DRB4*01:01 Has a Distinct Motif and Presents a Proinsulin Epitope That Is Recognized in Subjects with Type 1 Diabetes.

DRB4*01:01 (DRB4) is a secondary HLA-DR product that is part of the high-risk DR4/DQ8 haplotype that is associated with type 1 diabetes (T1D). DRB4 shares considerable homology with HLA-DR4 alleles that predispose to autoimmunity, including DRB1*04:01 and DRB1*04:04. However, the DRB4 protein sequence includes distinct residues that would be expected to alter the characteristics of its binding pockets.

Avidity-dependent programming of autoreactive T cells in T1D.

Fate determination for autoreactive T cells relies on a series of avidity-dependent interactions during T cell selection, represented by two general types of signals, one based on antigen expression and density during T cell development, and one based on genes that interpret the avidity of TCR interaction to guide developmental outcome. We used proinsulin-specific HLA class II tetramers to purify and determine transcriptional signatures for autoreactive T cells under differential selection in type 1 diabetes (T1D), in which insulin (INS) genotypes consist of protective and susceptible alleles that regulate the level of proinsulin expression in the thymus. Upregulation of steroid nuclear receptor family 4A (NR4A) and early growth response family genes in proinsulin-specific T cells was observed in individuals with susceptible INS-VNTR genotypes, suggesting a mechanism for avidity-dependent fate determination of the T cell repertoire in T1D.

Comparison of cryopreservation methods on T-cell responses to islet and control antigens from type 1 diabetic patients and controls.

Background: Type 1 diabetes (T1D) is a cell-mediated autoimmune disease characterized by destruction of the pancreatic islet cells. The use of cryopreserved cells is preferable to the use of freshly isolated cells to monitor clinical trials to decrease assay and laboratory variability.

Methods: The T-Cell Workshop Committee of the Immunology of Diabetes Society compared two widely accepted T-cell freezing protocols (warm and cold) to freshly isolated peripheral blood mononuclear cells from patients with T1D and controls in terms of recovery, viability, cell subset composition, and performance in functional assays currently in use in T1D-related research.

Immunology of Diabetes Society T-Cell Workshop: HLA class II tetramer-directed epitope validation initiative.

Background: Islet-antigen-specific CD4+ T cells are known to promote auto-immune destruction in T1D. Measuring T-cell number and function provides an important biomarker. In response to this need, we evaluated responses to proinsulin and GAD epitopes in a multicentre study.

Immunology of Diabetes Society T-Cell Workshop: HLA class I tetramer-directed epitope validation initiative T-Cell Workshop Report-HLA Class I Tetramer Validation Initiative.

Background: Identification of T-cell reactivity to β-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D).

Methods: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (<2 years) diagnosed HLA-A*02:01+ T1D patients using blind-coded HLA-A2 tetramers (TMrs) and pentamers (PMrs), encompassing two epitopes of preproinsulin (PPI; PPIA12-20 and PPIB10-18) and two epitopes of glutamic acid decarboxylase (GAD; GAD114-122 and GAD536-545).

Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society.

Autoimmune T cell responses directed against insulin-producing β cells are central to the pathogenesis of type 1 diabetes (T1D). Detection of such responses is therefore critical to provide novel biomarkers for T1D 'immune staging' and to understand the mechanisms underlying the disease. While different T cell assays are being developed for these purposes, it is important to optimize and standardize methods for processing human blood samples for these assays.

Current approaches to measuring human islet-antigen specific T cell function in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease caused by the T cell-mediated destruction of the pancreatic insulin-producing beta cells. Currently there are no widely accepted and standardized assays available to analyse the function of autoreactive T cells involved in T1D. The development of such an assay would greatly aid efforts to understand the pathogenesis of T1D and is also urgently required to guide the development of antigen-based therapies intended to prevent, or cure, T1D.

Human risk allele HLA-DRB1*0405 predisposes class II transgenic Ab0 NOD mice to autoimmune pancreatitis.

Background & Aims: Autoimmune pancreatitis (AIP) underlies 5%-11% of cases of chronic pancreatitis. An association between AIP and the human leukocyte antigen (HLA)-DRB1*0405/DQB1*0401 haplotype has been reported, but linkage disequilibrium has precluded the identification of predisposing HLA gene(s). We studied the role of single HLA genes in the development of AIP in transgenic mice.

