Regulatory T cells are an important component of the immune system that plays a key role in maintaining homeostasis. Identification of distinct regulatory T cell subsets is essential to understand their function. Mass cytometry or CyTOF is a technology that enables the simultaneous measurement of up to 50 markers in single cells by using antibodies tagged with heavy metals, which are then detected with time-of-flight mass spectrometry. This chapter describes a mass cytometry approach for phenotypic characterization of regulatory T cells and determination of their master transcription factor Foxp3.

Aims: Immunomodulation with autoantigens potentially constitutes a specific and safe treatment for type 1 diabetes (T1D). Studies with GAD-alum administrated subcutaneously have shown to be safe, but its efficacy has been inconclusive. Administration of GAD-alum into the lymph nodes, aimed to optimise antigen presentation, has shown promising results in an open-label clinical trial. Herein, we compared the immune response of the individuals included in the trial with a group who received GAD-alum subcutaneously in a previous study.

Materials and methods: Samples from T1D individuals collected 15 months after administration of either three doses 1 month apart of 4 mu g GAD-alum into lymph nodes (LN, n = 12) or two doses 1 month apart of 20 mu g subcutaneously (SC, n = 12) were studied. GADA, GADA subclasses, GAD(65)-induced cytokines, peripheral blood mononuclear cell proliferation, and T cells markers were analysed.

Results: Low doses of GAD-alum into the lymph nodes induced higher GADA levels than higher doses administrated subcutaneously. Immune response in the LN group was characterised by changes in GADA subclasses, with a relative reduction of IgG1 and enhanced IgG2, IgG3, and IgG4 proportion, higher GAD(65)-induced secretion of IL-5, IL-10, and TNF-alpha, and reduction of cell proliferation and CD8(+) T cells. These changes were not observed after subcutaneous (SC) injections of GAD-alum.

Conclusions: GAD-specific immune responses 15 months after lymph node injections of GAD-alum differed from the ones induced by SC administration of the same autoantigen.

Aim To evaluate the long-term effect of intra-lymphatic administration of GAD-alum and a booster dose 2.5 years after the first intervention (DIAGNODE Extension study) in patients with recent-onset type 1 diabetes. Methods DIAGNODE-1: Samples were collected from 12 patients after 30 months who had received 3 injections of 4 mu g GAD-alum into a lymph node with one-month interval. DIAGNODE Extension study: First in human, a fourth booster dose of autoantigen (GAD-alum) was given to 3 patients at 31.5 months, who were followed for another 12 months. C-peptide was measured during mixed meal tolerance tests (MMTTs). GADA, IA-2A, GADA subclasses, GAD(65)-induced cytokines, PBMCs proliferation and T cells markers were analyzed. Results After 30-month treatment, efficacy was still seen in 8/12 patients (good responders, GR). Partial remission (IDAA1c < 9) had decreased compared to 15 months, but did not differ from baseline, and HbA1c remained stable. GAD(65)-specific immune responses induced by the treatment started to wane after 30 months, and most changes observed at 15 months were undetectable. GADA subclasses IgG2, IgG3 and IgG4 were predominant in the GR along with IgG1. A fourth intra-lymphatic GAD-alum dose to three patients after 31.5 months gave no adverse events. In all three patients, C-peptide seemed to increase the first 6 months, and thereafter, C-peptide, HbA1c, insulin requirement and IDAA1c remained stable. Conclusion The effect of intra-lymphatic injections of GAD-alum had decreased after 30 months. Good responders showed a specific immune response. Administration of a fourth booster dose after 31.5 months was safe, and there was no decline in C-peptide observed during the 12-month follow-up.

