Antigen-Specific Chimeric Antigen Receptor-T Regulatory Cells Home to Human Islets, Suppress Cytotoxic T Lymphocytes and Reverse Type 1 Diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of beta cells by immune cells. The interactions among cells within the islets may be closely linked to the pathogenesis of T1D. In this study, we used single-cell RNA sequencing (scRNA-Seq) to analyze the cellular heterogeneity within the islets of a T1D mouse model. We established a T1D mouse model induced by streptozotocin and identified cell subpopulations using scRNA-Seq technology. Our results revealed 11 major cell types in the pancreatic islets of T1D mice, with heterogeneity observed in the alpha and beta cell subgroups, which may play a crucial role in the progression of T1D. Flow cytometry further confirmed a mature alpha and beta cell reduction in T1D mice. Overall, our scRNA-Seq analysis provided insights into the cellular heterogeneity of T1D islet tissue and highlighted the potential importance of alpha and beta cells in developing T1D.NEW & NOTEWORTHY In this study, we created a comprehensive single-cell atlas of pancreatic islets in a T1D mouse model using scRNA-Seq and identified 11 major cell types in the islets, highlighting the role of alpha and beta cells in T1D. This study revealed a significant reduction in the maturity alpha and beta cells in T1D mice through flow cytometry. It also demonstrated the heterogeneity of alpha and beta cells, potentially crucial for T1D progression. Overall, our scRNA-Seq analysis provided new insights for understanding and treating T1D by studying cell subtype changes and functions.

Type 1 diabetes (T1D) contributes to bone loss in humans as well as in both spontaneous and pharmacologicinduced T1D mouse models. The severity of complications of T1D, such as nephropathy, differs in different mouse strains. However, the contribution of genetics in modifying the extent of T1D-induced bone loss has not been fully addressed. Here, we compare the T1D-induced bone pathology between three commonly used inbred mouse strains: Balb/c, C57BL/6 and 129/Sv mice. All T1D mouse strains displayed a characteristic bone phenotype characterized by significant bone loss, decreased levels of osteoblast markers and increased marrow adiposity. However, straindependent differences were also observed. Most notably, 129/Sv T1D mice displayed the greatest magnitude of bone loss despite having the least disease severity (as indicated by blood glucose levels) of the three strains studied. These findings suggest the contribution of strain dependent/genetically associated factors to the degree of bone loss observed in T1D mice.

In the present study, we tested hypothesis that hyperglycemia impairs the neuro-protective function of cerebral microvessels. Type 1 diabetes (T1D) was induced by treatment of mice with streptozotocin (blood glucose >300 mg/dL for 2 months). Brain tissues and cerebral microvessels were isolated from diabetic and control mice. We observed significant decrease in the protein levels of soluble amyloid precursor protein-α (sAPPα, a product of α-cleavage of APP and a neurotrophic and neuroprotective molecule) in the total brain tissue of diabetic mice (n=5, P<0.05). Further examination of brain regions revealed that the reduction of protein levels of sAPPα was only detected in hippocampus and cortex but not in cerebellum (n=6, P<0.05). Our studies also revealed that there were no significant differences in the protein expression of APP and its non-amyloidogenic processing enzymes, α-secretases (a disintegrin and metalloprotease 10 [ADAM10], ADAM9, and ADAM17) in total brain tissue between T1D mice and control mice (n=5-6, P>0.05). The protein level of β-processing enzyme BACE1 was also not changed in T1D mouse brain. In contrast, the protein levels of APP and ADAM10 were significantly decreased in cerebral microvasculature of T1D mice, as compared to control mice (n=9-10, P<0.05). Immunofluorescent confocal microscopy also demonstrated that expressions of APP and ADAM10 in cerebral microvasculature (identified by co-staining with CD31) in hippocampus and cortex were reduced in T1D mice. Of note, cerebrovascular protein levels of ADAM9, ADAM17 and BACE1 were not affected by hyperglycemia (n=5-7, P>0.05). These results suggest that hyperglycemia impairs APP expression and α-processing in brain vasculature, thereby leading to the reduction of brain content of sAPPα. The selective loss of neuro-protective molecule sAPPα in hippocampus and cortex may be an important mechanism contributing to the development of cognitive dysfunction in diabetes.

