immune checkpoint molecules, such as TIGIT, can also negatively regulate self-reactive T cells. TIGIT negatively regulates the CD226 costimulatory pathway, T-cell receptor (TCR) signaling, and hence T-cell function. The phenotype and function of TIGIT expressing islet infiltrating T cells was studied in non-obese diabetic (NOD) mice using flow cytometry and single cell RNA sequencing. To determine if TIGIT restrains self-reactive T cells, we used a TIGIT blocking antibody alone or in combination with anti-PDL1 antibody. We show that TIGIT is highly expressed on activated islet infiltrating T cells in NOD mice. We identified a subset of stem-like memory CD8+ T cells expressing multiple immune checkpoints including TIGIT, PD1 and the transcription factor EOMES, which is linked to dysfunctional CD8+ T cells. A known ligand for TIGIT, CD155 was expressed on beta cells and islet infiltrating dendritic cells. However, despite TIGIT and its ligand being expressed, islet infiltrating PD1+TIGIT+CD8+ T cells were functional. Inhibiting TIGIT in NOD mice did not result in exacerbated autoimmune diabetes while inhibiting PD1-PDL1 resulted in rapid autoimmune diabetes, indicating that TIGIT does not restrain islet infiltrating T cells in autoimmune diabetes to the same degree as PD1. Partial inhibition of PD1-PDL1 in combination with TIGIT inhibition resulted in rapid diabetes in NOD mice. These results suggest that TIGIT and PD1 act in synergy as immune checkpoints when PD1 signaling is partially impaired. Beta cell specific stem-like memory T cells retain their functionality despite expressing multiple immune checkpoints and TIGIT is below PD1 in the hierarchy of immune checkpoints in autoimmune diabetes.

Fucoidan alleviated autoimmune diabetes in NOD mice by regulating pancreatic autophagy through the AMPK/mTOR1/TFEB pathway.

The present study investigated the effect and its underlying mechanisms of fucoidan on Type 1 diabetes mellitus (T1DM) in non-obese diabetic (NOD) mice. Twenty 7-week-old NOD mice were used in this study, and randomly divided into two groups (10 mice in each group): the control group and the fucoidan treatment group (600 mg/kg. body weight). The weight gain, glucose tolerance, and fasting blood glucose level in NOD mice were detected to assess the development of diabetes. The intervention lasted for 5 weeks. The proportions of Th1/Th2 cells from spleen tissues were tested to determine the anti-inflammatory effect of fucoidan. Western blot was performed to investigate the expression levels of apoptotic markers and autophagic markers. Apoptotic cell staining was visualized through TdT-mediated dUTP nick-end labeling (TUNEL). The results suggested that fucoidan ameliorated T1DM, as evidenced by increased body weight and improved glycemic control of NOD mice. Fucoidan down-regulated the Th1/Th2 cells ratio and decreased Th1 type pro-inflammatory cytokines' level. Fucoidan enhanced the mitochondrial autophagy level of pancreatic cells and increased the expressions of Beclin-1 and LC3B II/LC3B I. The expression of p-AMPK was up-regulated and p-mTOR1 was inhibited, which promoted the nucleation of transcription factor EB (TFEB), leading to autophagy. Moreover, fucoidan induced apoptosis of pancreatic tissue cells. The levels of cleaved caspase-9, cleaved caspase-3, and Bax were up-regulated after fucoidan treatment. Fucoidan could maintain pancreatic homeostasis and restore immune disorder through enhancing autophagy via the AMPK/mTOR1/TFEB pathway in pancreatic cells.

Arachidonic acid alleviates autoimmune diabetes in NOD mice

BackgroundArachidonic acid (AA) is considered to link nutrient metabolism, to inflammation and immunity, suggesting it may have a role in autoimmune diseases. Our previous study suggests that DPP-4 inhibitors (DPP-4i) might regulate AA – relative signaling in type 1 diabetes. AimsTo examine the effect of AA on autoimmune diabetes and its cross-talk with DPP-4i in The Non-Obese Diabetic (NOD) mice. MethodsThe NOD mice were divided randomly and equally into three groups: AA group, AA plus DPP-4i group and control group. The incidence of diabetes, blood glucose, insulitis and cytokine profiles were monitored. At the end of the experiment, pancreatic tissues were stained by H&E. Serum cytokine profiles were examined using a Mesco Scale Discovery multiplexed-assay kit. ResultsEven though AA or AA plus DPP-4i treatment has no effect on incidence of diabetes and weight, AA treatment reduces blood glucose, preserves islet morphology and alleviates inflammatory cell infiltration into pancreatic islets in NOD mice, accompanying with increased serum levels of IL-10, IL-1 β, IL-6, IL-5, KC/GRO and TNF-α and decreased serum levels of IL-2. ConclusionWe observed that AA treatment alleviates autoimmune diabetes in NOD mice by reducing hyperglycemia, alleviating insulitis and improving cytokine profiles. DPP-4i might alleviate the effect of AA by cross-talk. We provide evidence of AA treatment to alleviate type 1 diabetes in NOD mice, which may provide a novel therapeutic option for type 1 diabetes.

