GAD65- and proinsulin-specific CD4 C T-cells detected by MHC class II tetramers in peripheral blood of type 1 diabetes patients and at-risk subjects Viveka O ¨ ling a, * , Jane Marttila a , Jorma Ilonen a , William W. Kwok b , Gerald Nepom b , Mikael Knip c,d , Olli Simell e , Helena Reijonen b a Department of Virology, University of Turku, Medicity, Biocity 4 krs., Tykisto ¨katu 6 A, 20520 Turku, Finland b Benaroya Research Institute, Seattle, WA, USA c Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland d Department of Pediatrics, Tampere University Hospital, Tampere, Finland e Department of Pediatrics, University of Turku, Turku, Finland Received 10 June 2005; revised 10 June 2005; accepted 7 September 2005 Abstract In type 1 diabetes the major loss of insulin producing beta-cells is caused by autoreactive T-cells specific for antigens expressed by the pan- creatic islets. In this study we have analyzed the prevalence of glutamate decarboxylase 65 (GAD65)- and proinsulin-specific CD4 C T-cells in type 1 diabetes patients, at-risk subjects and in HLA-matched control children. Peripheral blood mononuclear cells were cultured in the presence of two different GAD65 peptides (555e567, 557I and 274e286) or with a proinsulin (B24eC36) peptide for 10e11 days. The autoreactive T- cells were detected using antigen specific-MHC class II tetramers by flow cytometry. Our results show that 11 of 18 (61%) type 1 diabetes pa- tients and 7 of the 20 (35%) at-risk subjects were positive for one of the three GAD65 or proinsulin-containing tetramers, whereas only 2 of 21 (9.5%) controls had tetramer binding cells ( p Z 0.0007 type 1 diabetes vs. controls and p Z 0.0488 at-risk subjects vs. controls, Chi-square test). Type 1 diabetes patients responded to all three peptides. At-risk subjects recognized also the GAD65 555e567 557I peptide, while none of the controls responded to it. In conclusion, type 1 diabetes patients and at-risk subjects have a significantly higher prevalence of GAD65- and proinsulin-specific CD4 C T-cells than the control subjects. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: GAD65; MHC class II tetramers; Proinsulin; Type 1 diabetes 1. Introduction Type 1 diabetes (T1D) is an autoimmune disease character- ized by destruction of the beta-cells of the pancreas. It can take years until most of the beta-cells have been damaged and in- sulin secretion becomes insufficient to sustain normoglycemia. Autoantibodies specific for insulin, glutamate decarboxylase 65 (GAD65) and protein-tyrosine phosphatase 2 (IA-2) appear often during the preclinical phase of the disease and have become a useful predictive marker for the progression of type 1 diabetes [1e3]. Less is known on the specificity of cel- lular responsiveness although the major loss of beta-cells is caused by autoreactive T-cells specific for antigens expressed in the pancreatic islets [4,5]. The development of T1D is also strongly associated with the major histocompatibility complex (MHC) class II genes. The genetic susceptibility in Caucasians correlates with the HLA-DR3-DQ201 and HLA-DR4-DQ302 haplotypes, where both HLA-DR and -DQ molecules affect the disease risk. Both these molecules may play an essential role in T1D through their presentation of islet cell specific- peptides to autoreactive CD4 C T-cells [6e8]. * Corresponding author. Tel.: C358 2 333 7010; fax: C358 2 333 7000. E-mail address: viveka.oling@utu.fi (V. O ¨ ling). 0896-8411/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jaut.2005.09.018 Journal of Autoimmunity 25 (2005) 235e243 www.elsevier.com/locate/issn/08968411