Disclosure: S. Imam: AdopTracell. S. Rafiqi: None. J.C. Jaume: AdopTracell. The onset of human autoimmune Type 1 Diabetes (T1D) requires two key factors: genetic susceptibility and the presence of target autoantigen. Once activated, the immune system deploys autoantigen-specific cytotoxic T cells that destroy insulin-producing beta cells. Regulatory T cell (Treg) responses are often insufficient to suppress immune activity at inflammation sites.While Treg therapy shows promise for autoimmune diseases, its clinical application remains limited due to the low availability of antigen-specific Tregs. The ability to engineer Tregs with specificity to known autoantigens could enable targeted immune suppression and provide effective treatments. One strategy to impart antigen specificity involves using chimeric antigen receptors (CARs), which endow T cells with antibody-like antigen recognition. Adoptive cell transfer (ACT) therapies using CAR-engineered cytotoxic T cells have achieved notable success in treating hematologic cancers. This same technology can potentially redirect Tregs to autoimmune sites, offering therapeutic benefit across a range of autoimmune and alloimmune disorders.To this end, we developed a novel CAR-Treg approach targeting Glutamic Acid Decarboxylase 65 (GAD65) for the prevention and treatment of T1D. GAD65 is an enzyme critical for gamma-aminobutyric acid (GABA) synthesis in beta cells and neurons. Within beta cells, GAD65 resides in a vesicular compartment distinct from insulin granules. It is mainly anchored to Golgi membranes and trafficked via small cytoplasmic synaptic-like microvesicles (SLMVs) that carry GABA to the extracellular space. To support paracrine GABA signaling, SLMVs containing membrane-bound GAD65 are secreted from beta cells into the intra-islet interstitial space. As a well-established autoantigen in T1D-and with autoantibodies to GAD65 often detectable years before clinical symptoms—we have selected it as the target for our CAR-Treg therapy.We developed GAD65-CAR-Tregs and tested their therapeutic potential in a 6-month preclinical study in T1D mice. Treatment resulted in significant improvements in fasting blood glucose, insulin concentrations, and glucose tolerance. Histological analysis showed retrieval of infiltration of effector T cells and signs of Treg-mediated immune modulation, leading to the preservation and regeneration of pancreatic islet architecture. Notably, GAD65-CAR-Tregs did not cross the blood-brain barrier, and no neurological symptoms or cytokine release syndrome (CRS) were observed—supporting a favorable safety profile.Collectively, our findings demonstrate that GAD65-CAR-Tregs can home to pancreatic islets, suppress diabetogenic T cells, and reverse T1D in a clinically relevant mouse model. These results suggest that GAD65-CAR-Treg therapy holds strong potential for restoring immune tolerance and promoting beta-cell recovery in T1D patients. Presentation: Sunday, July 13, 2025

Knockdown of IFNAR2 reduces the inflammatory response in mouse model of type 1 diabetes

People with type 1 diabetes (T1D) have a significantly elevated risk of stroke, but the mechanism through which T1D worsens ischemic stroke remains unclear. This study was aimed at investigating the roles of T1D-associated changes in the gut microbiota in aggravating ischemic stroke and the underlying mechanism. Fecal 16SrRNA sequencing indicated that T1D mice and mice with transplantation of T1D mouse gut microbiota had lower relative abundance of butyric acid producers, f_Erysipelotrichaceae and g_Allobaculum, and lower content of butyric acid in feces. After middle cerebral artery occlusion (MCAO), these mice had poorer neurological outcomes and more severe inflammation, but higher expression of myeloid differentiation factor 88 (MyD88) in the ischemic penumbra; moreover, the microglia were inclined to polarize toward the pro-inflammatory type. Administration of butyrate to T1D mice in the drinking water alleviated the neurological damage after MCAO. Butyrate influenced the response and polarization of BV2 and decreased the production of inflammatory cytokines via MyD88 after oxygen-glucose deprivation/reoxygenation. Knocking down MyD88 in the brain alleviated neurological outcomes and decreased the concentrations of inflammatory cytokines in the brain after stroke in mice with transplantation of T1D mouse gut microbiota. Poor neurological outcomes and aggravated inflammatory responses of T1D mice after ischemic stroke may be partly due to differences in microglial polarization mediated by the gut microbiota-butyrate-MyD88 pathway. These findings provide new ideas and potential intervention targets for alleviating neurological damage after ischemic stroke in T1D.

