T-cell responses to hybrid insulin peptides prior to type 1 diabetes development.

T-cell responses to posttranslationally modified self-antigens are associated with many autoimmune disorders. In type 1 diabetes, hybrid insulin peptides (HIPs) are implicated in the T-cell-mediated destruction of insulin-producing ß-cells within pancreatic islets. The natural history of the disease is such that it allows for the study of T-cell reactivity prior to the onset of clinical symptoms. We hypothesized that CD4 T-cell responses to posttranslationally modified islet peptides precedes diabetes onset. In a cohort of genetically at-risk individuals, we measured longitudinal T-cell responses to native insulin and hybrid insulin peptides. Both proinflammatory (interferon-γ) and antiinflammatory (interluekin-10) cytokine responses to HIPs were more robust than those to native peptides, and the ratio of such responses oscillated between pro- and antiinflammatory over time. However, individuals who developed islet autoantibodies or progressed to clinical type 1 diabetes had predominantly inflammatory T-cell responses to HIPs. Additionally, several HIP T-cell responses correlated to worsening measurements of blood glucose, highlighting the relevance of T-cell responses to posttranslationally modified peptides prior to autoimmune disease development.

Human islet T cells are highly reactive to preproinsulin in type 1 diabetes.

Cytotoxic CD8 T lymphocytes play a central role in the tissue destruction of many autoimmune disorders. In type 1 diabetes (T1D), insulin and its precursor preproinsulin are major self-antigens targeted by T cells. We comprehensively examined preproinsulin specificity of CD8 T cells obtained from pancreatic islets of organ donors with and without T1D and identified epitopes throughout the entire preproinsulin protein and defective ribosomal products derived from preproinsulin messenger RNA. The frequency of preproinsulin-reactive T cells was significantly higher in T1D donors than nondiabetic donors and also differed by individual T1D donor, ranging from 3 to over 40%, with higher frequencies in T1D organ donors with HLA-A*02:01. Only T cells reactive to preproinsulin-related peptides isolated from T1D donors demonstrated potent autoreactivity. Reactivity to similar regions of preproinsulin was also observed in peripheral blood of a separate cohort of new-onset T1D patients. These findings have important implications for designing antigen-specific immunotherapies and identifying individuals that may benefit from such interventions.

Screening children for type 1 diabetes-associated antibodies at community health fairs.

OBJECTIVE: The incidence of type 1 diabetes (T1D) is increasing, most notably in young children and in racial and ethnic minorities. Historically, screening for risk with T1D-associated antibodies has been limited to those with a family history, while up to 90% of newly diagnosed patients lack such a family history. To address the needs to screen diverse ethnic groups in the general population, we screened children for T1D-associated antibodies in the Denver, Colorado metro area at community health fairs. METHODS: Children attending health fairs from 2015 to 2018 were offered free T1D screening by measuring the four prototypical T1D-associated antibodies. A finger stick capillary puncture was performed to collect blood spots on filter paper. Dried blood spots (DBSs) were eluted and antibodies were measured using fluid-phase radio-binding assays. RESULTS: At 39 health fairs, children were educated on the signs and symptoms of diabetes, and screened for T1D-associated antibodies (n = 478), which represented 90% of those that attended. Median age was 9.0 years (range of 1-18) with diverse ethnic backgrounds: 37% Hispanic, 31% Caucasian, 20% African American, and 12% other. Nine children screened positive for antibodies, single n = 8 and multiple n = 1, and confirmation with serum samples showed excellent correlation to the measurements from DBSs for antibodies directed against GAD, IA-2, and ZnT8 (P < .01 for each). CONCLUSIONS: Screening for T1D risk at community health fairs using DBSs on filter paper is feasible and provides an avenue to screen children from ethnically diverse backgrounds.

Combinatorial detection of autoreactive CD8+ T cells with HLA-A2 multimers: a multi-centre study by the Immunology of Diabetes Society T Cell Workshop.