Adoptive transfer of immature dendritic cells with high HO-1 expression delays the onset of T1DM in NOD mice

AimsTo investigate the potential of imDCs with high expression of HO-1 in preventing or delaying the onset of Type 1 diabetes mellitus (T1DM) in non-obese diabetic (NOD) mice. Materials and methodsThe phenotypic features of DCs in each group were assessed using flow cytometry. Western blot analysis was used to confirm the high expression of HO-1 in imDCs induced with CoPP. Additionally, flow cytometry was used to evaluate the suppressive capacity of CoPP-induced imDCs on splenic lymphocyte proliferation. Finally, the preventive effect of CoPP-induced imDCs was tested in NOD mice. Key findingsCompared to imDCs, CoPP-induced imDCs exhibited a reduced mean fluorescence intensity (MFI) of the co-stimulatory molecule CD80 on their surface (P < 0.05) and significantly increased HO-1 protein expression (P < 0.05). Following LPS stimulation, the MFI of co-stimulatory molecules CD80 and CD86 on the surface of CoPP-induced imDCs remained at a lower level (P < 0.05). Furthermore, there was a reduced proliferation rate of lymphocytes stimulated with anti-CD3/28 antibodies. The adoptive transfer of CoPP-imDCs significantly reduced the incidence of T1DM (16.66 % vs. control group: 66.67 %, P = 0.004). Furthermore, at 15 weeks of age, the insulitis score was also decreased in the CoPP-induced imDC treatment group (P < 0.05). There were no significant differences in serum insulin levels among all groups. SignificanceImDCs induced with CoPP and exhibiting high expression of HO-1 demonstrate a robust ability to inhibit immune responses and effectively reduce the onset of diabetes in NOD mice. This finding suggests that CoPP-induced imDCs could potentially serve as a promising treatment strategy for T1DM.

The amphiregulin/EGFR axis has limited contribution in controlling autoimmune diabetes

Conventional immunosuppressive functions of CD4+Foxp3+ regulatory T cells (Tregs) in type 1 diabetes (T1D) pathogenesis have been well described, but whether Tregs have additional non-immunological functions supporting tissue homeostasis in pancreatic islets is unknown. Within the last decade novel tissue repair functions have been ascribed to Tregs. One function is production of the epidermal growth factor receptor (EGFR) ligand, amphiregulin, which promotes tissue repair in response to inflammatory or mechanical tissue injury. However, whether such pathways are engaged during autoimmune diabetes and promote tissue repair is undetermined. Previously, we observed that upregulation of amphiregulin at the transcriptional level was associated with functional Treg populations in the non-obese diabetic (NOD) mouse model of T1D. From this we postulated that amphiregulin promoted islet tissue repair and slowed the progression of diabetes in NOD mice. Here, we report that islet-infiltrating Tregs have increased capacity to produce amphiregulin, and that both Tregs and beta cells express EGFR. Moreover, we show that amphiregulin can directly modulate mediators of endoplasmic reticulum stress in beta cells. Despite this, NOD amphiregulin deficient mice showed no acceleration of spontaneous autoimmune diabetes. Taken together, the data suggest that the ability for amphiregulin to affect the progression of autoimmune diabetes is limited.

Treg-specific CD226 Deletion Reduces Diabetes Incidence in NOD Mice by Improving Regulatory T Cell Stability.

