Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation

CD25, a component of the IL-2 receptor, is important in T cell proliferation, activation induced cell death, as well as the actions of both regulatory (Treg) and effector (Teff) T cells. Recent genome wide association studies have implicated the CD25 locus as an important region for genetic susceptibility to a number of autoimmune disorders, with serum levels of soluble CD25 receptor (sCD25) serving as a potential phenotypic marker for this association. However, the functional impact of CD25 cleavage, as well as the influence of sCD25 on immunoregulatory activities, remain largely unknown and form the basis of this effort.The generation of sCD25 by Treg (CD4(+)CD25(+)) and Teff (CD4(+)CD25(-)) cells was examined during in vitro suppression assays, efforts that demonstrated constitutive and stable surface CD25 expression on Treg throughout the period of in vitro assessment. In contrast, Teff cells increased CD25 expression during the process of in vitro suppression, with supernatant sCD25 levels correlating to the amount of cellular proliferation. Interestingly, under serum-free conditions, Tregs partially lost their characteristic anergic and suppressive properties. sCD25 supplementation at physiological concentrations to serum free in vitro suppression assays reduced Teff proliferation without specifically influencing suppression. Indeed, sCD25 production within these cultures correlated with cell death.These results support the notion that sCD25 functions as both a surrogate marker of T cell activation as well as an indicator of subsequent cellular death. In addition, the role of CD25 in immunomodulation is likely dependent on the local inflammatory milieu, with molecules capable of modulating surface CD25 expression playing a key role in defining immune responsiveness

Failure to Preserve β-Cell Function With Mycophenolate Mofetil and Daclizumab Combined Therapy in Patients With New- Onset Type 1 Diabetes

OBJECTIVE This trial tested whether mycophenolate mofetil (MMF) alone or with daclizumab (DZB) could arrest the loss of insulin-producing β-cells in subjects with new-onset type 1 diabetes. RESEARCH DESIGN AND METHODS A multi-center, randomized, placebo-controlled, double-masked trial was initiated by Type 1 Diabetes TrialNet at 13 sites in North America and Europe. Subjects diagnosed with type 1 diabetes and with sufficient C-peptide within 3 months of diagnosis were randomized to either MMF alone, MMF plus DZB, or placebo, and then followed for 2 years. The primary outcome was the geometric mean area under the curve (AUC) C-peptide from the 2-h mixed meal tolerance test. RESULTS One hundred and twenty-six subjects were randomized and treated during the trial. The geometric mean C-peptide AUC at 2 years was unaffected by MMF alone or MMF plus DZB versus placebo. Adverse events were more frequent in the active therapy groups relative to the control group, but not significantly. CONCLUSIONS Neither MMF alone nor MMF in combination with DZB had an effect on the loss of C-peptide in subjects with new-onset type 1 diabetes. Higher doses or more targeted immunotherapies may be needed to affect the autoimmune process

Antithymocyte Globulin Plus G-CSF Combination Therapy Leads to Sustained Immunomodulatory and Metabolic Effects in a Subset of Responders With Established Type 1 Diabetes.

Low-dose antithymocyte globulin (ATG) plus pegylated granulocyte colony-stimulating factor (G-CSF) preserves β-cell function for at least 12 months in type 1 diabetes. Herein, we describe metabolic and immunological parameters 24 months following treatment. Patients with established type 1 diabetes (duration 4-24 months) were randomized to ATG and pegylated G-CSF (ATG+G-CSF) (N = 17) or placebo (N = 8). Primary outcomes included C-peptide area under the curve (AUC) following a mixed-meal tolerance test (MMTT) and flow cytometry. "Responders" (12-month C-peptide ≥ baseline), "super responders" (24-month C-peptide ≥ baseline), and "nonresponders" (12-month C-peptide < baseline) were evaluated for biomarkers of outcome. At 24 months, MMTT-stimulated AUC C-peptide was not significantly different in ATG+G-CSF (0.49 nmol/L/min) versus placebo (0.29 nmol/L/min). Subjects treated with ATG+G-CSF demonstrated reduced CD4+ T cells and CD4+/CD8+ T-cell ratio and increased CD16+CD56hi natural killer cells (NK), CD4+ effector memory T cells (Tem), CD4+PD-1+ central memory T cells (Tcm), Tcm PD-1 expression, and neutrophils. FOXP3+Helios+ regulatory T cells (Treg) were elevated in ATG+G-CSF subjects at 6, 12, and 18 but not 24 months. Immunophenotyping identified differential HLA-DR expression on monocytes and NK and altered CXCR3 and PD-1 expression on T-cell subsets. As such, a group of metabolic and immunological responders was identified. A phase II study of ATG+G-CSF in patients with new-onset type 1 diabetes is ongoing and may support ATG+G-CSF as a prevention strategy in high-risk subjects

