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.

Reduction in CD4 central memory T-cell subset in costimulation modulator abatacept-treated patients with recent-onset type 1 diabetes is associated with slower C-peptide decline.

We previously reported that continuous 24-month costimulation blockade by abatacept significantly slows the decline of ß-cell function after diagnosis of type 1 diabetes. In a mechanistic extension of that study, we evaluated peripheral blood immune cell subsets (CD4, CD8-naive, memory and activated subsets, myeloid and plasmacytoid dendritic cells, monocytes, B lymphocytes, CD4(+)CD25(high) regulatory T cells, and invariant NK T cells) by flow cytometry at baseline and 3, 6, 12, 24, and 30 months after treatment initiation to discover biomarkers of therapeutic effect. Using multivariable analysis and lagging of longitudinally measured variables, we made the novel observation in the placebo group that an increase in central memory (CM) CD4 T cells (CD4(+)CD45R0(+)CD62L(+)) during a preceding visit was significantly associated with C-peptide decline at the subsequent visit. These changes were significantly affected by abatacept treatment, which drove the peripheral contraction of CM CD4 T cells and the expansion of naive (CD45R0(-)CD62L(+)) CD4 T cells in association with a significantly slower rate of C-peptide decline. The findings show that the quantification of CM CD4 T cells can provide a surrogate immune marker for C-peptide decline after the diagnosis of type 1 diabetes and that costimulation blockade may exert its beneficial therapeutic effect via modulation of this subset.

Costimulation modulation with abatacept in patients with recent-onset type 1 diabetes: follow-up 1 year after cessation of treatment.

OBJECTIVE We previously reported that 2 years of costimulation modulation with abatacept slowed decline of ß-cell function in recent-onset type 1 diabetes (T1D). Subsequently, abatacept was discontinued and subjects were followed to determine whether there was persistence of effect. RESEARCH DESIGN AND METHODS Of 112 subjects (ages 6-36 years) with T1D, 77 received abatacept and 35 received placebo infusions intravenously for 27 infusions over 2 years. The primary outcome-baseline-adjusted geometric mean 2-h area under the curve (AUC) serum C-peptide during a mixed-meal tolerance test (MMTT) at 2 years-showed higher C-peptide with abatacept versus placebo. Subjects were followed an additional year, off treatment, with MMTTs performed at 30 and 36 months. RESULTS C-peptide AUC means, adjusted for age and baseline C-peptide, at 36 months were 0.217 nmol/L (95% CI 0.168-0.268) and 0.141 nmol/L (95% CI 0.071-0.215) for abatacept and placebo groups, respectively (P = 0.046). The C-peptide decline from baseline remained parallel with an estimated 9.5 months' delay with abatacept. Moreover, HbA1c levels remained lower in the abatacept group than in the placebo group. The slightly lower (nonsignificant) mean total insulin dose among the abatacept group reported at 2 years was the same as the placebo group by 3 years. CONCLUSIONS Costimulation modulation with abatacept slowed decline of ß-cell function and improved HbA1c in recent-onset T1D. The beneficial effect was sustained for at least 1 year after cessation of abatacept infusions or 3 years from T1D diagnosis.

Effect of abatacept on immunogenicity of vaccines in individuals with type 1 diabetes.

Abatacept delayed progression of type 1 diabetes (T1D) when administered soon after diagnosis. Its use in T1D is expanding to prevention trials and, therefore, it is important to fully characterize its immunosuppressive effect. We compared antibody responses to trivalent inactivated influenza vaccine (TIIV) administered during 2 consecutive seasons and to tetanus toxoid (TT) vaccine administered after 24 months of treatment in115 early onset T1D subjects randomly assigned to 24 months of abatacept (N=71) or placebo (N=34). Anti-influenza titers before TIIV were similar between the 2 treatment groups and both groups had significant increases after vaccination. Although the magnitude of antibody responses against some influenza serotypes was significantly lower (p<0.05) in abatacept compared with placebo recipients, no differences were observed in the proportion of subjects with protective titers against influenza after vaccination. The magnitude of antibody responses against TT also tended to be lower (p=0.06) in abatacept compared with placebo recipients, without affecting the proportion of subjects who achieved protective titers. We conclude that abatacept moderately decreases the magnitude of antibody responses to recall vaccination. Further studies are needed to assess its effect on primary immunization.