Aims Residual beta cell function in type 1 diabetes (T1D) is associated with lower risk of complications. Autoantigen therapy with GAD-alum (Diamyd) given in 3 intralymphatic injections with oral vitamin D has shown promising results in persons with T1D carrying the human leukocyte antigen (HLA) DR3-DQ2 haplotype in the phase 2b trial DIAGNODE-2. We aimed to explore the efficacy of intralymphatic GAD-alum on blood glucose recorded by continuous glucose monitoring (CGM). Methods DIAGNODE-2 (NCT03345004) was a multicenter, randomized, placebo-controlled, double-blind trial of 109 recent-onset T1D patients aged 12 to 24 years with GAD65 antibodies and fasting C-peptide > 0.12 nmol/L, which randomized patients to 3 intralymphatic injections of 4 mu g GAD-alum and oral vitamin D, or placebo. We report results for exploratory endpoints assessed by 14-day CGM at months 0, 6, and 15. Treatment arms were compared by mixed-effects models for repeated measures adjusting for baseline values. Results We included 98 patients with CGM recordings of sufficient quality (DR3-DQ2-positive patients: 27 GAD-alum-treated and 15 placebo-treated). In DR3-DQ2-positive patients, percent of time in range (TIR, 3.9-10 mmol/L) declined less between baseline and month 15 in GAD-alum-treated compared with placebo-treated patients (-5.1% and -16.7%, respectively; P = 0.0075), with reduced time > 13.9 mmol/L (P = 0.0036), and significant benefits on the glucose management indicator (P = 0.0025). No differences were detected for hypoglycemia. GAD-alum compared to placebo lowered the increase in glycemic variability (standard deviation) observed in both groups (P = 0.0219). Change in C-peptide was correlated with the change in TIR. Conclusions Intralymphatic GAD-alum improves glycemic control in recently diagnosed T1D patients carrying HLA DR3-DQ2.

Antigen-specific immunotherapy is an immunomodulatory strategy for autoimmune diseases, such as type 1 diabetes, in which patients are treated with autoantigens to promote immune tolerance, stop autoimmune β-cell destruction and prevent permanent dependence on exogenous insulin. In this study, human proinsulin peptide C19-A3 (known for its positive safety profile) was conjugated to ultrasmall gold nanoparticles (GNPs), an attractive drug delivery platform due to the potential anti-inflammatory properties of gold. We hypothesised that microneedle intradermal delivery of C19-A3 GNP may improve peptide pharmacokinetics and induce tolerogenic immunomodulation and proceeded to evaluate its safety and feasibility in a first-in-human trial. Allowing for the limitation of the small number of participants, intradermal administration of C19-A3 GNP appears safe and well tolerated in participants with type 1 diabetes. The associated prolonged skin retention of C19-A3 GNP after intradermal administration offers a number of possibilities to enhance its tolerogenic potential, which should be explored in future studies.

Neutrophil extracellular traps (NETs) and mitochondrial DNA (mtDNA) are inflammatory mediators involved in the development of type 1 diabetes (T1D). Pancreas-infiltrating neutrophils can release NETs, contributing to the inflammatory process. Levels of NETs are increased in serum from patients with T1D and mtDNA is increased in adult T1D patients. Our aim was to investigate extracellular DNA (NETs, mtDNA and nuclear DNA) in children with newly diagnosed T1D and in children at high risk of the disease. We also elucidated if extracellular DNA short after diagnosis could predict loss of endogenous insulin production. Samples were analysed for mtDNA and nuclear DNA using droplet digital PCR and NETs were assessed by a NET-remnants ELISA. In addition, in vitro assays for induction and degradation of NETs, as well as analyses of neutrophil elastase, HLA genotypes, levels of c-peptide, IL-1beta, IFN and autoantibodies (GADA, IA-2A, IAA and ZnT8A) were performed. In serum from children 10 days after T1D onset there was an increase in NETs (p=0.007), mtDNA (p<0.001) and nuclear DNA (p<0.001) compared to healthy children. The elevated levels were found only in younger children. In addition, mtDNA increased in consecutive samples short after onset (p=0.017). However, levels of extracellular DNA short after onset did not reflect future loss of endogenous insulin production. T1D serum induced NETs in vitro and did not deviate in the ability to degrade NETs. HLA genotypes and autoantibodies, except for ZnT8A, were not associated with extracellular DNA in T1D children. Serum from children with high risk of T1D showed fluctuating levels of extracellular DNA, sometimes increased compared to healthy children. Therefore, extracellular DNA in serum from autoantibody positive high-risk children does not seem to be a suitable biomarker candidate for prediction of T1D. In conclusion, we found increased levels of extracellular DNA in children with newly diagnosed T1D, which might be explained by an ongoing systemic inflammation.