AIMS/HYPOTHESIS: Validated biomarkers are needed to monitor the effects of immune intervention in individuals with type 1 diabetes. Despite their importance, few options exist for monitoring antigen-specific T cells. Previous reports described a combinatorial approach that enables the simultaneous detection and quantification of multiple islet-specific CD8+ T cell populations. Here, we set out to evaluate the performance of a combinatorial HLA-A2 multimer assay in a multi-centre setting. METHODS: The combinatorial HLA-A2 multimer assay was applied in five participating centres using centralised reagents and blinded replicate samples. In preliminary experiments, samples from healthy donors were analysed using recall antigen multimers. In subsequent experiments, samples from healthy donors and individuals with type 1 diabetes were analysed using beta cell antigen and recall antigen multimers. RESULTS: The combinatorial assay was successfully implemented in each participating centre, with CVs between replicate samples that indicated good reproducibility for viral epitopes (mean %CV = 33.8). For beta cell epitopes, the assay was very effective in a single-centre setting (mean %CV = 18.4), but showed sixfold greater variability across multi-centre replicates (mean %CV = 119). In general, beta cell antigen-specific CD8+ T cells were detected more commonly in individuals with type 1 diabetes than in healthy donors. Furthermore, CD8+ T cells recognising HLA-A2-restricted insulin and glutamate decarboxylase epitopes were found to occur at higher frequencies in individuals with type 1 diabetes than in healthy donors. CONCLUSIONS/INTERPRETATION: Our results suggest that, although combinatorial multimer assays are challenging, they can be implemented in multiple laboratories, providing relevant T cell frequency measurements. Assay reproducibility was notably higher in the single-centre setting, suggesting that biomarker analysis of clinical trial samples would be most successful when assays are performed in a single laboratory. Technical improvements, including further standardisation of cytometry platforms, will likely be necessary to reduce assay variability in the multi-centre setting.

Prevention of virus-induced type 1 diabetes with antibiotic therapy.

Microbes were hypothesized to play a key role in the progression of type 1 diabetes (T1D). We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced T1D to test the hypothesis that the intestinal microbiota is involved in the mechanism leading to islet destruction. Treating LEW1.WR1 rats with KRV and a combination of trimethoprim and sulfamethoxazole (Sulfatrim) beginning on the day of infection protected the rats from insulitis and T1D. Pyrosequencing of bacterial 16S rRNA and quantitative RT-PCR indicated that KRV infection resulted in a transient increase in the abundance of Bifidobacterium spp. and Clostridium spp. in fecal samples from day 5- but not day 12-infected versus uninfected animals. Similar alterations in the gut microbiome were observed in the jejunum of infected animals on day 5. Treatment with Sulfatrim restored the level of intestinal Bifidobacterium spp. and Clostridium spp. We also observed that virus infection induced the expression of KRV transcripts and the rapid upregulation of innate immune responses in Peyer's patches and pancreatic lymph nodes. However, antibiotic therapy reduced the virus-induced inflammation as reflected by the presence of lower amounts of proinflammatory molecules in both the Peyer's patches and pancreatic lymph nodes. Finally, Sulfatrim treatment reduced the number of B cells in Peyer's patches and downmodulated adaptive immune responses to KRV, but did not interfere with antiviral Ab responses or viral clearance from the spleen, pancreatic lymph nodes, and serum. The data suggest that gut microbiota may be involved in promoting virus-induced T1D in the LEW1.WR1 rat model.

The role of the intestinal microbiota in type 1 diabetes.

The digestive tract hosts trillions of bacteria that interact with the immune system and can influence the balance between pro-inflammatory and regulatory immune responses. Recent studies suggest that alterations in the composition of the intestinal microbiota may be linked with the development of type 1 diabetes (T1D). Data from the biobreeding diabetes prone (BBDP) and the LEW1.WR1 models of T1D support the hypothesis that intestinal bacteria may be involved in early disease mechanisms. The data indicate that cross-talk between the gut microbiota and the innate immune system may be involved in islet destruction. Whether a causal link between intestinal microbiota and T1D exists, the identity of the bacteria and the mechanism whereby they promote the disease remain to be examined. A better understanding of the interplay between microbes and innate immune pathways in early disease stages holds promise for the design of immune interventions and disease prevention in genetically susceptible individuals.

Protecting Stem Cell Derived Pancreatic Beta-Like Cells From Diabetogenic T Cell Recognition.

