Immune and Metabolic Effects of Antigen-Specific Immunotherapy Using Multiple ß-Cell Peptides in Type 1 Diabetes.

Type 1 diabetes is characterized by a loss of tolerance to pancreatic ß-cell autoantigens and defects in regulatory T-cell (Treg) function. In preclinical models, immunotherapy with MHC-selective, autoantigenic peptides restores immune tolerance, prevents diabetes, and shows greater potency when multiple peptides are used. To translate this strategy into the clinical setting, we administered a mixture of six HLA-DRB1*0401-selective, ß-cell peptides intradermally to patients with recent-onset type 1 diabetes possessing this genotype in a randomized placebo-controlled study at monthly doses of 10, 100, and 500 µg for 24 weeks. Stimulated C-peptide (measuring insulin functional reserve) had declined in all placebo subjects at 24 weeks but was maintained at ≥100% baseline levels in one-half of the treated group. Treatment was accompanied by significant changes in islet-specific immune responses and a dose-dependent increase in Treg expression of the canonical transcription factor FOXP3 and changes in Treg gene expression. In this first-in-human study, multiple-peptide immunotherapy shows promise as a strategy to correct immune regulatory defects fundamental to the pathobiology of autoimmune diabetes.

Metabolic and immune effects of immunotherapy with proinsulin peptide in human new-onset type 1 diabetes.

Immunotherapy using short immunogenic peptides of disease-related autoantigens restores immune tolerance in preclinical disease models. We studied safety and mechanistic effects of injecting human leukocyte antigen-DR4(DRB1*0401)-restricted immunodominant proinsulin peptide intradermally every 2 or 4 weeks for 6 months in newly diagnosed type 1 diabetes patients. Treatment was well tolerated with no systemic or local hypersensitivity. Placebo subjects showed a significant decline in stimulated C-peptide (measuring insulin reserve) at 3, 6, 9, and 12 months versus baseline, whereas no significant change was seen in the 4-weekly peptide group at these time points or the 2-weekly group at 3, 6, and 9 months. The placebo group's daily insulin use increased by 50% over 12 months but remained unchanged in the intervention groups. C-peptide retention in treated subjects was associated with proinsulin-stimulated interleukin-10 production, increased FoxP3 expression by regulatory T cells, low baseline levels of activated ß cell-specific CD8 T cells, and favorable ß cell stress markers (proinsulin/C-peptide ratio). Thus, proinsulin peptide immunotherapy is safe, does not accelerate decline in ß cell function, and is associated with antigen-specific and nonspecific immune modulation.

ß-cell-specific CD8 T cell phenotype in type 1 diabetes reflects chronic autoantigen exposure.

Autoreactive CD8 T cells play a central role in the destruction of pancreatic islet ß-cells that leads to type 1 diabetes, yet the key features of this immune-mediated process remain poorly defined. In this study, we combined high-definition polychromatic flow cytometry with ultrasensitive peptide-human leukocyte antigen class I tetramer staining to quantify and characterize ß-cell-specific CD8 T cell populations in patients with recent-onset type 1 diabetes and healthy control subjects. Remarkably, we found that ß-cell-specific CD8 T cell frequencies in peripheral blood were similar between subject groups. In contrast to healthy control subjects, however, patients with newly diagnosed type 1 diabetes displayed hallmarks of antigen-driven expansion uniquely within the ß-cell-specific CD8 T cell compartment. Molecular analysis of selected ß-cell-specific CD8 T cell populations further revealed highly skewed oligoclonal T cell receptor repertoires comprising exclusively private clonotypes. Collectively, these data identify novel and distinctive features of disease-relevant CD8 T cells that inform the immunopathogenesis of type 1 diabetes.

Blood and islet phenotypes indicate immunological heterogeneity in type 1 diabetes.