The autoimmune disease-associated SNP rs917997 of IL18RAP controls IFNγ production by PBMC

AbstractType 1 Diabetes (T1D) is an autoimmune disorder characterized by aberrant T cell responses. Innate immune activation defects may facilitate a T helper 1 (Th1) phenotype. The cytokine IL-18 synergizes with IL-12 to induce IFNγ production and Th1 differentiation. The IL-18R subunit (IL18RAP) SNP rs917997 has been linked to decreased IL18RAP gene expression. Prior reports link rs917997 allele A with protection from T1D, and conversely with susceptibility to Celiac disease. However, few studies have investigated the IL-18 pathway in T1D. In this study, we analyzed responsiveness to IL-18 in T1D, and the effect of rs917997 genotype on IL18RAP gene expression post-activation. Upon IL-12 and IL-18 treatment, peripheral blood mononuclear cells from subjects carrying susceptibility alleles at rs917997 produced higher levels of IFNγ than those with protective genotypes. Additionally, the SNP modified IL18RAP surface protein expression by NK cells and gene expression in activated T cells. Taken together, these data suggest that the disease-associated rs917997 allele G permits hyperresponsiveness to IL-18, providing a novel target for therapeutic intervention in T1D

Specific human leukocyte antigen DQ influence on expression of antiislet autoantibodies and progression to type 1 diabetes

Human leukocyte antigen (HLA) DQ haplotypes have the strongest genetic association with type 1 diabetes (T1DM) risk. OBJECTIVE: The objective of the study was to analyze whether HLA DQ alleles influence the development of antiislet autoantibodies, the progression to T1DM among autoantibody-positive relatives, or both. DESIGN: The Diabetes Prevention Trial-1 screened more than 90,000 nondiabetic relatives of patients for cytoplasmic islet-cell autoantibody (ICA) expression between 1994 and 2002. SETTING: The study was conducted in the general community. PARTICIPANTS: The Diabetes Prevention Trial-1 found 2817 ICA-positive relatives who were tested for biochemical autoantibodies (GAD65, ICA512, and insulin) and HLA-DQ haplotypes, and 2796 of them were followed up for progression to diabetes for up to 8 yr (median, 3.6 yr). MAIN OUTCOME MEASURE: Progression to T1DM was measured. RESULTS: High-risk DQ haplotypes and genotypes were associated with a higher percentage of relatives expressing multiple biochemical autoantibodies and higher T1DM risk (e.g., respectively, 59 and 36% at 5 yr for carriers of the DQA1*0301-DQB1*0302/DQA1*0501-DQB1*0201 genotype). The number of autoantibodies expressed significantly increased T1DM risk and across different DQ genotypes, autoantibody positivity directly correlated with diabetes risk. However, multivariate analyses indicated that the influence of most genotypes on T1DM risk was not independent from autoantibody expression, with the possible exception of DQA1*0102-DQB1*0602. Specific genotypic combinations conferred 5-yr diabetes risks significantly lower (e.g. 7%-DQA1*0201-DQB1*0201/DQA1*0501-DQB1*0201 and 14%-DQA1*0301-DQB1*0301/DQA1*0501-DQB1*0201) than when those haplotypes were found in other combinations. CONCLUSION: HLA DQ alleles determine autoantibody expression, which is correlated with diabetes progression. Among autoantibody-positive relatives, most HLA DQ genotypes did not further influence T1DM risk

Lactobacillus johnsonii N6.2 Mitigates the Development of Type 1 Diabetes in BB-DP Rats

BACKGROUND: The intestinal epithelium is a barrier that composes one of the most immunologically active surfaces of the body due to constant exposure to microorganisms as well as an infinite diversity of food antigens. Disruption of intestinal barrier function and aberrant mucosal immune activation have been implicated in a variety of diseases within and outside of the gastrointestinal tract. With this model in mind, recent studies have shown a link between diet, composition of intestinal microbiota, and type 1 diabetes pathogenesis. In the BioBreeding rat model of type 1 diabetes, comparison of the intestinal microbial composition of diabetes prone and diabetes resistant animals found Lactobacillus species were negatively correlated with type 1 diabetes development. Two species, Lactobacillus johnsonii and L. reuteri, were isolated from diabetes resistant rats. In this study diabetes prone rats were administered pure cultures of L. johnsonii or L. reuteri isolated from diabetes resistant rats to determine the effect on type 1 diabetes development. METHODOLOGY/PRINCIPAL: Findings Results Rats administered L. johnsonii, but not L. reuteri, post-weaning developed type 1 diabetes at a protracted rate. Analysis of the intestinal ileum showed administration of L. johnsonii induced changes in the native microbiota, host mucosal proteins, and host oxidative stress response. A decreased oxidative intestinal environment was evidenced by decreased expression of several oxidative response proteins in the intestinal mucosa (Gpx1, GR, Cat). In L. johnsonii fed animals low levels of the pro-inflammatory cytokine IFNgamma were correlated with low levels of iNOS and high levels of Cox2. The administration of L. johnsonii also resulted in higher levels of the tight junction protein claudin. CONCLUSIONS: It was determined that the administration of L. johnsonii isolated from BioBreeding diabetes resistant rats delays or inhibits the onset of type 1 diabetes in BioBreeding diabetes prone rats. Taken collectively, these data suggest that the gut and the gut microbiota are potential agents of influence in type 1 diabetes development. These data also support therapeutic efforts that seek to modify gut microbiota as a means to modulate development of this disorder

