Ivana Durinovic-Belló

Cellular Immune Response to Diverse Islet Cell Antigens in IDDM

In IDDM, T-cells are postulated to mediate the destruction of pancreatic β-cells. We analyzed peripheral blood mononuclear cell (PBMC) responses to human insulin, glutamate decarboxylase GAD65, tyrosine phosphatase ICA512, glucagon, membrane preparations of RIN cells and human pancreas, and three control antigens (La = nuclear cell antigen, tetanus toxoid, and phytohemagglutinin). A total of 28 patients with newly diagnosed IDDM, 9 antibody-positive (Ab+) first-degree relatives, and 16 healthy control subjects were included. Increased proliferative responses to pancreatic islet cell antigens were observed in diabetic patients and in Ab+ relatives compared with control subjects, whereas T-cell reactivity to nonpancreatic control antigens was similar between the study groups. The highest differences in the magnitude of proliferative responses were seen for ICA512, followed by membrane preparations of RIN cells, GAD65, and human pancreas. Few subjects reacted with insulin or glucagon. Interestingly, Ab+ relatives showed higher T-cell reactivity with respect to stimulation indexes and prevalences than newly diagnosed diabetic patients, and as many as 89% of Ab+ relatives showed proliferation to more than one islet cell antigen preparation in comparison to 43% of newly diagnosed diabetic patients and none of the control subjects. Statistical analysis revealed significant positive correlation of insulin autoantibody levels with the levels of insulin-specific T-cells in Ab+ relatives, but no relation of PBMC responses to age, sex, or HLA-DR haplotypes. Our results demonstrate the simultaneous existence of various autoreactive T-cells specific for islet cell antigens in the prediabetic period. These T-cells may play a significant role in the pathogenesis of the disease.

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

Aims/hypothesisPreproinsulin 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.MethodsA 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.ResultsIn CD4+ T cell subsets we identified immunodominant epitopes and cytokine production patterns that differed profoundly between patients, Ab+ subjects and non-diabetic HLA-matched control subjects. In Ab+ subjects, a C-peptide epitope C13–29 and insulin B-chain epitope B11–27 were preferentially recognised, whereas insulin-treated Type 1 diabetic patients reacted to native insulin and B-chain epitope B1–16. In peripheral blood lymphocytes of Ab+ subjects, an increase in T helper (Th) 1 (IFNγ, IL-2) and Th2 (IL-4) cytokines was detectable, wheras in CD45RA+ and CD45RA− subsets, IL-4 and IL-10 phenotypes dominated, compatible with the contribution of non-CD4 cells to IFNγ content. In insulin-treated Type 1 diabetic patients, naive and recently primed CD4+ cells were characterised by increasd IFNγ, TNFα, and IL-5.Conclusions/interpretationOur data show that T cell reactivity to preproinsulin in CD45RA subsets is Th2-dominant in Ab+ subjects, challenging the Th1 paradigm in Type 1 diabetes. Characteristic immunodominant epitopes and cytokine patterns distinguish diabetic patients and Ab+ subjects from HLA-matched healthy individuals. This could prove useful in monitoring of T-cell immunity in clinical diabetes intervention trials.

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-γ-producing memory T cells in 27 diabetic patients, 14 high risk individuals, and 15 partially HLA-matched healthy controls. Compared to the controls, both a higher number of determinants on the islet cell antigens were recognized and the frequencies of peptide specific cells were increased in patients and high risk individuals. Inclusion of signal enhancing anti-CD28 antibody further accentuated this difference. Considerable heterogeneity in peptide recognition was seen even in DRB1*04, DQB1*0302 matched individuals. Unlike its peptides, the GAD protein antigen did not recall a T cell memory response. The highly heterogeneous recognition of a multitude of peptide determinants on both autoantigens, occurring in the absence of protein recognition, and the low functional avidity of the memory cells involved jointly suggest that the autoimmune T cell repertoire in human type 1 diabetes primarily targets cryptic determinants engaged by determinant spreading.

HLA-DQ-Restricted, Islet-Specific T-Cell Clones of a Type I Diabetic Patient: T-Cell Receptor Sequence Similarities to Insulitis-Inducing T-Cells of Nonobese Diabetic Mice

We established a T-cell line and 20 CD4+ T-cell clones from the peripheral blood lymphocytes of a type I diabetic patient using a membrane preparation of a rat insulinoma cell line (beta membrane antigen [BMA]) as a source of antigen. The T-cell line and three selected clones proliferated specifically to stimulation with BMA and to membranes prepared from human islets, rat pancreas, and NOD pancreas, but not to control antigens. Proliferationinhibition studies using human leukocyte antigen (HLA)-specific monoclonal antibodies revealed HLA-DQw1-restricted recognition of BMA. An analysis of the T-cell receptor (TCR) repertoire of the T-cell line after 8 and 40 days of culture showed a prominent usage of the Val and Vα12 gene families. Although sequencing of the TCR Vα and Vβ chains of the three clones demonstrated that each used different Va and V(J gene segments, structural similarities were found in complementary-determining region 3 (CDR3), the region that is postulated to interact with the peptide component of the TCR ligand. When we compared these TCR sequences with published sequences of disease-inducing T-cells of NOD mice, highly related TCR Vβ families were detected. Furthermore, stretches of identical amino acids within the CDR3 region were found between two pairs of human and mouse T-cells. If one considers the species differences in TCR genes and sequence differences in the restriction elements for these cells (HLA-DQ vs. H-2I-Anod), these sequence similarities are striking and may be useful for pinpointing T-cells of primary importance in the development of disease.