Studies in type 1 diabetes indicate potential disease heterogeneity, notably in the rate of ß-cell loss, responsiveness to immunotherapies, and, in limited studies, islet pathology. We sought evidence for different immunological phenotypes using two approaches. First, we defined blood autoimmune response phenotypes by combinatorial, multiparameter analysis of autoantibodies and autoreactive T-cell responses in 33 children/adolescents with newly diagnosed diabetes. Multidimensional cluster analysis showed two equal-sized patient agglomerations characterized by proinflammatory (interferon-γ-positive, multiautoantibody-positive) and partially regulated (interleukin-10-positive, pauci-autoantibody-positive) responses. Multiautoantibody-positive nondiabetic siblings at high risk of disease progression showed similar clustering. Additionally, pancreas samples obtained post mortem from a separate cohort of 21 children/adolescents with recently diagnosed type 1 diabetes were examined immunohistologically. This revealed two distinct types of insulitic lesions distinguishable by the degree of cellular infiltrate and presence of B cells that we termed "hyper-immune CD20Hi" and "pauci-immune CD20Lo." Of note, subjects had only one infiltration phenotype and were partitioned by this into two equal-sized groups that differed significantly by age at diagnosis, with hyper-immune CD20Hi subjects being 5 years younger. These data indicate potentially related islet and blood autoimmune response phenotypes that coincide with and precede disease. We conclude that different immunopathological processes (endotypes) may underlie type 1 diabetes, carrying important implications for treatment and prevention strategies.

Circulating preproinsulin signal peptide-specific CD8 T cells restricted by the susceptibility molecule HLA-A24 are expanded at onset of type 1 diabetes and kill ß-cells.

Type 1 diabetes results from T cell-mediated ß-cell destruction. The HLA-A*24 class I gene confers significant risk of disease and early onset. We tested the hypothesis that HLA-A24 molecules on islet cells present preproinsulin (PPI) peptide epitopes to CD8 cytotoxic T cells (CTLs). Surrogate ß-cell lines secreting proinsulin and expressing HLA-A24 were generated and their peptide ligandome examined by mass spectrometry to discover naturally processed and HLA-A24-presented PPI epitopes. A novel PPI epitope was identified and used to generate HLA-A24 tetramers and examine the frequency of PPI-specific T cells in new-onset HLA-A*24(+) patients and control subjects. We identified a novel naturally processed and HLA-A24-presented PPI signal peptide epitope (PPI(3-11); LWMRLLPLL). HLA-A24 tetramer analysis reveals a significant expansion of PPI(3-11)-specific CD8 T cells in the blood of HLA-A*24(+) recent-onset patients compared with HLA-matched control subjects. Moreover, a patient-derived PPI(3-11)-specific CD8 T-cell clone shows a proinflammatory phenotype and kills surrogate ß-cells and human HLA-A*24(+) islet cells in vitro. These results indicate that the type 1 diabetes susceptibility molecule HLA-A24 presents a naturally processed PPI signal peptide epitope. PPI-specific, HLA-A24-restricted CD8 T cells are expanded in patients with recent-onset disease. Human islet cells process and present PPI(3-11), rendering themselves targets for CTL-mediated killing.

Peripheral and islet interleukin-17 pathway activation characterizes human autoimmune diabetes and promotes cytokine-mediated ß-cell death.

