provided critical feedback around the manuscript. without diabetes. Patients with a shorter disease duration had higher frequencies of insulin-reactive CD4+ T cells, with most of these cells being antigen experienced. We also demonstrate Gpc2 that the number of insulin tetramer+ effector memory cells is usually directly correlated with insulin antibody titers, suggesting insulin-specific T- and B-cell interactions. Notably, one of four control subjects with tetramer+ cells was a first-degree relative who had insulin-specific cells with an effector memory phenotype, potentially representing an early marker of T-cell autoimmunity. Our results suggest that studying InsB10C23:DQ8 reactive T-cell frequency and phenotype may provide a biomarker of disease activity in patients with T1D and those at risk. Introduction The strongest genetic risk factor associated with autoimmune type 1 diabetes (T1D) is usually genes within the HLA complex. The HLA-DR4-DQ8 haplotype in humans and MHC class II (MHCII) IAg7 in NOD mice, a spontaneous murine model of autoimmune diabetes, provide the strongest genetic risk for T1D, supporting a critical role for CD4+ T cells in disease development (1). CD4+ and CD8+ T cells, as well as B cells and dendritic cells, are important for the progression of T1D in mice and humans (2). CD8+ T cells mediate direct islet killing, whereas CD4+ T cells may play a critical role to initiate disease by providing help for CD8+ T cells and B cells (3). Interestingly, HLA-DQ8 and mouse IAg7 molecules share structural similarity and have comparable peptide binding preferences (4). Historically, the strongest biological indicator of future T1D onset is the presence of insulin autoantibodies (IAAs), because they can appear years before the clinical onset of T1D and almost all patients diagnosed with T1D aged younger than 6 years with the DR4-DQ8 haplotype are IAA positive (5). In addition, there is considerable evidence in mouse models that insulin is usually a major target during the development of diabetes (6C9). Using a transgenic NOD mouse model, Nakayama et al. (6) decided that a single amino acid substitution in a T-cell receptor contact site within the insulin B-chain (InsB) conferred complete T1D protection by masking the dominant immune peptide target. In separate studies, we as well as others decided that T cells specific for InsB amino acids 9C23 (InsB9C23) are critical for disease development in the spontaneous diabetes NOD mouse model (6, 10). Notably, the amino acid sequence of InsB9C23 is usually identical in mice and humans, which has led others to investigate T-cell reactivity to this epitope in humans. RN486 In a very recent report, InsB9C23Creactive CD4+ T cells were identified from the inflamed pancreatic islets of two organ donors with recent-onset T1D, indicating that these cells are relevant to human disease (11). In individual studies, InsB-specific T cells could be detected in the peripheral blood of patients with new-onset T1D with the use of indirect cytokine ELISAs (12) and expanded from the peripheral blood of patients with T1D with established disease (13). With these discoveries, it is now critical to understand the phenotype of these cells in the peripheral circulation, how the insulin-specific T-cell response relates to disease duration, and whether monitoring insulin-specific CD4+ T-cell responses could be a useful biomarker of disease activity. In the current study, we used peptide:HLAII tetramer staining to compare the frequency and phenotype of InsB-specific CD4+ T cells directly ex vivo within peripheral blood from HLA-DQ8+ patients with T1D and HLA-matched control subjects without diabetes. We found that 54% (20 of 37) of patients with T1D had detectable insulin tetramer+ cells compared with only 15% (4 of 26) of control subjects without diabetes. Within the patients with RN486 T1D, 64% of insulin tetramer+ cells were antigen experienced (CD45RO+). In fact, patients with the most tetramer+ effector memory cells (CD45RO+ CCR7?) had significantly higher insulin antibody titers and the shortest T1D duration. Importantly, tetramer+ cells were enumerated from several RN486 patients with new-onset T1D where RN486 insulin administration was shorter than 15 days, providing evidence that these cells are self-reactive. In one subject without diabetes, a genetically at-risk first-degree relative of a patient with T1D, we found effector memory tetramer+ cells in the absence of IAAs. Taken together, these.