Preclinical serum 25-hydroxyvitamin D levels and risk of type 1 diabetes in a cohort of US military personnel.

To determine whether serum levels of 25-hydroxyvitamin D (25(OH)D) in young adults are associated with risk of type 1 diabetes mellitus (T1D), we conducted a prospective, nested case-control study among US active-duty military personnel with serum in the US Department of Defense Serum Repository, identifying 310 T1D cases diagnosed between 1997 and 2009 with at least 2 serum samples collected before disease onset and 613 controls matched to cases on age, sex, race/ethnicity, branch of military service, and dates of serum collection. Conditional logistic regression was used to estimate rate ratios and 95% confidence intervals. Among non-Hispanic whites, those with average 25(OH)D levels of ≥ 100 nmol/L had a 44% lower risk of developing T1D than those with average 25(OH)D levels < 75 nmol/L (rate ratio = 0.56, 95% confidence interval: 0.35, 0.90, P for trend = 0.03) over an average follow-up of 5.4 years. In quintile analyses, T1D risk was highest among individuals whose 25(OH)D levels were in the lowest 20% of those measured. There was no association between 25(OH)D levels and risk of T1D among non-Hispanic blacks or Hispanics. Low 25(OH)D levels may predispose healthy, young, non-Hispanic white adults to the development of T1D.

A longitudinal study of GAD65 and ICA512 autoantibodies during the progression to type 1 diabetes in Diabetes Prevention Trial-Type 1 (DPT-1) participants.

OBJECTIVE: We examined changes in GAD65 and ICA-512 autoantibodies (GADA and IA-2A) during progression to type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS: Diabetes Prevention Trial-Type 1 (DPT-1) participants were assessed for changes in positivity and titers of GADA and IA-2A during the progression to T1D. RESULTS: Among 99 progressors to T1D with GADA and IA-2A measurements at baseline and diagnosis (mean interval = 3.3 ± 1.5 years), GADA positivity changed little and GADA titers decreased (P < 0.01). In contrast, both IA-2A positivity and titers increased substantially (P < 0.001). Even among those positive at baseline, IA-2A titers increased from baseline to diagnosis (n = 57; P < 0.001), whereas GADA titers decreased (n = 80; P < 0.01). The same patterns of change were also evident among those positive for both autoantibodies (n = 48) at baseline. CONCLUSIONS: IA-2A titers increase during the years before the diagnosis of T1D, even among those positive for IA-2A. In contrast, GADA titers tend to decline during those years.

Co-stimulation modulation with abatacept in patients with recent-onset type 1 diabetes: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: The immunopathogenesis of type 1 diabetes mellitus is associated with T-cell autoimmunity. To be fully active, immune T cells need a co-stimulatory signal in addition to the main antigen-driven signal. Abatacept modulates co-stimulation and prevents full T-cell activation. We evaluated the effect of abatacept in recent-onset type 1 diabetes. METHODS: In this multicentre, double-blind, randomised controlled trial, patients aged 6-45 years recently diagnosed with type 1 diabetes were randomly assigned (2:1) to receive abatacept (10 mg/kg, maximum 1000 mg per dose) or placebo infusions intravenously on days 1, 14, 28, and monthly for a total of 27 infusions over 2 years. Computer-generated permuted block randomisation was used, with a block size of 3 and stratified by participating site. Neither patients nor research personnel were aware of treatment assignments. The primary outcome was baseline-adjusted geometric mean 2-h area-under-the-curve (AUC) serum C-peptide concentration after a mixed-meal tolerance test at 2 years' follow-up. Analysis was by intention to treat for all patients for whom data were available. This trial is registered at ClinicalTrials.gov, NCT00505375. FINDINGS: 112 patients were assigned to treatment groups (77 abatacept, 35 placebo). Adjusted C-peptide AUC was 59% (95% CI 6·1-112) higher at 2 years with abatacept (n=73, 0·378 nmol/L) than with placebo (n=30, 0·238 nmol/L; p=0·0029). The difference between groups was present throughout the trial, with an estimated 9·6 months' delay (95% CI 3·47-15·6) in C-peptide reduction with abatacept. There were few infusion-related adverse events (36 reactions occurred in 17 [22%] patients on abatacept and 11 reactions in six [17%] on placebo). There was no increase in infections (32 [42%] patients on abatacept vs 15 [43%] on placebo) or neutropenia (seven [9%] vs five [14%]). INTERPRETATION: Co-stimulation modulation with abatacept slowed reduction in ß-cell function over 2 years. The beneficial effect suggests that T-cell activation still occurs around the time of clinical diagnosis of type 1 diabetes. Yet, despite continued administration of abatacept over 24 months, the decrease in ß-cell function with abatacept was parallel to that with placebo after 6 months of treatment, causing us to speculate that T-cell activation lessens with time. Further observation will establish whether the beneficial effect continues after cessation of abatacept infusions. FUNDING: US National Institutes of Health.

