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Type 1 diabetes results from autoimmune destruction of insulin producing pancreatic β-cells. We have shown that treatment with alum-formulated glutamic acid decarboxylase 65 (GAD-alum) preserved residual insulin secretion and induced antigen-specific responses in children with recent onset type 1 diabetes. The aim of this study was to further investigate the immunomodulatory effect of GAD-alum, focusing on CD4(+)CD25(high) cells and their association to cytokine secretion. Samples obtained 21 and 30months after the initial injection of GAD-alum or placebo were included in the present study. GAD(65)-stimulation enhanced the percentage of CD4(+)CD25(high)FOXP3(+) cells, but reduced the percentage of CD4(+)CD25(+) cells, in samples from the GAD-alum treated group. Further, the GAD(65)-induced secretion of IL-5, -10, and -13 correlated with the expression of CD4(+)CD25(high)FOXP3(+) cells, but inversely with CD4(+)CD25(+) cells. These new data suggest that GAD-alum treatment induced GAD(65)-specific T cells with regulatory features., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Regulatory T (Treg) cells are important tools with the purpose to control and regulate the immune system. These cells use FOXP3, TGF-beta, CTLA-4 and sCTLA-4 to regulate other T-cells. Since cryopreservation of peripheral blood mononuclear cells (PBMC) is a convenient way to handle samples, we investigated whether these cells will change their mRNA expression of Treg associated markers, as well as secretion of TGF-beta1, after cryopreservation. Additionally, we aimed to investigate the mRNA expression after two different time intervals of in vitro antigen stimulation. PBMC from healthy adults were stimulated either fresh (48/96 h) or after cryopreservation (48 h), with PHA, tetanus toxoid, beta-lactoglobulin, ovalbumin or in culture medium exclusively (spontaneous). The mRNA expression of FOXP3, TGF-beta, CTLA-4 and sCTLA-4 were studied with multiplex real-time RT-PCR and TGF-beta1 with ELISA. Cryopreserved PBMC were appropriate for detection of the Treg associated markers FOXP3, TGF-beta, CTLA-4 and sCTLA-4 expressed spontaneously. Also antigen-induced mRNA expression of CTLA-4, sCTLA-4 and TGF-beta and secreted TGF-beta1, with the exception of FOXP3, preserved a stable transcription activity after cryopreservation. Further, a stimulation period of 48 h in general revealed the highest mRNA expression of the markers studied. In conclusion, cryopreserved cells are in general suitable for studying Treg associated markers.

Background: Type 1 diabetes (T1D) is an autoimmune disease dominated by loss of self-tolerance resulting in depletion of the beta-cells. This study aims to confirm previous observations of a dominant T-helper (Th)1-like profile during the period close to onset of disease. Further, to follow the immune response from onset to 2 years duration, the study focused on spontaneous as well as autoantigen-induced immune profile., Methods: Peripheral blood mononuclear cells were collected 4 days and 1 and 2 years after diagnosis of T1D children, from healthy children carrying the human leukocyte antigen-risk genes and from high-risk children (ICA > or = 20 IJDF units). Peripheral blood mononuclear cells were stimulated with glutamic acid decarboxylase (GAD(65)) and phytohaemagglutinin (PHA). Cytokines and chemokines were detected in cell-culture supernatants by protein microarray (naive T-cells; interleukin (IL)-7, Th1; interferon-gamma, tumour necrosis factor-beta, Th2; IL-5, Th3; transforming growth factor-beta, T-regulatory cell type 1; IL-10 and inflammatory cytokines; tumour necrosis factor-alpha, IL-6 and chemokines; monocyte chemoattractant protein-1, monokine upregulated by IFN-gamma) in relation to clinical outcome (C-peptide)., Results: High-risk children showed a dominant Th1-associated profile with high spontaneous and GAD(65)-induced secretion. The mitogen PHA instead induced a Th2-associated response exclusively in high-risk children. In contrast, newly diagnosed T1D children showed a pronounced Th3-associated cytokine profile as well as a burst of inflammatory cytokines and chemokines secreted both spontaneously and by GAD(65) and PHA stimulation. The immune response to GAD(65) and PHA, however, diminished with duration of disease., Conclusion: A dominant Th1-associated immune profile was observed during the pre-diabetic phase. This Th1 dominance, however, diminished in favour of a temporary increase in a Th3-associated and inflammatory immune profile at the onset of disease.

