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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Dysregulated Toll-like receptor-induced interleukin-1ß and interleukin-6 responses in subjects at risk for the development of type 1 diabetes.

We tested the hypothesis that altered Toll-like receptor (TLR) signaling may be involved in early stages of type 1 diabetes (T1D). To do so, we analyzed TLR-induced interleukin (IL)-1ß and IL-6 responses in freshly isolated peripheral blood mononuclear cells (PBMNCs) from seropositive compared with seronegative subjects. Similar frequencies of myeloid dendritic cells (mDCs), plasmacytoid DCs (pDCs), and monocytes were observed in seropositive and seronegative subjects. Subjects with autoantibodies had increased proportions of monocytes expressing IL-1ß ex vivo. Activating PBMNCs with TLR3, TLR4, or TLR7/8 agonists in vitro led to increased percentages of IL-1ß-expressing monocytes and mDCs from seropositive versus seronegative subjects. TLR ligation also resulted in a diminished IL-6 response in seropositive individuals as lower frequencies of IL-6-expressing monocytes and mDCs were induced. The dysregulated TLR-induced IL-1ß and IL-6 pathways were more readily detectable in children aged <11 years and from 11 to <21 years, respectively, and did not involve altered HbA(1c) or the presence of one or more autoantibodies. Finally, subjects with autoantibodies had lower amounts of serum chemokine (C-X-C motif) ligand 10 compared with autoantibody-negative subjects. Our data may imply that alterations in innate immune pathways are detectable in genetically susceptible individuals and could be linked with the early course of T1D.