Type 1 diabetes results from an autoimmune attack directed at pancreatic beta cells predominantly mediated by T cells. Transplantation of stem cell derived beta-like cells (sBC) have been shown to rescue diabetes in preclinical animal models. However, how sBC will respond to an inflammatory environment with diabetogenic T cells in a strict human setting has not been determined. This is due to the lack of model systems that closely recapitulates human T1D. Here, we present a reliable in vitro assay to measure autologous CD8 T cell stimulation against sBC in a human setting. Our data shows that upon pro-inflammatory cytokine exposure, sBC upregulate Human Leukocyte Antigen (HLA) class I molecules which allows for their recognition by diabetogenic CD8 T cells. To protect sBC from this immune recognition, we utilized genome engineering to delete surface expression of HLA class I molecules and to integrate an inducible overexpression system for the immune checkpoint inhibitor Programmed Death Ligand 1 (PD-L1). Genetically engineered sBC that lack HLA surface expression or overexpress PD-L1 showed reduced stimulation of diabetogenic CD8 T cells when compared to unmodified cells. Here, we present evidence that manipulation of HLA class I and PD-L1 receptors on sBC can provide protection from diabetes-specific immune recognition in a human setting.

Prevalence of SARS-CoV-2 Antibodies in Children and Adults with Type 1 Diabetes.

Objective: As diabetes is a risk factor for severe symptoms, hospitalization, and death with COVID-19 disease, we aimed to assess the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in children and adults with and without type 1 diabetes in Colorado during 2020. Research Design and Methods: We developed a highly sensitive and specific test for antibodies against SARS-CoV-2 and measured the antibodies in children and adults with new-onset (n = 129) and established type 1 diabetes (n = 94) seen for routine diabetes care at our center between January and October 2020. The antibodies were also measured in 562 children and 102 adults from the general population of Colorado. Results: The prevalence of SARS-CoV-2 antibodies in persons with new-onset type 1 diabetes (0.8%; 95% confidence interval 0.1%-4.2%) or those with established disease (4.3%; 1.7%-10.4%) did not differ from that in the general population children (2.8%; 1.8%-4.6%) or adults (3.9%; 1.5%-9.7%). In a subset of individuals with positive antibodies (n = 31), antibodies remained positive for up to 9 months, although the levels decreased starting 3 months after the infection (P = 0.007). Conclusions: From January to October 2020, the prevalence of SARS-CoV-2 antibodies were not different in children and adults with and without type 1 diabetes in Colorado. We found no evidence for increased prevalence of COVID-19 infections among youth with newly diagnosed type 1 diabetes. (COMIRB Protocol 20-1007).

Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation Reporter-Based Detection System.

Recent advancements in single cell sequencing technologies allow for identification of numerous immune-receptors expressed by T cells such as tumor-specific and autoimmune T cells. Determining antigen specificity of those cells holds immense therapeutic promise. Therefore, the purpose of this study was to develop a method that can efficiently test antigen reactivity of multiple T cell receptors (TCRs) with limited cost, time, and labor. Nuclear factor of activated T cells (NFAT) is a transcription factor involved in producing cytokines and is often utilized as a reporter system for T cell activation. Using a NFAT-based fluorescent reporter system, we generated T-hybridoma cell lines that express intensely fluorescent proteins in response to antigen stimulation and constitutively express additional fluorescent proteins, which serve as identifiers of each T-hybridoma expressing a unique TCR. This allows for the combination of multiple T-hybridoma lines within a single reaction. Sensitivity to stimulation is not decreased by adding fluorescent proteins or multiplexing T cells. In multiplexed reactions, response by one cell line does not induce response in others, thus preserving specificity. This multiplex assay system will be a useful tool for antigen discovery research in a variety of contexts, including using combinatorial peptide libraries to determine T cell epitopes.

Failed Genetic Protection: Type 1 Diabetes in the Presence of HLA-DQB1*06:02.