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

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.

Preproinsulin-Specific CD8+ T Cells Secrete IFNγ in Human Type 1 Diabetes

Abstract: 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 IFNγ reactivity against PPI in human T1D. Further investigations defining epitope specificity, cytokine secretion, and cytotoxic capacity are important to clarify their role in T1D development.

Th2 Dominance of T Helper Cell Response to Preproinsulin in Individuals with Preclinical Type 1 Diabetes

Abstract: 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‐α, IFN‐γ, 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. In Ab+ individuals our results reveal higher IL‐4 levels in CD45RO+ memory cells and higher IL‐5 levels in CD45RA+ naive/resting cells, while higher IL‐2 production was found in PBMCs. In contrast, in PBMCs of T1D patients higher IFN‐γ and IL‐10 secretion was found. Our data delineate characteristic cytokine patterns in peripheral T lymphocytes from patients at different stages of the T1D development.

Relationship between T and B Cell Responses to Proinsulin in Human Type 1 Diabetes

Abstract: 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. We correlated T and B cell responses to insulin in subjects with increased genetic risk (HLA‐DRB1*04, DQB1*0302) for diabetes with or without islet autoantibodies (Ab+ subjects and controls, respectively) and HLA‐matched patients. Peripheral blood mononuclear cells were stimulated with 15 overlapping proinsulin peptides (16‐mer), and proinflammatory Th1 (IFNγ) and anti‐inflammatory Th2 (IL‐4) cytokines were analyzed. We observed a simultaneous increase in IL‐4 and IFNγ secretion in early islet autoimmunity of Ab+ subjects, but not in insulin‐treated T1D patients. Furthermore, the increase in IL‐4 secretion in Ab+ subjects was associated with insulin autoantibody responses. There was no correlation of either IFNγ or IL‐4 secretion with insulin antibody responses in patients already treated with exogenous insulin. In conclusion, our findings reveal that quantification of cytokine responses to proinsulin in peripheral blood may prove to be a promising specific marker of diabetes progression and could, in addition to insulin autoantibodies, be used in the prediction of type 1 diabetes.

THE T-CELL RECEPTOR BETA CHAIN CDR3 REGION OF BV8S1/BJ1S5 TRANSCRIPTS IN TYPE 1 DIABETES

Abstract We recently described the T-cell receptor (TCR) β chain CDR3 motif S-SDRLG-NQPQH (BV8S1-BJ1S5) in an islet-specific T-cell clone (K2.12) from a type 1 diabetic patient (AS). A similar motif (RLGNQ) was also reported in a T-cell clone of non-obese diabetic (NOD) mice by others. In order to determine the frequency of our motif in selected and unselected T-cell populations, we cloned and sequenced the CDR3 region of BV8S1-BJ1S5 transcripts. These transcripts were derived from unstimulated peripheral blood T lymphocytes from two type 1 diabetic patients (AS and FS) and their non-diabetic sibling (WS), as well as from an islet-specific T-cell line of one of the patients. In addition, we compared the structure and composition of the CDR3 region in BV8S1-BJ1S5 transcripts from peripheral blood T cells between the patients and their non-diabetic sibling (>50 sequences each). We found that 30% of the islet-specific T-cell line cDNA clones expressed the entire sequence-motif, whereas it was absent in the clones of unstimulated peripheral blood T cells from both patients and their non-diabetic sibling. The average length of the CDR3 region was shorter in the patients (mean AS 9.9, FS 9.9, versus WS 10.7, p = 0.0037) and the number of inserted nucleotides in N nucleotide addition at the DJ-junction lower (mean AS 3.5, FS 3.2, versus WS 5.2, P = <10–4) as compared with their non-diabetic sibling. Moreover, the pattern of amino acid usage in the CDR3 region was dissimilar at positions 5 and 6, where polar amino acids predominated in both diabetic siblings. In contrast, basic amino acids are preferentially used at position 5 in the clones of the non-diabetic sibling. These data provide information on the general structure of the TCR(BV8S1-BJ1S5) CDR3 region in type 1 diabetes and may indicate differences in the amino and nucleic acid composition of the TCR β chain CDR3 region between two type 1 diabetic patients and their non-diabetic sibling.

Experimental Autoimmune Diabetes: A New Tool to Study Mechanisms and Consequences of Insulin-Specific Autoimmunity

Abstract: (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. Based on these findings we have developed experimental autoimmune diabetes (EAD), a new mouse model characterized by (1) CD4+/CD8+ insulitis, induced by (2) (prepro)insulin DNA vaccination, leading to (3) beta cell damage and insulin deficiency in (4) RIP‐B7.1 transgenic mice (H‐2b). EAD develops rapidly in 60‐95% of mice after intramuscular, but not intradermal (“gene gun”), vaccination; and DNA plasmids expressing insulin or the insulin analogues glargine, aspart, and lispro are equally potent to induce EAD. Similar to NOD mice, diabetes is adoptively transferred into syngeneic recipients by spleen cell transplantation in a dose‐dependent fashion. We have devised a two‐stage concept of EAD in which T cell activation and expansion is driven by in vivo autoantigen expression, followed by islet damage that requires beta cell expression of costimulatory B7.1 for disease manifestation. Taken together, EAD is a novel, genetically defined animal model of type 1 diabetes suitable to analyze mechanisms and consequences of insulin‐specific T cell autoimmunity.