Co-stimulation serves as a critical checkpoint for T cell activation, and several genetic variants affecting co-stimulatory pathways confer risk for autoimmune diseases. A single nucleotide polymorphism (rs763361) in the CD226 gene encoding a co-stimulatory receptor increases susceptibility to multiple autoimmune diseases, including type 1 diabetes. We previously found that Cd226 knockout protected non-obese diabetic (NOD) mice from disease, but the impact of CD226 on individual immune subsets remained unclear. Our prior reports implicate regulatory T cells (Treg), as human CD226+ Tregs exhibit reduced suppressive function. Hence, we hypothesized that genomic Cd226 gene deletion would increase Treg stability, and Treg-specific Cd226 deletion would inhibit diabetes in NOD mice. Indeed, crossing NOD.Cd226-/- and a NOD Treg-lineage tracing strain resulted in decreased pancreatic Foxp3-deficient "ex-Tregs." We generated a novel Treg-conditional knockout (TregΔCd226) strain that displayed decreased insulitis and diabetes incidence. CD226-deficient pancreatic Tregs had increased expression of the coinhibitory counter-receptor TIGIT. Moreover, NOD splenocytes treated with a TIGIT-Fc fusion protein exhibited reduced T cell proliferation and IFN-β production following anti-CD3/CD28 stimulation. This study demonstrates that a CD226/TIGIT imbalance contributes to Treg instability in NOD mice and highlights the potential for therapeutic targeting this co-stimulatory pathway to halt autoimmunity.

Delayed graft rejection in autoimmune islet transplantation via biomaterial immunotherapy

The induction of operational immune tolerance is a major goal in beta-cell replacement strategies for the treatment of type 1 diabetes. Our group previously reported long-term efficacy via biomaterial-mediated programmed death ligand 1 (PD-L1) immunotherapy in islet allografts in nonautoimmune models. In this study, we evaluated autoimmune recurrence and allograft rejection during islet transplantation in spontaneous nonobese diabetic (NOD) mice. Graft survival and metabolic function were significantly prolonged over 60 days in recipients of syngeneic islets receiving the biomaterial-delivered immunotherapy, but not in control animals. The biomaterial-mediated PD-L1 immunotherapy resulted in delayed allograft rejection in diabetic NOD mice compared with controls. Discrimination between responders and nonresponders was attributed to the enriched presence of CD206+ program death 1+ macrophages and exhausted signatures in the cytotoxic T cell compartment in the local graft microenvironment. Notably, draining lymph nodes had similar remodeling in innate and adaptive immune cell populations. This work establishes that our biomaterial platform for PD-L1 delivery can modulate immune responses to transplanted islets in diabetic NOD mice and, thus, can provide a platform for the development of immunologic strategies to curb the allo- and autoimmune processes in beta-cell transplant recipients.

743-P: Delayed Diabetes with Proinsulin mRNA Vaccines in NOD Mice

Objective: In type 1 diabetes, an immunomodulatory, antigen-specific therapy may abrogate autoimmunity and support immune tolerance to prevent β-cell destruction and disease progression. This study aims to assess the efficacy of a novel proinsulin II mRNA multi-lamellar liposome vaccine in preventing diabetes in the non-obese diabetic (NOD) mouse. We hypothesized that proinsulin mRNA vaccines would restore immunotolerance and delay diabetes. Methods: Proinsulin II was cloned into plasmids for in vitro RNA transcription. mRNA was loaded into DOTAP liposomes. mRNA expression was validated in HEK293 cell line transfection. NOD/ShiLtJ strain mice were obtained at 4 weeks of age. Group 1 received three IV tail vein injections of mRNA vaccines every other day at 8 weeks and one dose at 23 weeks of age. Group 2 remained untreated. Mice were checked weekly for diabetes (glucose ≥250 mg/dl on two measurements one day apart). Kaplan-Meier survival curves, hazard ratio, median time to diabetes, and t-tests were used to compare time to diabetes. Results: The diabetes rate by 32 weeks was 64.2% (9/14) in group 1 compared to 78.6% (11/14) in group 2 (Hazard ratio 1.60 95% CI 0.66-3.86). By 15 weeks, the treatment group had no diabetes compared to 35.7% untreated (p=0.04). Median time to diabetes was 23 weeks in the treatment group and 21 weeks in untreated. Conclusions: Proinsulin II mRNA multi-lamellar liposome vaccines temporarily delay diabetes in NOD mice. Disclosure T.P.Foster: None. M.J.Haller: Board Member; SAB Biotherapeutics, Inc., Consultant; Sanofi, MannKind Corporation. D.Schatz: Advisory Panel; Medtronic, Avotres Inc., QuLab Medical Ltd., Kriya Therapeutics. C.Wasserfall: None. E.Sayour: None. Funding Endocrine Fellows Foundation

