Peripheral blood biomarkers of cell-specific autoimmunity. Studies in children at increased risk for type 1 diabetes

Abstract: Objective While the incidence of children suffering from autoimmune type 1 diabetes (T1D) is increasing in Sweden and worldwide, the underlying etiology and cellular mechanisms behind this remain unknown. The predisposition of the high-risk HLA DR-DQ genotype and as yet unknown environmental triggers lead to autoimmunity and the onset of T1D, which is preceded by islet beta-cell autoantibodies acting as markers for ongoing autoimmunity. This study aims to identify peripheral blood biomarkers to predict and explain cellular autoimmune processes leading to beta-cell loss before and after seroconversion. We also investigate whether immune tolerance treatment with GAD-alum affects T-cells in nondiabetic children at increased genetic risk of T1D prospectively followed in longitudinal studies. Methods Children participating in the Swedish TEDDY cohort with or without islet beta-cell autoantibodies were studied. Complete blood count in these children was analyzed and related to autoantibody status, gender, HLA genotype, and glucose metabolism measures. HbA1c, a predictive biomarker for a subsequent autoantibody or T1D, was analyzed in the TEDDY cohort from Finland, Germany, Sweden, and the US. HbA1c trajectories were also studied in the progression from developing a single autoantibody to diagnosing T1D. Children aged 4–17.99 years at enrollment participating in the DiAPREV-IT2 clinical trial were studied and different T-cells were immunophenotyped to investigate the immune tolerance treatment with GAD-alum. Results A reduction in neutrophil counts primarily in boys and children with the HLA-DR3-DQ2/DR4-DQ8 genotype, and a reduction of red blood cell counts, hemoglobin, and hematocrit primarily in girls and in children with HLA-DR3-DQ2/DR4-DQ8 were inversely associated with autoimmunity and the number of beta-cell autoantibodies. A reduction in red blood cell indices (MCH and MCV) was associated with increased HbA1c, by increased number of beta-cell autoantibodies. Reduction in red blood cell count, hemoglobin, and hematocrit levels were associated with increased fasting blood glucose. Increased red blood cell counts and hemoglobin, hematocrit, and MCH were associated with increased fasting insulin. Increased HbA1c was associated with an increased risk of T1D regardless of the number and type of autoantibodies. The development of IA-2A as a second or fourth autoantibody was associated with decreased HbA1c levels. The HbA1c trajectories presented a more rapid increase of HbA1c as the number of autoantibodies increased from one to three. GAD-alum-treated children had lower T-helper cell (CD3+ CD4+ T-cells ) and cytotoxic T-cell (CD3+ CD8+ T-cells) levels 18–24 months after two immunizations with GAD-alum. Conclusion Reductions in neutrophil levels, red blood cells, and red blood cell parameters and increased levels of HbA1c are all associated with multiple autoantibodies, reflecting a prominent islet autoimmune burden. The reduction in different complete blood counts with increasing numbers of beta-cell autoantibodies may suggest an unknown effect of impaired beta-cell function on hematopoiesis. Predicted trajectories of HbA1c could be used to further develop a model to predict the time to T1D diagnosis in children with multiple autoantibodies. The decrease in HbA1c associated with the appearance of IA-2A may be a consequence of aggressive autoimmune destruction of beta-cells leading to insulin leakage into the bloodstream. These results should prove helpful for understanding the pathogenesis of T1D and better predicting the onset of T1D in seroconverted children. Immunization with GAD-alum has a long-term effect on T-cells 18–24 months after treatment.