Association of serum microRNAs with islet autoimmunity, disease progression and metabolic impairment in relatives at risk of type 1 diabetes.

AIMS/HYPOTHESIS: MicroRNAs (miRNAs) are key regulators of gene expression and novel biomarkers for many diseases. We investigated the hypothesis that serum levels of some miRNAs would be associated with islet autoimmunity and/or progression to type 1 diabetes. METHODS: We measured levels of 93 miRNAs most commonly detected in serum. This retrospective cohort study included 150 autoantibody-positive and 150 autoantibody-negative family-matched siblings enrolled in the TrialNet Pathway to Prevention Study. This was a young cohort (mean age = 11 years), and most autoantibody-positive relatives were at high risk because they had multiple autoantibodies, with 39/150 (26%, progressors) developing type 1 diabetes within an average 8.7 months of follow-up. We analysed miRNA levels in relation to autoantibody status, future development of diabetes and OGTT C-peptide and glucose indices of disease progression. RESULTS: Fifteen miRNAs were differentially expressed when comparing autoantibody-positive/negative siblings (range -2.5 to 1.3-fold). But receiver operating characteristic (ROC) analysis indicated low specificity and sensitivity. Seven additional miRNAs were differentially expressed among autoantibody-positive relatives according to disease progression; ROC returned significant AUC values and identified miRNA cut-off levels associated with an increased risk of disease in both cross-sectional and survival analyses. Levels of several miRNAs showed significant correlations (r values range 0.22-0.55) with OGTT outcomes. miR-21-3p, miR-29a-3p and miR-424-5p had the most robust associations. CONCLUSIONS/INTERPRETATION: Serum levels of selected miRNAs are associated with disease progression and confer additional risk of the development of type 1 diabetes in young autoantibody-positive relatives. Further studies, including longitudinal assessments, are warranted to further define miRNA biomarkers for prediction of disease risk and progression.

Immune profiling by multiple gene expression analysis in patients at-risk and with type 1 diabetes.

There is a need for biomarkers to monitor the development and progression of type 1 DM. We analyzed mRNA expression levels for granzyme B, perforin, fas ligand, TNF-α, IFN-γ, Foxp3, IL-10, TGF-ß, IL-4, IL-6, IL-17, Activation-induced cytidine deaminase (AID) and Immunoglobulin G gamma chain (IgG) genes in peripheral blood of at-risk, new-onset and long-term type 1 DM , and healthy controls. The majority of the genes were suppressed in long-term type 1 DM compared to controls and new-onset patients. IFN-γ, IL-4 and IL-10 mRNA levels were significantly higher in new-onset compared to at-risk and long-term groups. There was decreased mRNA expression for AID and IgG and up-regulation of IFN-γ with age in controls. Data suggest an overall depressed immunity in long-term type 1 DM. Increased gene expression levels for IFN-γ, IL-4 and IL-10 in new-onset patients from at-risk patients might be used as potential markers for progression of the disease.

Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T-cells.

OBJECTIVE: To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients. RESEARCH DESIGN AND METHODS: We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays. RESULTS: Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within approximately 1 year from hyperglycemia recurrence and revealed beta-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell-directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell-directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed beta-cell loss in mice receiving autoreactive T-cells but not control T-cells. CONCLUSIONS: We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating beta-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.

Retrospective assessment of islet cell autoantibodies in pancreas organ donors.

OBJECTIVE: Of deceased pancreas donors, 3-4% may have autoantibodies (AAb) to pancreatic islet cell antigens; these autoantibodies are well-established markers of type 1 diabetes. We investigated whether donor AAb positivity could affect the outcome of pancreas transplantation. RESEARCH DESIGN AND METHODS: We retrospectively tested AAb in 135 donors whose pancreata and kidneys were transplanted in type 1 diabetes patients. We measured AAb to glutamic acid decarboxylase (GAD-AAb), the tyrosine-phosphatase-like protein IA2 (IA2-AAb), and insulin (insulin-AAb). We then evaluated pancreas transplant outcome data. RESULTS: Four of 135 (2.96%) donors were AAb positive: three donors had GAD-AAb, and one donor had insulin-AAb. Their respective recipients became insulin independent on follow-up. Three of the four recipients had normal, insulin-producing grafts 3-5.8 years after transplant. The recipient of the insulin-AAb-positive donor pancreas developed chronic rejection following discontinuation of immunosuppression 3.3 years after transplant. CONCLUSIONS: Single AAb positivity did not affect the outcome of pancreas transplantation in our study.

Dendritic cells in human thymus and periphery display a proinsulin epitope in a transcription-dependent, capture-independent fashion.

The natural expression of tissue-specific genes in the thymus, e.g., insulin, is critical for self-tolerance. The transcription of tissue-specific genes is ascribed to peripheral Ag-expressing (PAE) cells, which discordant studies identified as thymic epithelial cells (TEC) or CD11c+ dendritic cells (DC). We hypothesized that, consistent with APC function, PAE-DC should constitutively display multiple self-epitopes on their surface. If recognized by Abs, such epitopes could help identify PAE cells to further define their distribution, nature, and function. We report that selected Abs reacted with self-epitopes, including a proinsulin epitope, on the surface of CD11c+ cells. We find that Proins+ CD11c+ PAE cells exist in human thymus, spleen, and also circulate in blood. Human thymic Proins+ cells appear as mature DC but express CD8alpha, CD20, CD123, and CD14; peripheral Proins+ cells appear as immature DC. However, DC derived in vitro from human peripheral blood monocytes include Proins+ cells that uniquely differentiate and mature into thymic-like PAE-DC. Critically, we demonstrate that human Proins+ CD11c+ cells transcribe the insulin gene in thymus, spleen, and blood. Likewise, we show that mouse thymic and peripheral CD11c+ cells transcribe the insulin gene and display the proinsulin epitope; moreover, by using knockout mice, we show that the display of this epitope depends upon insulin gene transcription and is independent of Ag capturing. Thus, we propose that PAE cells include functionally distinct DC displaying self-epitopes through a novel, transcription-dependent mechanism. These cells might play a role in promoting self-tolerance, not only in the thymus but also in the periphery.

Thymic expression of peripheral tissue antigens in humans: a remarkable variability among individuals.

The majority of maturing T lymphocytes that recognize self-antigens is eliminated in the thymus upon exposure to their target antigens. This physiological process of negative selection requires that tissue-specific antigens be expressed by thymic cells, a phenomenon that has been well studied in experimental animals. Here, we have examined the expression in human thymi of four retinal antigens, that are capable of inducing autoimmune ocular disease retinal S-antigen (S-Ag), recoverin, RPE65 and inter-photoreceptor retinoid-binding protein (IRBP)], as well as four melanocyte-specific antigens, two of which are used as targets for melanoma immunotherapy [gp100, melanoma antigen recognized by T cells 1, tyrosinase-related protein (TRP)-1 and TRP-2]. Using reverse transcription (RT)-PCR, we found that all thymic samples from the 18 donors expressed mRNA transcripts of most or all the eight tested tissue antigens. Yet, the expression of the transcripts varied remarkably among the individual thymic samples. In addition, S-Ag, RPE65 and IRBP were detected by immunostaining in rare cells in sections of human thymi by antibodies against these proteins. Quantitative real-time RT-PCR analysis revealed that the retinal antigen transcripts in the human thymus are present at trace levels, that are lower by approximately five orders of magnitude than those in the retina. Our observations thus support the notions that thymic expression is a common feature for all tissue-specific antigens and that the levels of expression play a role in determining the susceptibility to autoimmunity against these molecules.