Antigen-based therapy with glutamic acid decarboxylase (GAD) vaccine in patients with recent-onset type 1 diabetes: a randomised double-blind trial.

BACKGROUND: Glutamic acid decarboxylase (GAD) is a major target of the autoimmune response that occurs in type 1 diabetes mellitus. In animal models of autoimmunity, treatment with a target antigen can modulate aggressive autoimmunity. We aimed to assess whether immunisation with GAD formulated with aluminum hydroxide (GAD-alum) would preserve insulin production in recent-onset type 1 diabetes. METHODS: Patients aged 3-45 years who had been diagnosed with type 1 diabetes for less than 100 days were enrolled from 15 sites in the USA and Canada, and randomly assigned to receive one of three treatments: three injections of 20 µg GAD-alum, two injections of 20 µg GAD-alum and one of alum, or 3 injections of alum. Injections were given subcutaneously at baseline, 4 weeks later, and 8 weeks after the second injection. The randomisation sequence was computer generated at the TrialNet coordinating centre. Patients and study personnel were masked to treatment assignment. The primary outcome was the baseline-adjusted geometric mean area under the curve (AUC) of serum C-peptide during the first 2 h of a 4-h mixed meal tolerance test at 1 year. Secondary outcomes included changes in glycated haemoglobin A(1c) (HbA(1c)) and insulin dose, and safety. Analysis included all randomised patients with known measurements. This trial is registered with ClinicalTrials.gov, number NCT00529399. FINDINGS: 145 patients were enrolled and treated with GAD-alum (n=48), GAD-alum plus alum (n=49), or alum (n=48). At 1 year, the 2-h AUC of C-peptide, adjusted for age, sex, and baseline C-peptide value, was 0·412 nmol/L (95% CI 0·349-0·478) in the GAD-alum group, 0·382 nmol/L (0·322-0·446) in the GAD-alum plus alum group, and 0·413 nmol/L (0·351-0·477) in the alum group. The ratio of the population mean of the adjusted geometric mean 2-h AUC of C-peptide was 0·998 (95% CI 0·779-1·22; p=0·98) for GAD-alum versus alum, and 0·926 (0·720-1·13; p=0·50) for GAD-alum plus alum versus alum. HbA(1c), insulin use, and the occurrence and severity of adverse events did not differ between groups. INTERPRETATION: Antigen-based immunotherapy therapy with two or three doses of subcutaneous GAD-alum across 4-12 weeks does not alter the course of loss of insulin secretion during 1 year in patients with recently diagnosed type 1 diabetes. Although antigen-based therapy is a highly desirable treatment and is effective in animal models, translation to human autoimmune disease remains a challenge. FUNDING: US National Institutes of Health.

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.

Prevention of Type 1 Diabetes Mellitus using a Novel Vaccine.