Imbalance between different types of T lymphocytes, such as T helper (Th) and regulatory T cells (Tregs), has been reported to play a part in the pathogenesis behind such autoimmune diseases as type 1 diabetes (T1D) and celiac disease (CD). Defects in Tregs are proposed to at least partly explain the imbalance of Th cells found in children with immunologic diseases. Peripheral blood mononuclear cells from 24 children with T1D and/or CD, and reference children (that is, those without any of these diseases) were stimulated with disease-associated antigens (insulin, gluten, transglutaminase [tTG]), and phytohemagglutinin (PHA). The mRNA expression of the Treg-associated marker FOXP3 was analyzed with multiplex real-time RT-PCR. Children with T1D showed both a low spontaneous (P < 0.05) and PHA-induced (P < 0.01) expression of FOXP3 mRNA compared to children with CD. Children with T1D also had a low PHA-induced FOXP3 mRNA expression compared to the group of children diagnosed with both T1D and CD (P < 0.05). Spontaneous (P < 0.05) and PHA-induced (P < 0.05) FOXP3 mRNA expression was high in children with CD compared to reference children. In contrast, stimulation with insulin tended to induce high FOXP3 mRNA expression in T1D children compared to reference children (P= 0.057). In conclusion, children with only T1D generally showed a lower FOXP3 mRNA expression than did children with CD, or with T1D in combination with CD, which suggests impaired regulation of the immune system in children with T1D.

Cryopreserved peripheral blood mononuclear cells (PBMC) are commonly used when assessing immune responses in clinical trials, both for practical reasons and to minimize interassay variation, as samples are often collected and studied over time. This study investigated the effect of cryopreservation on cytokine and chemokine secretion, and on expression of regulatory T-cell associated markers, in samples from children with type 1 diabetes. PBMC were cultured before and after cryopreservation either with GAD(65) or PHA. Secretion of cytokines (IL-5, -6, -10, -12, -13 -17, IFN-gamma and TNF-alpha) and chemokines (IP-10, MCP-1, MIP-1alpha, MIP-1beta and RANTES) was analysed in cell supernatants using multiplex fluorochrome technique (Luminex). Expression of FOXP3 and TGF-beta mRNA was detected by multiplex real-time RT-PCR. Increased spontaneous secretion of IL-6, -10, -12, -13, IFN-gamma and MCP-1, and mRNA expression of FOXP3 and TGF-beta, was detected after cryopreservation. Stimulation with GAD(65) induced higher levels of IL-6, IFN-gamma, TNF-alpha and MIP-1alpha, whereas lower secretion was found for IL-10 and IL-13 in cryopreserved PBMC. Stimulation with PHA induced lower secretion of IP-10, MCP-1 and RANTES and FOXP3 mRNA expression after cryopreservation. Thus, cryopreserved PBMC were suitable to assess the immunological markers included in this study, even though their expression could differ from freshly handled cells.

Background: Type 1 diabetes (T1D) is suggested to be of T-helper (Th)1-like origin. However, recent reports indicate a diminished interferon (IFN)-gamma secretion at the onset of the disease. We hypothesize that there is a discrepancy in subsets of Th-cells between children with a high risk of developing T1D, children newly diagnosed with T1D and healthy children., Methods: Peripheral blood mononuclear cells (PBMC) were collected from children at high risk for T1D (islet cells antibodies [ICA] >/= 20 IJDF-U), those newly diagnosed and healthy children carrying the HLA-risk gene DQB1*0302 or DQB1*0201 and DQA1*0501. Th1- (IFN-gamma, tumour necrosis factor [TNF]-beta, interleukin [IL]-2), Th2- (IL-4,-5,-13), Th3- (transforming growth factor [TGF-beta], IL-10) and inflammatory associated cytokines (TNF-alpha, IL-1alpha,-6) and chemokines (monocyte chemoattractant protein [MCP]-1,-2,-3, Monokine unregulated by IFN-gamma [MIG], Regulated on Activation, Normal T-cell Expressed and Secreted [RANTES], IL-7,-8,-15) were detected in cell-culture supernatants of PBMC, stimulated with glutamic acid decarboxylase 65 (GAD(65)) and phytohaemagglutinin (PHA), by protein micro array and enzyme linked immunospot (ELISPOT) technique., Results: The Th1 cytokines IFN-gamma and TNF-beta, secreted both spontaneously and by GAD(65)- and mitogen stimulation, were seen to a higher extent in high-risk children than in children newly diagnosed with T1D. In contrast, TNF-alpha and IL-6, classified as inflammatory cytokines, the chemokines RANTES, MCP-1 and IL-7 as well as the Th3 cytokines TGF-beta and IL-10 were elevated in T1D children compared to high-risk children., Conclusion: High Th-1 cytokines were observed in children with high risk of developing TID, whereas in children newly diagnosed with T1D Th3 cytokines, inflammatory cytokines and chemokines were increased. Thus, an inverse relation between Th1-like cells and markers of inflammation was shown between children with high risk and those newly diagnosed with T1D.