Certain HLA class II genes increase the risk for type 1 diabetes (T1D) development while others provide protection from disease development. HLA class II alleles encode MHC proteins on antigen-presenting cells, which function to present peptides and activate CD4 T cells. The DRB1*15:01 (DR15)-DQA1*01:02-DQB1*06:02 (DQ6) haplotype provides dominant protection across all stages of T1D and is a common haplotype found in Caucasians. However, it is present in <1% of people with T1D. Knowing which metabolic, immunologic, and genetic features are unique to individuals who fail genetic protection and develop T1D is important for defining the underlying mechanisms of DQB1*06:02-mediated protection. We describe a T1D cohort with DQB1*06:02 (n = 50) and compare them to individuals with T1D and without DQB1*06:02 (n = 2,759) who were identified over the last 26 years at the Barbara Davis Center for Diabetes. The age at diagnosis was similar between the cohorts and normally distributed throughout childhood and early adulthood. The average hemoglobin A1c was 10.8 ± 2.8% (95 ± 7 mmol/mol) at diagnosis in those DQB1*06:02 positive. The majority of T1D DQB1*06:02 + individuals were positive for one or more islet autoantibodies; however, there was a greater proportion who were islet autoantibody negative compared with those T1D DQB1*06:02 - individuals. Interestingly, DQB1*03:02, which confers significant T1D risk, was present in only those DQB1*06:02 + individuals with islet autoantibodies. This is one of the largest studies examining patients presenting with clinical T1D in the presence of DQB1*06:02, which provides a population to study the mechanisms of failed genetic protection against T1D.

Biomarker Discovery in Pre-Type 1 Diabetes; Th40 Cells as a Predictive Risk Factor.

CONTEXT: The incidence of type 1 diabetes (T1D) is increasing worldwide. The quest to understand T1D etiology and how to predict diabetes is ongoing; and, in many ways, those goals intertwine. Although genetic components associate with T1D, not all individuals with T1D have those components, and T1D does not develop in all subjects with those components. OBJECTIVE: More robust methods for prediction of T1D are needed. We investigated if high CD4+CD40+ T-cell (Th40) levels can be used as a biomarker. METHODS: Th40 levels were assessed along with other parameters in blood collected from prediabetic subjects in TrialNet. RESULTS: In prediabetic subjects stratified according to Th40 cell level, patterns paralleled those seen between control subjects and those with T1D. Cytokine patterns were significantly different between those with high Th-40 levels (Th40-high) and those with low levels, and a CD4/CD8 double-positive population was more represented in Th40-high groups. Subjects experiencing impaired glucose tolerance had a significantly higher Th40 level than did control subjects. HLA DR4/DR4 and DQ8/DQ8 were more likely found among Th40-high subjects. Interestingly, HLA DR4/DR4 subjects were significantly older compared with all other subjects, suggesting that this haplotype, together with a high Th40 level, may represent someone in whom T1D will develop after age 30 years, which is reported for 42% of T1D cases. CONCLUSION: Considering the differences found in relation to prediabetic Th40 cell level, it may be possible to devise methods that more accurately predict who will proceed toward diabetes and, possibly, indicate prediabetic stage.

PD-1 Inhibitor Immune-Related Adverse Events in Patients With Preexisting Endocrine Autoimmunity.

Context: Immune checkpoint inhibitors, including monoclonal antibodies directed against programmed cell death protein 1 (PD-1) and its ligand, have emerged as beneficial cancer immunotherapies. These therapies are known to cause immune-related side effects; however, their role in patients with a preexisting autoimmune disease is not clear. Case Description: We describe two cases of anti-PD-1 immune-related adverse events. A 52-year-old male with longstanding type 1 diabetes (T1D), long-term stable kidney transplant, and hypothyroidism received two separate anti-PD-1 monoclonal antibodies for metastatic melanoma. The patient developed acute kidney graft rejection requiring hemodialysis and worsening of autoimmune hypothyroidism 3 weeks after starting treatment. He continued anti-PD-1 treatments and remained on hemodialysis and increased levothyroxine dosage. The second case is a 62-year-old male with no previous history of diabetes who received anti-PD-1 treatment and developed severe diabetic ketoacidosis (DKA) 5 days following the start of therapy. Further laboratory testing revealed high titer antibodies directed against glutamic acid decarboxylase. These antibodies, which were of the IgG isotype and involved in memory immune responses, were likely present before anti-PD-1 treatment. He also had human leukocyte antigen genes that confer T1D genetic risk. Despite normal pretreatment blood glucose levels and HbA1c, the patient requires permanent exogenous insulin treatment. Conclusion: Patients with preexisting endocrine autoimmunity may have more frequent and severe immune-related side effects with anti-PD-1 treatment. Given the morbidity and mortality associated with solid organ transplant rejection and DKA, clinicians caring for patients receiving these state-of-the-art therapies need to be aware of the potential adverse events.