Rituximab, B-lymphocyte depletion, and preservation of beta-cell function

BACKGROUND: The immunopathogenesis of type 1 diabetes mellitus is associated with T-lymphocyte autoimmunity. However, there is growing evidence that B lymphocytes play a role in many T-lymphocyte-mediated diseases. It is possible to achieve selective depletion of B lymphocytes with rituximab, an anti-CD20 monoclonal antibody. This phase 2 study evaluated the role of B-lymphocyte depletion in patients with type 1 diabetes. METHODS: We conducted a randomized, double-blind study in which 87 patients between 8 and 40 years of age who had newly diagnosed type 1 diabetes were assigned to receive infusions of rituximab or placebo on days 1, 8, 15, and 22 of the study. The primary outcome, assessed 1 year after the first infusion, was the geometric mean area under the curve (AUC) for the serum C-peptide level during the first 2 hours of a mixed-meal tolerance test. Secondary outcomes included safety and changes in the glycated hemoglobin level and insulin dose. RESULTS: At 1 year, the mean AUC for the level of C peptide was significantly higher in the rituximab group than in the placebo group. The rituximab group also had significantly lower levels of glycated hemoglobin and required less insulin. Between 3 months and 12 months, the rate of decline in C-peptide levels in the rituximab group was significantly less than that in the placebo group. CD19+ B lymphocytes were depleted in patients in the rituximab group, but levels increased to 69% of baseline values at 12 months. More patients in the rituximab group than in the placebo group had adverse events, mostly grade 1 or grade 2, after the first infusion. The reactions appeared to be minimal with subsequent infusions. There was no increase in infections or neutropenia with rituximab. CONCLUSIONS: A four-dose course of rituximab partially preserved beta-cell function over a period of 1 year in patients with type 1 diabetes. The finding that B lymphocytes contribute to the pathogenesis of type 1 diabetes may open a new pathway for exploration in the treatment of patients with this condition

An Anti-CD3 Antibody, Teplizumab, in Relatives at Risk for Type 1 Diabetes.

BackgroundType 1 diabetes is a chronic autoimmune disease that leads to destruction of insulin-producing beta cells and dependence on exogenous insulin for survival. Some interventions have delayed the loss of insulin production in patients with type 1 diabetes, but interventions that might affect clinical progression before diagnosis are needed.MethodsWe conducted a phase 2, randomized, placebo-controlled, double-blind trial of teplizumab (an Fc receptor-nonbinding anti-CD3 monoclonal antibody) involving relatives of patients with type 1 diabetes who did not have diabetes but were at high risk for development of clinical disease. Patients were randomly assigned to a single 14-day course of teplizumab or placebo, and follow-up for progression to clinical type 1 diabetes was performed with the use of oral glucose-tolerance tests at 6-month intervals.ResultsA total of 76 participants (55 [72%] of whom were ≤18 years of age) underwent randomization - 44 to the teplizumab group and 32 to the placebo group. The median time to the diagnosis of type 1 diabetes was 48.4 months in the teplizumab group and 24.4 months in the placebo group; the disease was diagnosed in 19 (43%) of the participants who received teplizumab and in 23 (72%) of those who received placebo. The hazard ratio for the diagnosis of type 1 diabetes (teplizumab vs. placebo) was 0.41 (95% confidence interval, 0.22 to 0.78; P = 0.006 by adjusted Cox proportional-hazards model). The annualized rates of diagnosis of diabetes were 14.9% per year in the teplizumab group and 35.9% per year in the placebo group. There were expected adverse events of rash and transient lymphopenia. KLRG1+TIGIT+CD8+ T cells were more common in the teplizumab group than in the placebo group. Among the participants who were HLA-DR3-negative, HLA-DR4-positive, or anti-zinc transporter 8 antibody-negative, fewer participants in the teplizumab group than in the placebo group had diabetes diagnosed.ConclusionsTeplizumab delayed progression to clinical type 1 diabetes in high-risk participants. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01030861.)