Type 1 diabetes (T1D) is correlated with osteopenia primarily due to low bone formation. Parathyroid hormone (PTH) is a known anabolic agent for bone, the anabolic effects of which are partially mediated through the Wnt/β-catenin signaling pathway. In the present study, we first determined the utility of intermittent PTH treatment in a streptozotocin-induced T1D mouse model. It was shown that the PTH-induced anabolic effects on bone mass and bone formation were attenuated in T1D mice compared with nondiabetic mice. Further, PTH treatment failed to activate β-catenin signaling in osteoblasts of T1D mice and was unable to improve osteoblast proliferation and differentiation. Next, the Col1-3.2 kb-CreERTM; β-cateninfx(ex3) mice were used to conditionally activate β-catenin in osteoblasts by injecting tamoxifen, and we addressed whether or not preactivation of β-catenin boosted the anabolic action of PTH on T1D-related bone loss. The results demonstrated that pretreatment with activation of osteoblastic β-catenin followed by PTH treatment outperformed PTH or β-catenin activation monotherapy and led to greatly improved bone structure, bone mass, and bone strength in this preclinical model of T1DM. Further analysis demonstrated that osteoblast proliferation and differentiation, as well as osteoprogenitors in the marrow, were all improved in the combination treatment group. These findings indicated a clear advantage of developing β-catenin as a target to improve the efficacy of PTH in the treatment of T1D-related osteopenia.

Applied photocatalysis – at last !!

Alzheimer's disease and type 2 diabetes-related alterations in brain mitochondria, autophagy and synaptic markers

Hyperglycemia worsens stroke, yet rigorous glycemic control does not improve neurologic outcome. An alternative is to target downstream molecular mediators triggered by hyperglycemia. Soluble epoxide hydrolase (sEH) is a potential mediator of ischemic injury via its metabolism of neuroprotective epoxyeicosatrienoic acids (EETs). We previously demonstrated that sEH mRNA is overexpressed in type 1 diabetic (T1D) mice, and specific sEH blockade protects the brain from the deleterious effect of T1D on stroke. We tested the hypothesis that type 2 diabetes (T2D) exacerbates injury following middle cerebral artery occlusion (MCAO) in part by up-regulating expression of EPHX2 (gene encoding for sEH) and decreasing brain concentrations of neuroprotective EETs. T2D was produced by combined high-fat diet, nicotinamide and streptozotocin in male C57BL/6J mice. T2D and control mice were treated with vehicle or the sEH inhibitor trans-4-[4-(3-Adamantan-1-yl-ureido) -cyclohexyloxy]-benzoic acid (t-AUCB; 1mg/kg, i.p., 7 days), then subjected to 60-min MCAO. Compared to normal chow-fed mice, high fat diet-fed mice exhibited a 1.7 fold upregulation of EPHX2 mRNA in brain (p<0.05, n=7). T2D mice had increased blood glucose levels compared to control mice before, during and after MCAO (p<0.001, n=4-5). Relative laser-Doppler perfusion of the MCA territory after reperfusion was decreased in T2D mice compared to controls (p<0.05, n= 4-5). Vehicle-treated T2D mice sustained larger cortical infarcts than vehicle-treated control mice (p<0.05, n=5-7). t-AUCB decreased fasting glucose levels at baseline and throughout ischemia (p<0.001, n=4-5) and improved cortical perfusion after MCAO (p<0.001, n=4-5) in T2D mice. In line with these improvements, t-AUCB significantly reduced infarct size in T2D mice (p<0.05 vs. T2D vehicle, n= 5-7). We conclude that increasing EETs bioavailability via sEH inhibition improves stroke outcome in T2D in part by improving glycemic status and improving post-ischemic reperfusion in the ischemic territory.