Insulin gene VNTR genotype associates with frequency and phenotype of the autoimmune response to proinsulin.

Immune responses to autoantigens are in part controlled by deletion of autoreactive cells through genetically regulated selection mechanisms. We have directly analyzed peripheral CD4+ proinsulin (PI) 76-90 (SLQPLALEGSLQKRG)-specific T cells using soluble fluorescent major histocompatibility complex class II tetramers. Subjects with type I diabetes and healthy controls with high levels of peripheral proinsulin-specific T cells were characterized by the presence of a disease-susceptible polymorphism in the insulin variable number of tandem repeats (INS-VNTR) gene.

Visualizing antigen specific CD4+ T cells using MHC class II tetramers.

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry. This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor. While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.

The sentinel role of CD8 T cells in regulating CD4 T cell responses to proinsulin in beta-islet cell autoimmunity.

Circulating CD4 T cells specific for peptide epitopes of proinsulin and other autoantigens are markers of autoimmune beta cell destruction in type 1 diabetes, while the role of CD8 T cells is still largely unknown. Here we show that CD8 T cells of a diabetic patient--after rechallange with proinsulin peptides--secrete IFNgamma and granzyme B, markers of their effector capacity. On the other hand, CD8 T cells of the same patient in a "cross-talk" with proinsulin-specific CD4 T cells suppress their proliferation.

Class III alleles at the insulin VNTR polymorphism are associated with regulatory T-cell responses to proinsulin epitopes in HLA-DR4, DQ8 individuals.

A variable number of tandem repeats (VNTR) polymorphism upstream of the insulin promoter is strongly associated with type 1 diabetes. The short class I alleles are predisposing and the long class III alleles are protective. As a possible mechanism for this effect, we previously reported a two- to threefold higher insulin transcription from class III than from class I chromosomes in thymus where insulin is expressed at low levels, presumably for the purpose of self-tolerance.

Immunological mechanisms associated with long-term remission of human type 1 diabetes.

Background: Preservation of beta cell function is a central goal in type 1 diabetes (type 1 DM) immune intervention. The characterization of individuals with recovery from established type 1 DM should provide insight into regulatory mechanisms of beta cell autoimmunity.

Methods: We studied a patient with antibody-positive type 1 DM with complete recovery of beta cell function for an observation period of 60 months.

Islet-cell antigen-reactive T cells show different expansion rates and Th1/Th2 differentiation in type 1 diabetic patients and healthy controls.

The low frequency of islet-cell antigen-reactive T cells in type 1 diabetes makes their direct measurement difficult. Commonly used in vitro expansion could alter in vivo frequencies and Th1/Th2 differentiation states. Using IFN-gamma/IL-4 double color ELISPOT, we tested longitudinally the reactivity of PBMC from HLA-matched diabetic patients and healthy controls to GAD65, IA-2, and proinsulin peptides ex vivo and after in vitro culture.

Experimental autoimmune diabetes: a new tool to study mechanisms and consequences of insulin-specific autoimmunity.

(Prepro)insulin is considered a central antigenic determinant in diabetic autoimmunity. Insulin has been used to modify diabetes development in NOD mice and prediabetic individuals. We have recently shown that (prepro)insulin can adversely promote diabetes development in murine type 1 diabetes.

Preproinsulin-specific CD8+ T cells secrete IFNgamma in human type 1 diabetes.

In animal models autoreactive CD8(+) T cells are crucial in the development of type 1 diabetes (T1D); however, their role in human T1D is still not known. To address the role of CD81 T cells we performed a pilot study by investigating CD8(+) T cell-mediated cytokine secretion after in vitro stimulation with 94 preproinsulin (PPI) peptides. We were able to show that CD8(+) T cells contribute to a strong IFNgamma reactivity against PPI in human T1D.

Increased in vivo frequency of IA-2 peptide-reactive IFNgamma+/IL-4- T cells in type 1 diabetic subjects.

Active T cell recognition of islet antigens has been postulated as the pathogenic mechanism in human type 1 diabetes, but evidence is scarce. If T cells are engaged, they are expected to display increased clonal size and exhibit a T helper (Th)1/Th2 differentiation state. We used a peptide library that covers tyrosine phosphatase IA-2, a target antigen expressed in pancreatic beta cells, to probe 8 diabetic patients and 5 HLA-matched controls.