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

PAHSAs reduce cellular senescence and protect pancreatic beta cells from metabolic stress through regulation of Mdm2/p53

Senescence in pancreatic beta cells plays a major role in beta cell dysfunction, which leads to impaired glucose homeostasis and diabetes. Therefore, prevention of beta cell senescence could reduce the risk of diabetes. Treatment of nonobese diabetic (NOD) mice, a model of type 1 autoimmune diabetes (T1D), with palmitic acid hydroxy stearic acids (PAHSAs), a novel class of endogenous lipids with antidiabetic and antiinflammatory effects, delays the onset and reduces the incidence of T1D from 82% with vehicle treatment to 35% with PAHSAs. Here, we show that a major mechanism by which PAHSAs protect islets of the NOD mice is by directly preventing and reversing the initial steps of metabolic stress-induced senescence. In vitro PAHSAs increased Mdm2 expression, which decreases the stability of p53, a key inducer of senescence-related genes. In addition, PAHSAs enhanced expression of protective genes, such as those regulating DNA repair and glutathione metabolism and promoting autophagy. We demonstrate the translational relevance by showing that PAHSAs prevent and reverse early stages of senescence in metabolically stressed human islets by the same Mdm2 mechanism. Thus, a major mechanism for the dramatic effect of PAHSAs in reducing the incidence of type 1 diabetes in NOD mice is decreasing cellular senescence; PAHSAs may have a similar benefit in humans.

Calcitonin gene-related peptide is a potential autoantigen for CD4 T cells in type 1 diabetes.

The calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide with critical roles in the development of peripheral sensitization and pain. One of the CGRP family peptides, islet amyloid polypeptide (IAPP), is an important autoantigen in type 1 diabetes. Due to the high structural and chemical similarity between CGRP and IAPP, we expected that the CGRP peptide could be recognized by IAPP-specific CD4 T cells. However, there was no cross-reactivity between the CGRP peptide and the diabetogenic IAPP-reactive T cells. A set of CGRP-specific CD4 T cells was isolated from non-obese diabetic (NOD) mice. The T-cell receptor (TCR) variable regions of both α and β chains were highly skewed towards TRAV13 and TRBV13, respectively. The clonal expansion of T cells suggested that the presence of activated T cells responded to CGRP stimulation. None of the CGRP-specific CD4 T cells were able to be activated by the IAPP peptide. This established that CGRP-reactive CD4 T cells are a unique type of autoantigen-specific T cells in NOD mice. Using IAg7-CGRP tetramers, we found that CGRP-specific T cells were present in the pancreas of both prediabetic and diabetic NOD mice. The percentages of CGRP-reactive T cells in the pancreas of NOD mice were correlated to the diabetic progression. We showed that the human CGRP peptide presented by IAg7 elicited strong CGRP-specific T-cell responses. These findings suggested that CGRP is a potential autoantigen for CD4 T cells in NOD mice and probably in humans. The CGRP-specific CD4 T cells could be a unique marker for type 1 diabetes. Given the ubiquity of CGRP in nervous systems, it could potentially play an important role in diabetic neuropathy.

10-OR: Combination Therapy with Anti-CD3 Antibody and Harmine plus Exendin-4 Enhances Remission from Recent-Onset Type 1 Diabetes.