NLRP3 Inflammasome is a platform that regulates inflammatory responses by caspase-1 activation and processing of pro-IL-1β and pro-IL-18 to mature cytokines. NLRP3 is activated by several mechanisms including mitochondrial DNA (mitDNA). Circulating mitDNA is increased in diabetes, a condition associated with NLRP3 activation. We tested the hypothesis that mitDNA release is increased in type 1 diabetes (T1D) leading to NLRP3 activation and contributing to vascular inflammatory and oxidative processes. Wild type (WT) and NLRP3-deficient (NLRP3 -/- ) mice were treated with vehicle or streptozotocin (40 mg/kg), i.p. for 5 days. Vascular reactivity was determined in mesenteric resistance arteries (MA). Cultured vascular smooth muscle cells (VSMC) were stimulated with mitDNA of T1D (dmDNA) and control (cmDNA) mice. Caspase-1 and IL-1β activation was evaluated by western blot analysis and reactive oxygen species (ROS) by fluorescence to DHE. DNA was extracted, purified and amplified by real-time-PCR. Data are presented as mean ± standard error of mean, Veh vs. T1D. NLRP3 -/- T1D mice exhibited attenuated hyperglycemia vs. WT T1D mice [mg/dL, 241.0±27.7 vs. 337.6±18.1, p<0.05]. MA from T1D mice exhibited decreased ACh-induced dilatation vs. Veh [E max , 46.6±4.0 vs . 91.5±2.8, p<0.05], which was not observed in NLRP3 -/- T1D mice. Diabetes increased vascular caspase-1 [arbitrary units (a.u.), 1.2±0.1 vs. 0.8±0.1, p<0.05)] and IL-1β activation [4.8±1.1 vs. 0.8±0.5 p <0.05], but this activation was attenuated in NLRP3 -/- T1D. T1D mice exhibited increased NLRP3 activation and mitDNA release in pancreatic cells and increased circulating mitDNA. dmDNA, but not cmDNA, increased NLRP3 activation in VSMC (i.e. activated caspase-1 and increased IL-1β levels) [a.u., 4.2±0.1 vs. 1.9±0.1; 2.3±0.1 vs. 0.7±0.1, p<0.05]. NLRP3 activation was attenuated in NLRP3 -/- VMSC, but not in WT VSMC incubated with a TLR-9 antagonist. Increased ROS generation was observed in response to dmDNA, which was prevented by a mitochondrial uncoupler. Our data show that T1D increases mitDNA release, which promotes vascular NLRP3 activation via mitochondrial superoxide production, contributing to T1D-associated vascular dysfunction. Financial Support: FAPESP, CNPq.

Characterisation of immune responses to mycobacterial infections in a murine model of type 2 diabetes

Pancreatic islet transplantation is a promising potential cure for type 1 diabetes (T1D). Islet allografts can survive long term in the liver parenchyma. Here we show that liver NK1.1+ cells induce allograft tolerance in a T1D mouse model. The tolerogenic effects of NK1.1+ cells are mediated through IL-22 production, which enhances allograft survival and increases insulin secretion. Increased expression of NKG2A by liver NK1.1+ cells in islet allograft-transplanted mice is involved in the production of IL-22 and in the reduced inflammatory response to allografts. Vaccination of T1D mice with a CpG oligonucleotide TLR9 agonist (ODN 1585) enhances expansion of IL-22-producing CD3-NK1.1+ cells in the liver and prolongs allograft survival. Our study identifies a role for liver NK1.1+ cells, IL-22 and CpG oligonucleotides in the induction of tolerance to islet allografts in the liver parenchyma.

Recent studies have suggested that hyperglycaemia influences the bile acid profile and concentrations of secondary bile acids in the gut. This study aimed to measure changes in the bile acid profile in the gut, tissues, and faeces in type 1 Diabetes (T1D) and Type 2 Diabetes (T2D). T1D and T2D were established in a mouse model. Twenty-one seven-weeks old balb/c mice were randomly divided into three equal groups, healthy, T1D and T2D. Blood, tissue, urine and faeces samples were collected for bile acid measurements. Compared with healthy mice, T1D and T2D mice showed lower levels of the primary bile acid, chenodeoxycholic acid, in the plasma, intestine, and brain, and higher levels of the secondary bile acid, lithocholic acid, in the plasma and pancreas. Levels of the bile acid ursodeoxycholic acid were undetected in healthy mice but were found to be elevated in T1D and T2D mice. Bile acid profiles in other organs were variably influenced by T1D and T2D development, which suggests similarity in effects of T1D and T2D on the bile acid profile, but these effects were not always consistent among all organs, possibly since feedback mechanisms controlling enterohepatic recirculation and bile acid profiles and biotransformation are different in T1D and T2D.

Inhibitory effects of type 2 diabetes serum components in P450 inhibition assays can potential diagnose asymptomatic diabetic mice