Methyldopa blocks MHC class II binding to disease-specific antigens in autoimmune diabetes.

Major histocompatibility (MHC) class II molecules are strongly associated with many autoimmune disorders. In type 1 diabetes (T1D), the DQ8 molecule is common, confers significant disease risk, and is involved in disease pathogenesis. We hypothesized that blocking DQ8 antigen presentation would provide therapeutic benefit by preventing recognition of self-peptides by pathogenic T cells. We used the crystal structure of DQ8 to select drug-like small molecules predicted to bind structural pockets in the MHC antigen-binding cleft. A limited number of the predicted compounds inhibited DQ8 antigen presentation in vitro, with 1 compound preventing insulin autoantibody production and delaying diabetes onset in an animal model of spontaneous autoimmune diabetes. An existing drug with a similar structure, methyldopa, specifically blocked DQ8 in patients with recent-onset T1D and reduced inflammatory T cell responses to insulin, highlighting the relevance of blocking disease-specific MHC class II antigen presentation to treat autoimmunity.

Islet Autoantibody Measurements from Dried Blood Spots on Filter Paper Strongly Correlate to Serum Levels.

Type 1 diabetes (T1D) is increasing in incidence and predictable with measurement of serum islet autoantibodies (iAb) years prior to clinical disease onset. Identifying iAb positive individuals reduces diabetic ketoacidosis and identifies individuals for T1D prevention trials. However, large scale screening for iAb remains challenging as assays have varying sensitivities and specificities, insulin autoantibodies remain difficult to measure and venipuncture is generally required to obtain serum. We developed an approach to reliably measure all four major iAb, including insulin autoantibodies, from dried blood spots (DBS) on filter-paper. By spiking iAb positive serum into iAb negative whole blood in a dose titration, we optimized the conditions for autoantibody elution from filter paper as measured by fluid phase radioimmunoassays. After assessing stability of measuring iAb from DBS over time, we then screened iAb from DBS and the corresponding serum in new-onset T1D (n = 52), and controls (n = 72) which included first-degree relatives of T1D patients. iAb measured from eluted DBS in new-onset T1D strongly correlated with serum measurements (R2 = 0.96 for mIAA, GADA = 0.94, IA-2A = 0.85, ZnT8A = 0.82, p<0.01 for each autoantibody). There were no false positives in control subjects, and 5/6 with previously unknown iAb positivity in sera were detected using DBS. With further validation, measuring iAb from DBS can be a reliable method to screen for T1D risk.

Alterations in Intestinal Microbiota Correlate With Susceptibility to Type 1 Diabetes.

We tested the hypothesis that alterations in the intestinal microbiota are linked with the progression of type 1 diabetes (T1D). Herein, we present results from a study performed in subjects with islet autoimmunity living in the U.S. High-throughput sequencing of bacterial 16S rRNA genes and adjustment for sex, age, autoantibody presence, and HLA indicated that the gut microbiomes of seropositive subjects differed from those of autoantibody-free first-degree relatives (FDRs) in the abundance of four taxa. Furthermore, subjects with autoantibodies, seronegative FDRs, and new-onset patients had different levels of the Firmicutes genera Lactobacillus and Staphylococcus compared with healthy control subjects with no family history of autoimmunity. Further analysis revealed trends toward increased and reduced abundances of the Bacteroidetes genera Bacteroides and Prevotella, respectively, in seropositive subjects with multiple versus one autoantibody. Canonical discriminant analysis suggested that the gut microbiomes of autoantibody-positive individuals and seronegative FDRs clustered together but separate from those of new-onset patients and unrelated healthy control subjects. Finally, no differences in biodiversity were evident in seropositive versus seronegative FDRs. These observations suggest that altered intestinal microbiota may be associated with disease susceptibility.