Type 1 diabetes (T1D) results from loss of both immune tolerance and functional β-cells. Administration of harmine (H) plus exendin-4 (E) markedly induces human β-cell expansion. Anti-CD3 antibody treatment reduces C-peptide loss in T1D patients. Here, we tested whether combination therapy with anti-CD3 antibody and H+E enhances T1D remission in non-obese diabetic (NOD) mice. First, we tested whether H+E protects human β-cells against inflammation and ER stress. We found that H+E, but not the drugs alone, significantly reduced both thapsigargin- and cytokine-induced human β-cell apoptosis. Single-cell RNAseq of human islets treated with cytokines and H+E showed reduced IL1β and IFNγ signaling in β-cells. Apoptosis genes such as CYLD and RIPK1 were downregulated and prosurvival genes such as HIF1A and VEGFA were upregulated in β-cells of H+E-treated islets. Next, treatment of early-onset diabetic NOD mice with H+E (daily ip) for 8 weeks improved glucose homeostasis but failed to induce long-lasting immune tolerance and T1D remission. Therefore, we next treated early-onset diabetic NOD mice with low-dose anti-CD3 antibody daily for 3 days (40µg/day) followed by 8 weeks treatment with H+E or vehicle (V) (Alzet pumps) . Treatment with anti-CD3 and H+E eliminated hyperglycemia in 100% of the mice vs. only 20% remission in anti-CD3 and V-treated mice. CD3+ T cell numbers were significantly and similarly reduced (∼50%) in mice treated with H+E or V. Preliminary results indicate that activated T cells in pancreatic lymph nodes (PLNs) and islet insulitis were significantly reduced, and T regulatory cells in spleen and PLNs were increased in H+E-treated mice. TUNEL+ β-cells were decreased and Ki67+ β-cells were increased in the pancreas of H+E-treated mice. Collectively, these results indicate that combination therapy with low-dose anti-CD3 antibody and H+E enhances T1D remission in diabetic NOD mice by increasing β-cell viability and favoring immune tolerance. Disclosure G.Lu: None. R.Kang: None. Y.Li: None. P.Wang: None. C.Rosselot: None. R.J.Devita: None. A.F.Stewart: None. A.Garcia-ocana: Consultant; Sun Pharmaceutical Industries Ltd. Funding NIH DK105015

Administration of Human Derived Upper gut Commensal Prevotella histicola delays the onset of type 1 diabetes in NOD mice

BackgroundType 1 diabetes (T1D) is an autoimmune disease that is increasing in prevalence worldwide. One of the contributing factors to the pathogenesis of T1D is the composition of the intestinal microbiota, as has been demonstrated. in T1D patients, with some studies demonstrating a deficiency in their levels of Prevotella. We have isolated a strain of Prevotella histicola from a duodenal biopsy that has anti-inflammatory properties, and in addition, alters the development of autoimmune diseases in mouse models. Therefore, our hypothesis is that the oral administration of P. histicola might delay the development of T1D in the non-obese diabetic (NOD) mice. To assess this, we used the following materials and methods. Female NOD mice (ages 5–8 weeks) were administered every other day P. histicola that was cultured in-house. Blood glucose levels were measured every other week. Mice were sacrificed at various time points for histopathological analysis of the pancreas. Modulation of immune response by the commensal was tested by analyzing regulatory T-cells and NKp46+ cells using flow cytometry and intestinal cytokine mRNA transcript levels using quantitative RT-PCR. For microbial composition, 16 s rRNA gene analysis was conducted on stool samples collected at various time points.ResultsAdministration of P. histicola in NOD mice delayed the onset of T1D. Beta diversity in the fecal microbiomes demonstrated that the microbial composition of the mice administered P. histicola was different from those that were not treated. Treatment with P. histicola led to a significant increase in regulatory T cells with a concomitant decrease in NKp46+ cells in the pancreatic lymph nodes as compared to the untreated group after 5 weeks of treatment.ConclusionsThese observations suggest that P. histicola treatment delayed onset of diabetes by increasing the levels of regulatory T cells in the pancreatic lymph nodes. This preliminary work supports the rationale that enteral exposure to a non pathogenic commensal P. histicola be tested as a future therapy for T1D.

P.159: Investigating the Role of Cathepsin L in Islet Graft Rejection

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disorder in which beta-cell specific destruction leads to life-long exogenous insulin dependence. While islet transplantation can restore euglycemia in these patients, both allogeneic and autoimmune rejection of the islet graft pose challenges limiting graft longevity. CD4+ T cells play a major role in driving T1D autoimmune development and islet graft rejection. Recent discovery of hybrid insulin peptides (HIPs), a fusion of insulin peptides with other beta-cell granule containing peptides, were found to be cognate autoantigens for autoreactive CD4+ T cells in mouse models and in patients with T1D. HIPs are formed within the beta-cell during crinophagy, a process of protein degradation or turnover where insulin granules fuse with lysosomes. By reducing the formation of HIPs, autoimmune-mediated islet graft rejection may be delayed. Cathepsin L (CatL) is a lysosomal endopeptidase enzyme involved in protein degradation and is implicated in the generation of HIPs. Non-Obese Diabetic (NOD) mice lacking CatL (NOD.CatL-/-) exhibited a delay in spontaneous T1D development. Immunophenotyping of islet-infiltrating immune cells from NOD.CatL-/- mice displayed reductions in effector CD4+ T cells and tetramer-specific autoreactive BDC-2.5, BDC-6.9, and insulin T cells compared to NOD mice. We hypothesize that islets deficient in CatL will delay immune-mediated rejection due to the absence of antigenic HIPs following transplantation. CatL-deficient islets may improve islet graft survival due to reduced release of antigenic peptides and passenger CD4+ T cells when transplanted into recipient mice. Isolated islets (250 islets/recipient) from NOD.CatL-/-, NOD.CatL+/-, or NOD.Rag mice, were transplanted under the kidney capsule of streptozotocin-treated and spontaneously diabetic NOD mice and monitored for restoration and maintenance of euglycemia. Both NOD.CatL-/- and NOD.CatL+/- islets restored euglycemia similarly as NOD.Rag islets. Future studies will investigate the impact of CatL-deficiency on long-term graft survival and effects on immune cell infiltration and activation. This work was supported by an NIH/NIDDK R01 award (DK099550), JDRF award SRA-2016-270-S-B, JDRF award 2-SRA-2019-692-S-B, NIH/NIDDK R01 award (DK126456), NIH/NIDDK R01 award (DK127497), NIH/NIGMS (T32GM008111) Cell, Molecular, and Developmental Biology T32 training grant.