Type 1 diabetes mellitus (T1DM) affects 1 in 300 people and the incidence of the disease is rising worldwide. T1DM is caused by chronic autoimmune destruction of the insulin-producing ß-cells. The exact etiology and primary auto-antigen are not yet known. The autoimmune, chronic, and progressive nature of the disease raises the possibility of intervention, preferably by slowing down or stopping the destruction of the ß-cells as early as the prediabetic stage. Since the 1980s, several attempts have been made to maintain ß-cell function using immunosuppressive agents, immune modulation such as plasmapheresis, cytokine therapy, or antibody treatment. These agents were not diabetes specific; the occasionally observed beneficial effect did not compensate for the often very severe side effects. According to the latest assumption, the administration of diabetes-specific auto-antigens can elicit tolerance, which can prevent the destruction of the ß-cells, hopefully without serious side effects. The authors summarize current understanding of the immunology of T1DM, review the trials on prevention, and discuss their vaccination study.

TGF-beta induces IL-9 production from human Th17 cells.

The secretion of IL-9, initially recognized as a Th2 cytokine, was recently attributed to a novel CD4 T cell subset termed Th9 in the murine system. However, IL-9 can also be secreted by mouse Th17 cells and may mediate aspects of the proinflammatory activities of Th17 cells. Here we report that IL-9 is secreted by human naive CD4 T cells in response to differentiation by Th9 (TGF-beta and IL-4) or Th17 polarizing conditions. Yet, these differentiated naive cells did not coexpress IL-17 and IL-9, unless they were repeatedly stimulated under Th17 differentiation-inducing conditions. In contrast to the naive cells, memory CD4 T cells were induced to secrete IL-9 by simply providing TGF-beta during stimulation, as neither IL-4 nor proinflammatory cytokines were required. Furthermore, the addition of TGF-beta to the Th17-inducing cytokines (IL-1beta, IL-6, IL-21, IL-23) that induce memory cells to secrete IL-17, resulted in the marked coexpression of IL-9 in IL-17 producing memory cells. The proinflammatory cytokine mediating TGF-beta-dependent coexpression of IL-9 and IL-17 was identified to be IL-1beta. Moreover, circulating monocytes were potent costimulators of IL-9 production by Th17 cells via their capacity to secrete IL-1beta. Finally, to determine whether IL-9/IL-17 coproducing CD4 cells were altered in an inflammatory condition, we examined patients with autoimmune diabetes and demonstrated that these subjects exhibit a higher frequency of memory CD4 cells with the capacity to transition into IL-9(+)IL-17(+) cells. These data demonstrate the presence of IL-17(+)IL-9(+) CD4 cells induced by IL-1beta that may play a role in human autoimmune disease.

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).

Pancreatic islet autoantibodies as predictors of type 1 diabetes in the Diabetes Prevention Trial-Type 1.

OBJECTIVE: There is limited information from large-scale prospective studies regarding the prediction of type 1 diabetes by specific types of pancreatic islet autoantibodies, either alone or in combination. Thus, we studied the extent to which specific autoantibodies are predictive of type 1 diabetes. RESEARCH DESIGN AND METHODS: Two cohorts were derived from the first screening for islet cell autoantibodies (ICAs) in the Diabetes Prevention Trial-Type 1 (DPT-1). Autoantibodies to GAD 65 (GAD65), insulinoma-associated antigen-2 (ICA512), and insulin (micro-IAA [mIAA]) were also measured. Participants were followed for the occurrence of type 1 diabetes. One cohort (Questionnaire) included those who did not enter the DPT-1 trials, but responded to questionnaires (n = 28,507, 2.4% ICA(+)). The other cohort (Trials) included DPT-1 participants (n = 528, 83.3% ICA(+)). RESULTS: In both cohorts autoantibody number was highly predictive of type 1 diabetes (P < 0.001). The Questionnaire cohort was used to assess prediction according to the type of autoantibody. As single autoantibodies, ICA (3.9%), GAD65 (4.4%), and ICA512 (4.6%) were similarly predictive of type 1 diabetes in proportional hazards models (P < 0.001 for all). However, no subjects with mIAA as single autoantibodies developed type 1 diabetes. As second autoantibodies, all except mIAA added significantly (P < 0.001) to the prediction of type 1 diabetes. Within the positive range, GAD65 and ICA autoantibody titers were predictive of type 1 diabetes. CONCLUSIONS: The data indicate that the number of autoantibodies is predictive of type 1 diabetes. However, mIAA is less predictive of type 1 diabetes than other autoantibodies. Autoantibody number, type of autoantibody, and autoantibody titer must be carefully considered in planning prevention trials for type 1 diabetes.