Histone deacetylase inhibitor suppresses virus-induced proinflammatory responses and type 1 diabetes.

UNLABELLED: Microbial infections are hypothesized to play a key role in the mechanism leading to type 1 diabetes (T1D). We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced islet destruction to better understand how virus infection triggers T1D. Inoculation of the LEW1.WR1 rat with KRV results in systemic inflammation followed by insulitis and islet destruction 2-4 weeks post-infection. In this study, we evaluated the effect of treatment with the anti-inflammatory histone deacetylase inhibitor (HDACi) ITF-2357 on KRV-induced immunity and disease progression. Administering LEW1.WR1 rats with KRV plus ITF-2357 on 14 consecutive days beginning on the day of infection protected animals from islet infiltration and T1D. ITF-2357 reversed KRV-induced T and B cell accumulation in the spleen or pancreatic lymph nodes on day 5 following infection. Moreover, ITF-2357 reduced the expression level of KRV-induced p40 subunit of IL-12/IL-23 in spleen cells in vitro and in the peripheral blood in vivo. ITF-2357 suppressed the KRV-induced expression of transcripts for IRF-7 in the rat INS-1 beta cell line. ITF-2357 increased the virus-induced IL-6 gene expression in the spleen, but did not alter the ability of LEW1.WR1 rats to develop normal KRV-specific humoral and cellular immune responses and clear the virus from the pancreatic lymph nodes, spleen, and serum. Finally, ITF-2357 reversed virus-induced modulation of bacterial communities in the intestine early following infection. The data suggest that targeting innate immune pathways with inhibitors of HDAC might represent an efficient therapeutic strategy for preventing T1D. KEY MESSAGE: Microbial infections have been implicated in triggering type 1 diabetes in humans and animal models. The LEW1.WR1 rat develops inflammation and T1D following infection with Kilham rat virus. The histone deacetylase inhibitor ITF-2357 suppresses virus-induced inflammation and prevents diabetes. ITF-2357 prevents T1D without altering virus-specific adaptive immunity or virus clearance. ITF-2357 therapy may be an efficient approach to prevent T1D in genetically susceptible individuals.

Kilham Rat Virus-induced type 1 diabetes involves beta cell infection and intra-islet JAK-STAT activation prior to insulitis.

We used the LEW1.WR1 rat model of Kilham Rat Virus (KRV)-induced type 1 diabetes (T1D) to test the hypothesis that disease mechanisms are linked with beta cell infection and intra-islet immune activation prior to insulitis. KRV induces genes involved in type I and type II interferon pathways in islet cell lines in vitro and in islets from day-5-infected animals in vivo via mechanisms that do not involve insulitis, beta cell apoptosis, or impaired insulin expression. Immunohistochemistry studies indicated that KRV protein is expressed in beta cells 5 days following infection. KRV induces the phosphorylation of Janus Kinase 1/2 (JAK1/2) and signal transducer and activator of transcription 1 (STAT-1) in islet cells via a mechanism that could involve TLR9 and NF-κB pathways. These data demonstrate for the first time that KRV-induced islet destruction is associated with beta cell infection and intra-islet innate immune upregulation early in the disease process.

Modulation of virus-induced innate immunity and type 1 diabetes by IL-1 blockade.

We used the LEW1.WR1 model of Kilham rat virus (KRV)-induced type 1 diabetes (T1D) to test the hypothesis that blocking IL-1 pathways early in the course of the disease can modulate virus-induced innate immunity and prevent disease progression. Administering KRV plus IL-1 receptor antagonist (Anakinra) for 14 d prevented insulitis and T1D. Anakinra reversed the KRV-induced systemic inflammation evidenced by the accumulation of T cells in the spleen and pancreatic lymph nodes on d 5 post-infection. Blocking IL-1 modulated the level of IRF-7 and IL-6 gene expression in the spleen and the p40 subunit of IL-12 and IL-23 in the serum. Anakinra did not interfere with the ability of LEW1.WR1 rats to clear the virus from the spleen, pancreatic lymph nodes or serum. Consistent with these data, normal levels of KRV-specific adaptive immune responses were detected in in the spleen and peripheral blood of the treated animals. Finally, blocking IL-1 pathways reversed the KRV-induced modulation of gut bacterial communities. The data may imply that IL-1 pathways are directly linked with early mechanisms whereby KRV infection leads to islet destruction, raising the hypothesis that blocking IL-1 pathways early in the course of the disease could be a useful therapeutic approach for disease prevention.