Suppressive role of E3 ubiquitin ligase FBW7 in type I diabetes in non-obese diabetic mice through mediation of ubiquitination of EZH2

The current study tried to uncover the molecular mechanism of E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBW7) in a heritable autoimmune disease, type I diabetes (T1D). After streptozotocin-induced T1D model establishment in non-obese diabetic (NOD) mouse, the protein expression of FBW7, enhancer of zeste homolog 2 (EZH2), and Zinc finger and BTB domain containing 16 (ZBTB16) was quantified. Next, splenocytes and pancreatic beta cells were isolated to measure the production of pro-inflammatory cytokines in splenocytes, as well as islet beta-cell apoptosis. Additionally, the stability of EZH2 induced by FBW7 was analyzed by cycloheximide chase assay. The binding affinity of FBW7 and EZH2 and the consequence of ubiquitination were monitored by co-immunoprecipitation assay. Last, a chromatin immunoprecipitation assay was employed to analyze the accumulation of EZH2 and H3K27me3 at the ZBTB16 promoter region. Our study demonstrated downregulated FBW7 and ZBTB16 and upregulated EZH2 in diabetic NOD mice. Overexpression of FBW7 in the NOD mice inhibited pro-inflammatory cytokine release in the splenocytes and the apoptosis of islets beta cells. FBW7 destabilized EZH2 and accelerated ubiquitin-dependent degradation. EZH2 and H3K27me3 downregulated the ZBTB16 expression by accumulating in the ZBTB16 promoter and methylation. FBW7 upregulates the expression of ZBTB16 by targeting histone methyltransferase EZH2 thus reducing the occurrence of T1D.

Revisiting definition and assessment of intestinal trans-epithelial passage

This study aims to remind that Intestinal Passage (IP) measurement is a complex task that cannot be achieved by a unique measure of an orally given exogenous marker in blood or urine. This will be illustrated in the case of NOD mice. Indeed, various methods have been proposed to measure IP. Among them ex vivo measurement in Ussing chambers of luminal to serosal fluxes of exogenous markers and in vivo measurement of exogenous markers in blood or urine after oral gavage are the more commonly used. Even though they are commonly used indifferently, they do not give the same information and can provide contradictory results. Published data showed that diabetic status in female Non Obese Diabetic (NOD) mice increased FD4 concentration in blood after gavage but did not modify FD4 fluxes in Ussing chamber. We observed the same results in our experimental conditions and tracked FD4 concentrations in blood over a kinetic study (Area Under the Curve—AUC). In vivo measurements are a dynamic process and address not only absorption (IP and intestinal surface) but also distribution, metabolism and excretion (ADME). Diabetic status in NOD mice was associated with an increase of intestinal length (absorptive surface), itself positively correlated with AUC of FD4 in blood. We concluded that increased intestinal length induced by diabetic status will extend the absorptive surface and increase FD4 concentration in plasma (in vivo measurement) despite no modification on IP of FD4 (ex vivo measurement). In addition, this study characterized intestinal function in diabetic NOD mice. Diabetic status in NOD female mice increases intestinal length and decreases paracellular IP (FSS) without affecting transcellular IP (HRP, FD4). Histological studies of small and large intestine did not show any modification of intestinal circumference nor villi and crypt size. Finally, diabetic status was not associated with intestinal inflammation (ELISA).