T cells recognize multiple GAD65 and proinsulin epitopes in human type 1 diabetes, suggesting determinant spreading.

Human type 1 diabetes is thought to be mediated by autoreactive T cells specific for antigens expressed by pancreatic beta cells. However, it is unclear which autoantigens and determinants thereof are the targets of the autoimmune attack. Using comprehensive peptide libraries that cover the entire sequence of two major candidate autoantigens, GAD65 and proinsulin, we measured the in vivo frequencies of peptide-specific, IFN-gamma-producing memory T cells in 27 diabetic patients, 14 high risk individuals, and 15 partially HLA-matched healthy controls.

CD28 costimulation enhances the sensitivity of the ELISPOT assay for detection of antigen-specific memory effector CD4 and CD8 cell populations in human diseases.

The frequencies of antigen-specific memory cells are often low in chronic disease states related to infection and autoimmunity, making detection of such populations difficult, even with high sensitivity assays such as the cytokine enzyme-linked immunospot (ELISPOT). The spectrum and function of antigen presenting cells (APC) in the peripheral compartment can differ considerably from the inflamed target organ. In order to approximate the costimulatory environment of the target organ, we measured T cell responses with and without the addition of agonistic anti-CD28 antibody in the ELISPOT assay.

Pro- and anti-inflammatory cytokine production by autoimmune T cells against preproinsulin in HLA-DRB1*04, DQ8 Type 1 diabetes.

Aims/hypothesis: Preproinsulin is a target T cell autoantigen in human Type 1 diabetes. This study analyses the phenotype and epitope recognition of preproinsulin reactive T cells in subjects with a high genetic risk of diabetes [HLA-DRB1*04, DQ8 with Ab+ (autoantibody-positive) or without islet autoantibodies (control subjects)], and in HLA-matched diabetic patients.

Methods: A preproinsulin peptide library approach was used to screen for cytokine profiles and epitope specificities in human peripheral blood lymphocytes, and CD4(+)CD45RA(-) and CD4(+)CD45RA(+) T cell subfractions, representing memory and naive and recently primed T cells respectively.

Relationship between T and B cell responses to proinsulin in human type 1 diabetes.

In type 1 diabetes, humoral and cell-mediated responses to insulin and proinsulin are detectable. Autoantibodies to insulin are associated with impending disease in young individuals and are used as predictive markers to determine disease risk. The aim of this study was to investigate whether different cytokine patterns of cellular reactivity to insulin might serve as additional specific markers of disease maturation and might improve disease prediction in individuals at risk.

Th2 dominance of T helper cell response to preproinsulin in individuals with preclinical type 1 diabetes.

In human type 1 diabetes (T1D) autoantibodies to insulin precede clinical disease, while little is known about the contribution of insulin-specific T lymphocytes-in particular, T helper (Th) subsets. Here we have studied the in vivo primed cytokine response to preproinsulin in peripheral blood mononuclear cells (PBMCs) and two major Th cell subsets-CD45RO+ memory cells and CD45RA+ naive/resting cells-in 35 individuals with HLA-DRB1*04, DQB1*0302 diabetes risk marker: 12 patients with T1D, 12 autoantibody-positive (Ab+) individuals, and 11 healthy controls. Cytokine secretion (TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, and IL-10) was measured in the supernatants of the cultures stimulated with 21 overlapping preproinsulin peptides as well as proinsulin and insulin.

Predominantly recognized proinsulin T helper cell epitopes in individuals with and without islet cell autoimmunity.

Antibody response to insulin and to its precursor ProInsulin is associated with increased risk for type 1 diabetes (T1D), though little is known about T cell reactivity to this molecule. In the present study from peripheral blood mononuclear cells (PBMC), in vivo primed CD45RO+ memory T helper (Th) cells were enriched and their reactivity to eight overlapping ProInsulin peptides and to protein was analyzed. Individuals with high risk HLA-DRB1*04, DQB1*0302 alleles were investigated: relatives of patients with T1D having humoral markers of islet cell autoimmunity (autoantibody positive, Ab+; n=11), patients with T1D (n=8), and healthy control individuals (n=16).