α1-Antitrypsin therapy downregulates toll-like receptor-induced IL-1ß responses in monocytes and myeloid dendritic cells and may improve islet function in recently diagnosed patients with type 1 diabetes.

CONTEXT: Recent studies have implicated proinflammatory responses in the mechanism of type 1 diabetes (T1D). OBJECTIVE: Our objective was to evaluate the safety and effects of therapy with the anti-inflammatory serum protein α1-antitrypsin (AAT) on islet function and innate immunity in recent-onset patients. DESIGN AND SETTING: This was an open-label phase I trial at the Barbara Davis Center for Childhood Diabetes, University of Colorado Denver. PATIENTS: Twelve recently diagnosed subjects with T1D with detectable C-peptides were included in the study. INTERVENTION: Eight consecutive weekly infusions of 80 mg/kg of AAT were given. MAIN OUTCOME MEASURES: PATIENTS were monitored for adverse effects of AAT therapy, C-peptide responses to a mixed-meal tolerance test, and toll-like receptor (TLR)-induced cellular IL-1ß in monocytes and myeloid dendritic cells (mDCs). RESULTS: No adverse effects were detected. AAT led to increased, unchanged, or moderately reduced levels of C-peptide responses compared with baseline in 5 patients. The total content of TLR4-induced cellular IL-1ß in monocytes at 12 months after AAT therapy was 3-fold reduced compared with baseline (P < .05). Furthermore, at baseline, 82% of monocytes produced IL-1ß, but at 12 months after therapy, the level decreased to 42%. Similar reductions were observed using TLR7/8 and TLR3 agonists in monocytes and mDCs. Unexpectedly, the reduction in cellular IL-1ß was observed only 9 and 12 months after treatment but not in untreated diabetics. Improved ß-cell function in the 5 AAT-treated individuals correlated with lower frequencies of monocytes and mDCs producing IL-1ß compared with subjects without improvement of islet function (P < .04 and P < .02, respectively). CONCLUSIONS: We hypothesize that AAT may have a beneficial effect on T1D in recently diagnosed patients that is associated with downmodulation of IL-1ß.

Induction of diabetes in the RIP-B7.1 mouse model is critically dependent on TLR3 and MyD88 pathways and is associated with alterations in the intestinal microbiome.

RIP-B7.1 transgenic mice express B7.1 costimulatory molecules in pancreatic islets and develop diabetes after treatment with polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA and agonist of Toll-like receptor (TLR) 3 and retinoic acid-inducible protein I. We used this model to investigate the role of TLR pathways and intestinal microbiota in disease progression. RIP-B7.1 mice homozygous for targeted disruption of TLR9, TLR3, and myeloid differentiation factor-88 (MyD88), and most of the wild-type RIP-B7.1 mice housed under normal conditions remained diabetes-free after poly I:C administration. However, the majority of TLR9-deficient mice and wild-type animals treated with poly I:C and an antibiotic developed disease. In sharp contrast, TLR3- and MyD88-deficient mice were protected from diabetes following the same treatment regimen. High-throughput DNA sequencing demonstrated that TLR9-deficient mice treated with antibiotics plus poly I:C had higher bacterial diversity compared with disease-resistant mice. Furthermore, principal component analysis suggested that TLR9-deficient mice had distinct gut microbiome compared with the diabetes-resistant mice. Finally, the administration of sulfatrim plus poly I:C to TLR9-deficient mice resulted in alterations in the abundance of gut bacterial communities at the phylum and genus levels. These data imply that the induction of diabetes in the RIP-B7.1 model is critically dependent on TLR3 and MyD88 pathways, and involves modulation of the intestinal microbiota.