Monocytes from patients with type 1 diabetes spontaneously secrete proinflammatory cytokines inducing Th17 cells.

Autoimmune diseases including type 1 diabetes (T1D) are thought to have a Th1/Th17 bias. The underlying mechanisms driving the activation and differentiation of these proinflammatory T cells are unknown. We examined the monocytes isolated directly from the blood of T1D patients and found they spontaneously secreted the proinflammatory cytokines IL-1beta and IL-6, which are known to induce and expand Th17 cells. Moreover, these in vivo-activated monocytes from T1D subjects induced more IL-17-secreting cells from memory T cells compared with monocytes from healthy control subjects. The induction of IL-17-secreting T cells by monocytes from T1D subjects was reduced in vitro with a combination of an IL-6-blocking Ab and IL-1R antagonist. In this study, we report a significant although modest increase in the frequency of IL-17-secreting cells in lymphocytes from long-term patients with T1D compared with healthy controls. These data suggest that the innate immune system in T1D may drive the adaptive immune system by expanding the Th17 population of effector T cells.

Concurrent detection of secreted products from human lymphocytes by microengraving: cytokines and antigen-reactive antibodies.

Cell surface determinants, cytokines and antibodies secreted by hematopoietic cells are used to classify their lineage and function. Currently available techniques are unable to elucidate multiple secreted proteins while also assigning phenotypic surface-displayed markers to the individual living cells. Here, a soft lithographic method, microengraving, was adapted for the multiplexed interrogation of populations of individual human peripheral blood mononuclear cells for secreted cytokines (IFN-gamma and IL-6), antigen-specific antibodies, and lineage-specific surface-expressed markers. Application of the method to a clinical sample from a recent-onset Type 1 diabetic subject with a positive titer of anti-insulin antibodies showed that approximately 0.58% of circulating CD19(+) B cells secreted proinsulin-reactive antibodies of the IgG isotype and 2-3% of circulating cells secreted IL-6. These data demonstrate the utility of microengraving for interrogating multiple phenotypes of single human cells concurrently and for detecting rare populations of cells by their secreted products.

Pathology of an islet transplant 2 years after transplantation: evidence for a nonimmunological loss.

BACKGROUND: We report the immunological and pathological findings of a 52-year-old woman, who died two years after the second of two islet transplants performed using the Edmonton protocol. After each islet transplant, she gradually lost insulin independence while maintaining low levels of C-peptide secretion. METHODS: A complete autopsy was performed including pathological and immunohistochemical analysis of hepatic allogeneic islets and native pancreatic islets to identify rejection or autoimmunity. Elispots assays for allogeneic sensitization and autoantibody assays for autoimmunity were performed antemortem after her islet transplantations to test in vitro for evidence of allogeneic sensitization or autoimmunity. RESULTS: The cause of death was a hypertensive stroke. Small numbers of islets without inflammation were identified within portal venules and stained with insulin. The atrophic pancreas contained small numbers of islets, which stained for insulin, and lacked any inflammation within or adjacent to the islets. In vitro assays for alloantibodies were negative, and Elispots assays failed to identify allogeneic sensitization. In vitro assays for diabetic associated autoantibodies did not identify autoimmune resensitization. The allografted kidney showed only early changes of recurrent diabetic nephropathy, and no evidence of rejection. CONCLUSIONS: In summary, no evidence was found to support an immunological basis (either allo or autoimmunity) for the slow loss of intrahepatic islets, which may, therefore, be related to nonimmunological anatomic and physiological abnormalities of islets infused into the portal veins or to drug toxicity.

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.

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.

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

CONTEXT: 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.