OBJECTIVE: CD4 T-cells secreting interleukin (IL)-17 are implicated in several human autoimmune diseases, but their role in type 1 diabetes has not been defined. To address the relevance of such cells, we examined IL-17 secretion in response to ß-cell autoantigens, IL-17A gene expression in islets, and the potential functional consequences of IL-17 release for ß-cells. RESEARCH DESIGN AND METHODS: Peripheral blood CD4 T-cell responses to ß-cell autoantigens (proinsulin, insulinoma-associated protein, and GAD65 peptides) were measured by IL-17 enzyme-linked immunospot assay in patients with new-onset type 1 diabetes (n = 50). mRNA expression of IL-17A and IFNG pathway genes was studied by qRT-PCR using islets obtained from subjects who died 5 days and 10 years after diagnosis of disease, respectively, and from matched control subjects. IL-17 effects on the function of human islets, rat ß-cells, and the rat insulinoma cell line INS-1E were examined. RESULTS: A total of 27 patients (54%) showed IL-17 reactivity to one or more ß-cell peptides versus 3 of 30 (10%) control subjects (P = 0.0001). In a single case examined close to diagnosis, islet expression of IL17A, RORC, and IL22 was detected. It is noteworthy that we show that IL-17 mediates significant and reproducible enhancement of IL-1ß/interferon (IFN)-γ-induced and tumor necrosis factor (TNF)-α/IFN-γ-induced apoptosis in human islets, rat ß-cells, and INS-1E cells, in association with significant upregulation of ß-cell IL17RA expression via activation of the transcription factors STAT1 and nuclear factor (NF)-κB. CONCLUSIONS: Circulating IL-17(+) ß-cell-specific autoreactive CD4 T-cells are a feature of type 1 diabetes diagnosis. We disclose a novel pathway to ß-cell death involving IL-17 and STAT1 and NF-κB, rendering this cytokine a novel disease biomarker and potential therapeutic target.

Cardiac valve disease and low-dose dopamine agonist therapy: an artefact of reporting bias?

INTRODUCTION: Chronic low-dose cabergoline treatment for microprolactinoma may cause cardiac valve pathology, but the evidence is contradictory. We investigated whether the expectation of the echocardiographer could influence the report. METHODS: Transthoracic echocardiograms from 40 patients aged 49·3 ± 9·6 (mean ± SD) years (Men:Women 7:33) on long-term cabergoline and bromocriptine therapy (duration 9·94 ± 4·5 years) were randomly assigned to two groups of echocardiographers so that each echocardiogram was reported twice. One group was told that 'the patients were control subjects' (Group A) and the other that 'the patients were on dopamine agonist therapy which is known to cause valve disease' (Group B). An observer who was blind to the group scored the reports for regurgitation at each valve (scores 0-4; max 16 per case). RESULTS: Mean total regurgitation score was significantly higher in Group B (1·43 ± 1·28; P = 0·014) than in Group A (0·73 ± 1·30). The difference was mainly from reporting trivial regurgitation: (mitral 16 vs 5, P = 0·005; tricuspid 17 vs 6, P = 0·007 and pulmonary 8 vs 1, P = 0·013). Mild regurgitation was uncommon (mitral 1 vs 1 and tricuspid 3 vs 6). Moderate regurgitation occurred in only one case and was associated with restriction of the leaflets consistent with the effects of cabergoline. Valve thickening was not reported in Group A, but in 9 (23%) mitral and 4 (10%) aortic valves in Group B. CONCLUSION: Long-term, low-dose dopamine agonist therapy rarely causes cardiac valve disease, but operator bias can lead to over-reporting of both valve thickening and trivial regurgitation.

The influence of the route of oestrogen administration on serum levels of cortisol-binding globulin and total cortisol.

