A Novel Quantitative Approach to Staging and Assessing Recovery from Type 1 Diabetes Mellitus: The Type 1 Diabetes Mellitus Metabolic Recovery Index.

Discovery of insulin in 1921 changed the lives of patients with type 1 diabetes (T1DM) forever. What had been a death sentence became a manageable, albeit chronic, disease. Insulin did not cure the disease, as it did not address the actual disease process, but instead treated its sequelae, namely elevated blood sugars. Importantly, insulin administration fails to ensure normoglycaemia. Even with the most sophisticated 'near closed-loop' methods, glucose homeostasis is not restored to normal. T1DM patients face complications, both short-term, such as hypo- and hyperglycaemia, and long-term, with increased glycosylation of proteins leading to eye, kidney, nervous system and other sequelae. These complications are associated with significant morbidity and mortality even after intensive insulin treatment. Nearly 100 years after the discovery of insulin, we continue to face the challenge of addressing the disease process itself, in order to fundamentally improve the life of these patients. There are major efforts to achieve just that: to completely arrest the autoimmune process destroying the insulin-producing cells in the pancreas, or at least significantly slow the process to blunt and delay short- and long-term complications. The aim of this Communication is to propose a novel assessment tool that would serve as a quantitative outcome measure by which therapies, short of clinical cure, may be compared and their true benefit to the treatment of diabetes assessed.

Characterization of human invariant natural killer T cells expressing FoxP3.

Recently described forkhead box protein 3 (FoxP3) transcription factor is a key molecule in CD4+ CD25hi+ T-cell characterization. Invariant NK T (iNKT) cells are also characterized as regulatory cells modulating the immune response by rapidly producing T(h)1 and T(h)2 cytokines. We aimed to analyze cellular markers important in regulatory features of human iNKT cells and to study their role in functional assays. iNKT cells were single cell sorted from peripheral mononuclear cells of healthy individuals after immunostaining of invariant TCR α-chain. We found FoxP3 expression in human iNKT clones. Randomly selected iNKT cell clones (CD4+, double negative, CD8+) expressed FoxP3 mRNA and protein at different levels upon stimulation as supported by various approaches. FoxP3 mRNA and protein expression was detected in unstimulated iNKT cells as well. Furthermore, different stimulations changed the FoxP3 expression in iNKT cells over time and the most dramatic changes were observed upon anti-CD3 stimulation. Both the supernatant of iNKT cells and iNKT cells themselves exerted similar stimulation effects on PBMC proliferation in functional assays and these stimulations showed a negative correlation with FoxP3 expression. Our data indicate that the FoxP3 expression in iNKT cells may be a key transcriptional factor in controlling the regulatory function of the iNKT cells.

Autoantigen-specific regulatory T cells induced in patients with type 1 diabetes mellitus by insulin B-chain immunotherapy.

There is a growing body of evidence to suggest that the autoimmunity observed in type 1 diabetes mellitus (T1DM) is the result of an imbalance between autoaggressive and regulatory cell subsets. Therapeutics that supplement or enhance the existing regulatory subset are therefore a much sought after goal in this indication. Here, we report the results of a double blind, placebo controlled, phase I clinical trial of a novel antigen-specific therapeutic in 12 subjects with recently diagnosed T1DM. Our primary objective was to test its safety. The study drug, human insulin B-chain in incomplete Freund's adjuvant (IFA) was administered as a single intramuscular injection, with subjects followed for 2 years. All subjects completed therapy and all follow-up visits. The therapy was generally safe and well-tolerated. Mixed meal stimulated C-peptide responses, measured every 6 months, showed no statistical differences between arms. All patients vaccinated with the autoantigen, but none who received placebo, developed robust insulin-specific humoral and T cell responses. Up to two years following the single injection, in peripheral blood from subjects in the experimental arm, but not the control arm, insulin B-chain-specific CD4+ T cells could be isolated and cloned that showed phenotypic and functional characteristics of regulatory T cells. The induction of a lasting, robust immune response generating autoantigen-specific regulatory T cells provides strong justification for further testing of this therapy in type 1 diabetes. (clinicaltrials.gov identifier NCT00057499).

Reduced CD4+ subset and Th1 bias of the human iNKT cells in Type 1 diabetes mellitus.

Invariant NKT (iNKT) cells are considered to be important in some autoimmune diseases including Type 1 diabetes mellitus (T1DM). So far, the published data are contradictory in regard to the role of iNKT cells in T1DM. We aimed to study iNKT cell frequency and the function of different iNKT cell subgroups in T1DM. We compared the results of four subject groups: healthy (H), long-term T2DM (ltT2DM; more than 1 year), newly diagnosed T1DM (ndT1DM; less than 3 months), and ltT1DM (more than 1 year) individuals. We measured the iNKT cell frequencies by costaining for the invariant TCR alpha-chain with 6B11-FITC and Valpha24-PE. After sorting the Valpha24+6B11+ cells, the generated iNKT clones were characterized. We tested CD4, CD8, and CD161 expression and IL-4 and IFN-gamma production on TCR stimulation. The CD4+ population among the iNKT cells was decreased significantly in ltT1DM versus ndT1DM, ltT2DM, or H individuals. The T1DM iNKT cell cytokine profile markedly shifted to the Th1 direction. There was no difference in the frequency of iNKT cells in PBMC among the different patient groups. The decrease in the CD4+ population among the iNKT cells and their Th1 shift indicates dysfunction of these potentially important regulatory cells in T1DM.

Reduced CD4+ T-cell-specific gene expression in human type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) in humans is characterized by the T-cell-dependent destruction of the insulin producing pancreatic beta cells; however, the precise pathogenesis of the disease, especially the initiation of pathologic immune response, is still largely unknown. We hypothesized that the function of human CD4+ T cells is altered in T1DM and analyzed unstimulated human peripheral blood CD4+ T-cell gene expression. We used a novel three-way comparison of DNA microarray data of CD4+ T cells isolated from patients with new onset T1DM, patients with long-term Type 2 diabetes (T2DM), and from healthy control subjects in order to eliminate any possible influence of glucose homeostasis on our findings. We analyzed the T1DM specific gene-expression changes and their functional relevance to T1DM autoimmunity. Our genetic and functional data show that T1DM CD4+ T cells are down-regulated specifically affecting key immune functions and cell cycle. Histone deacetylase gene expression, a key regulator of epigenetic modification is also reduced. The CD4+ T cells showed impaired function, including an abnormal immune response, which may be a key element that leads to the breakdown of self-tolerance.