Background: Type 1 diabetes (T1D) is an autoimmune disease associated with the destruction of pancreatic beta cells and genetically linked to human leukocyte antigen (HLA) class II DR3-DQ2 and DR4-DQ8 haplotypes. The +49A/G polymorphism of the immunoregulatory cytotoxic T-lymphocyte antigen 4 (CTLA-4) gene is also associated with T1D. Genetic and environmental risk factors precede the onset of T1D, which is characterized by a T helper 1 cell-dominating cytokine response to diabetes-related autoantigens., Aim: To investigate immunological differences between healthy children with and without CTLA-4 +49A/G and HLA genetic susceptibility for T1D., Study Design: Young, 7-15 years of age, healthy subjects (n = 58) were investigated to test whether CTLA-4 +49A/G genotype was associated with enzyme-linked immunospot assay T-cell responses to T1D-related autoantigens. Because T1D is primarily HLA-DQ associated, we stratified the healthy subjects by HLA genotypes associated with the disease., Results: Peptide of heat shock protein 60 induced a higher interferon-gamma (IFN-gamma) response in subjects with risk-associated CTLA-4 polymorphism (GG genotype) (p = 0.02) while glutamic acid decarboxylase 65-induced interleukin-4 (IL-4) secretion was lower in GG genotype subjects (p = 0.02)., Conclusion: The increased IFN-gamma response and lower IL-4 response toward diabetes-related autoantigens shown in CTLA-4 +49 GG risk subjects show a possible mechanism for the association between CTLA-4 and T1D.

The exact role of T-helper (Th) cells that precede the clinical manifestation of type 1 diabetes remains unclear. The aim of this investigation was to study the Th1- and Th2-like profile in children and adults with high risk of developing the disease. Peripheral blood mononuclear cells were collected from high-risk children and adults and from healthy individuals matched for age and gender. Using the sensitive enzyme-linked immunospot (ELISPOT) technique to divide Th1- from Th2-like lymphocytes, secretion of interferon-gamma (IFN-gamma) and interleukin-4 was analysed from lymphocytes spontaneously and after in vitro stimulation with different antigens, based on present paradigms regarding the pathogenesis of type 1 diabetes. Compared to the response observed in healthy individuals, we found that individuals with a high risk of developing type 1 diabetes, especially children, responded with less IFN-gamma secretion to the three autoantigens glutamic acid decarboxylase 65 (GAD65), insulin and tyrosinphosphatase (IA-2). Thus, a diminished Th1-like response by in vitro autoantigen stimulation was observed in especially children with a high risk of developing type 1 diabetes. Reduced Th1/Th2 response was related to signs of beta cell exhaustion.

Type 1 diabetes is an autoimmune disease with an inflammatory process directed against the beta cells in pancreas. This investigation aimed at studying the immune response during the first 3 months after the diagnosis of type 1 diabetes, with focus on the balance of T-helper 1 (Th1)- and Th2-like cytokines, produced spontaneously and in response to relevant autoantigens. Peripheral blood mononuclear cells (PBMCs) were collected from type 1 diabetic children (10-17 years) at 5, 20, 35 and 90 days after diagnosis. Expression of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) mRNA were detected by real-time reverse transcriptase polymerase chain reaction and IFN-gamma, IL-10 and IL-13 by enzyme-linked immunosorbent assay in cell supernatant after stimulation with a glutamic acid decarboxylase 65 (GAD(65))-peptide [amino acid (a.a.) 247-279], insulin, the ABBOS-peptide (a.a. 152-169), phytohaemagglutinin and keyhole limpet haemocyanin. Spontaneous and antigen-induced expression and secretion of cytokines were low at the diagnosis of type 1 diabetes. During the first month, after diagnosis, the GAD(65)-peptide caused an increased ratio of IFN-gamma/IL-4 mRNA expression (P < 0.05) and increased secretion of IFN-gamma (P = 0.07). Expression of IFN-gamma mRNA did also increase from stimulation with insulin (P < 0.05), even though cytokine secretion remained low. Thus, duration after diagnosis as well as metabolic state should be carefully considered both in studies of the pathogenesis of type 1 diabetes and in immune intervention studies at onset.

The identification of sensitive assay formats capable of distinguishing islet autoreactive T cells directly ex vivo in blood is a major goal in type 1 diabetes research. Recently, much interest has been shown in the cytokine enzyme linked immunospot assay (CK ELISpot), an assay potentially capable of fulfilling these difficult criteria. To address the utility of this assay in detecting autoreactive T cells, a 'wet' workshop was organized using the same fresh blood sample and coded antigens. Five different laboratories participated, using three distinct CK ELISpot assay formats. Samples from two subjects were pre-tested for responses to sub-optimal concentrations of tetanus toxoid, representing a low frequency recall response, and peptides from diabetes associated autoantigens GAD65, IA-2 and HSP60. All participants measured interferon-gamma production and combinations of interleukins-4, -5, -10 and -13. In the workshop 4 of 5 laboratories detected low frequency recall responses in both subjects and 3 of 5 detected at least one of the autoreactive peptide responses concordant with pre-testing. Significant assay format related differences in sensitivity and signal-to-noise ratio were observed. The results demonstrate the potential for detection of low-level autoreactive T cell responses and identify assay characteristics that will be useful for studies in type 1 diabetes.