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+ (auto-antibody-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. Results. In 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 insulintreated Type 1 diabetic patients, naive and recently primed CD4 + cells were characterised by increasd IFNγ, TNFα, and IL-5. Conclusions/interpretation. Our 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 HLAmatched healthy individuals. This could prove useful in monitoring of T-cell immunity in clinical diabetes intervention trials. [Diabetologia (2004) 47:439-450]

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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 (IFNgamma) and anti-inflammatory Th2 (IL-4) cytokines were analyzed. We observed a simultaneous increase in IL-4 and IFNgamma 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 IFNgamma 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.