OBJECTIVES: Oral oestrogen preparations increase total cortisol concentration by increasing circulating cortisol-binding globulin (CBG) levels. Transdermal oestrogen treatments are being used increasingly in clinical practice. These topical preparations may have less of an effect on CBG and hence on total serum cortisol levels by reducing hepatic oestrogen exposure. The purpose of this study was to compare the effects of oral and topical oestrogen treatments on CBG, total serum cortisol and salivary cortisol levels. DESIGN AND PATIENTS: This was a single-centre, cross-sectional study of 37 women aged 33 +/- 6 years (mean +/- SD). Fourteen women were using oral oestrogen therapy, eight were using transdermal therapy and 15 were oestrogen-naïve control subjects. MEASUREMENTS: Following a screening visit, the subjects attended the endocrine investigation unit following an overnight fast. Blood and salivary samples were taken from 0830 to 0930 h between days 10 and 18 of the menstrual cycle (where appropriate). RESULTS: Total serum cortisol concentrations were 67% higher in those receiving oral oestrogen when compared to control subjects (660.9 +/- 89.9 vs. 395.4 +/- 53.2 nmol/l, P < 0.001). Values in those receiving transdermal oestrogen (334.7 +/- 72.0 nmol/l) were no different from the control group. CBG levels were higher in those on oral oestrogen therapy (110.9 +/- 19.6 mg/l, P < 0.001) when compared with either those on transdermal oestrogen (51.0 +/- 5.4 mg/l) or the control population (49.0 +/- 11.8 mg/l). Similar salivary cortisol concentrations were recorded in the three groups (controls 13.8 +/- 2.6 nmol/l, oral oestrogen 15.5 +/- 2.6 nmol/l, transdermal oestrogen 15.7 +/- 3.9 nmol/l). CONCLUSIONS: Oral oestrogen-containing preparations increase total cortisol levels by increasing circulating CBG concentration. These effects were not seen in patients using transdermal oestrogen replacement. Although further studies are indicated, it is probably unnecessary to routinely discontinue transdermal oestrogen replacement when performing an assessment of the hypothalamic-pituitary-adrenal (HPA) axis or evaluating adequacy of hydrocortisone replacement.

Circulating nucleic acids and diabetic complications.

Diabetes mellitus is a major health problem across the world. Diabetic retinopathy (DR) and nephropathy are two of the major complications of diabetes. DR is the leading cause of blindness and diabetic nephropathy is the leading cause of end-stage renal failure. We have examined the potential value of circulating nucleic acids in the detection and monitoring of these two complications of diabetes. mRNA for nephrin was significantly higher in all diabetics compared to healthy controls and it was significantly higher in normoalbuminuric patients compared to healthy controls. This may indicate progression to microalbuminuric stage. Circulating rhodopsin mRNA was detectable in healthy subjects and in diabetic patients. It was significantly raised in diabetic patients with retinopathy. Higher rhodopsin mRNA in diabetic patients without retinopathy suggests that some of them may go on to develop it or already have it subclinically. Circulating nucleic acids have the potential to be noninvasive molecular tests for diabetic complications.

Real-time quantitative PCR measurement of circulatory rhodopsin mRNA in healthy subjects and patients with diabetic retinopathy.

Diabetic retinopathy is the commonest complication of diabetes and is the biggest single cause of registered blindness in the UK. No biochemical tests exist to determine the precise state and rate of change of the eyes in the diabetic patient. In the present study, using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we measured mRNA encoding the retina-specific pigment protein rhodopsin (RHO) in the peripheral blood of healthy individuals (n = 20) and diabetic patients (n = 46) with and without retinopathy. Beta-actin mRNA was also assayed and results are expressed as a ratio of RHO to beta-actin mRNA. Peripheral blood was taken by venipucture directly into PAXgene Blood RNA collection tubes and RNA extracted by use of the PAXgene Blood RNA extraction kit, as per the manufacturer's (Qiagen) instructions. Diabetic patients were divided into three groups defined by the severity of retinopathy as assessed by fundoscopy: A, diabetic without retinopathy; B, background retinopathy; and C, preproliferative retinopathy. Medians of the ratios between groups were compared. RHO mRNA was successfully detected and quantified in peripheral blood in all healthy and diabetic groups, with levels shown to be significantly higher in diabetic patients than in healthy controls (2.54 x 10(-5) vs. 1.29 x 10(-5); P = 0.002). Significant differences in RHO mRNA levels were also seen between healthy control subjects and diabetic groups A (2.52 x 10(-5); P = 0.022), B (1.98 x 10(-5); P = 0.028), and C (5.08 x 10(-5); P = 0.002). The results suggest that there is an increase in circulatory RHO mRNA with the severity of diabetic retinopathy.