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BACKGROUND: This study aimed to analyse data from two different studies (Phase II and Phase III) regarding the safety and efficacy of treatment with alum formulated glutamic acid decarboxylase GAD65 (GAD-alum), 30 months after administration to children and adolescents with Type 1 diabetes (T1D).

METHODS: The Phase II trial was a double-blind, randomized placebo-controlled study, including 70 children and adolescents which were followed for 30 months. Participants received a subcutaneous injection of either 20 µg of GAD-alum or placebo at baseline and one month later. During a subsequent larger European Phase III trial including three treatment arms, participants received two or four subcutaneous injections of either 20 µg of GAD-alum and/or placebo at baseline, 1, 3 and 9 months. The Phase III trial was prematurely interrupted at 15 months, but of the 148 Swedish patients, a majority completed the 21 months follow-up and 45 patients completed the trial at 30 months. Both studies included GADA-positive patients with fasting C-peptide ≥0.10 nmol/l. We have now combined the results of these two trials.

RESULTS: There were no treatment related adverse events. In patients treated with 2 GAD-alum doses, stimulated C-peptide AUC had decreased significantly less (9 m: p < 0.037; 15 m p < 0.032; 21 m p < 0.003 and 30 m p < 0.004) and a larger proportion of these patients were also able to achieve a peak stimulated C-peptide >0.2 nmol/l (p < 0.05), as compared to placebo.

CONCLUSION: Treatment with two doses of GAD-alum in children and adolescents with recent-onset T1D shows no adverse events and preserves residual insulin secretion. This article is protected by copyright. All rights reserved.

Aims/Hypothesis: Islet amyloid polypeptide (IAPP) is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes. Results: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (n = 224) were analysed and concentrations exceeding 100 pmol/L (127.2 - 888.7 pmol/L) were found in 11% (25/224). The IAPP increase did not correlate with C-peptide levels. Conclusions/Interpretation: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes.

We studied the effects of probiotic treatment on the innate immune system during infancy. The study included a subgroup of infants recruited to the pilot study testing the feasibility of probiotics intervention in infants with genetic risk of type 1 diabetes (T1D). A mixture of Lactobacillus rhamnosus GG (5 x 109 cfu), Lactobacillus rhamnosus LC705 (5 x 109 cfu), Bifidobacterium breve Bbi99 (2 x 108 cfu) and Propionibacterium freudenreichii ssp. Shermani JS (2 x 109 cfu) was given to the infants beginning one to three weeks after birth until the age of 6 months. Blood samples were drawn at the age of 6, 12 and 24 months for the analyses of beta-cell autoantibodies and the phenotype and stimulation response of monocytes with flow-cytometry, including surface markers on circulating CD14+ monocytes and expression of co-stimulatory markers on CD14+ monocytes as response to stimulation with lipoteichoic acid (LTA) and lipopolysaccharide (LPS). Also gene expression of toll-like receptor (TLR) signalling molecules was studied in the peripheral blood mononuclear cell (PBMC) population.

In the children who received probiotics the number of circulating CD14+ monocytes expressing CD58 was reduced at the age of 6 months, and a tendency for a decreased induction of CCR5, CD80 and CD58 expressing monocytes as response to LTA was seen when compared to the children who received placebo. At the age of 12 months, the number of monocytes expressing CCR5 was decreased in the probiotic group, and a decreased spontaneous expression of TNFRSF1A and an increased spontaneous expression of TLR9 was observed in the PBMC from the children treated with probiotics. In the whole study group, the numbers of circulating monocytes expressing CD80 increased with age as well as the induction of CCR5, CD80 and CD58 on monocytes as response to stimulation. By the age of 24 months one child in both groups developed multiple autoantibodies.

We demonstrated that probiotics modulated the activation stage and stimulation response of monocytes, and that prolonged effects of the treatment were seen at the age of 12 months. The findings suggest that early microbial exposure may program the function of the innate immune system for later life.