Combined unsupervised and semi-automated supervised analysis of flow cytometry data reveals cellular fingerprint associated with newly diagnosed pediatric type 1 diabetes

Type 1 diabetes is a chronic autoimmune disease resulting in an immune-mediated loss of pancreatic β-cells; however, an unbiased and reproducible profiling of type 1 diabetes-specific circulating immunome at disease onset has yet to be explored. In this study, fresh whole blood was collected from a pediatric cohort of 107 patients with new-onset type 1 diabetes, 85 relatives of patients with type 1 diabetes with 0-1 islet autoantibodies, 58 patients with celiac disease or autoimmune thyroiditis and 76 healthy controls. Up to 6 mL of blood was collected from each subject into a VACUETTE® TUBE 6 ml ACD-B (Greiner). Fresh whole blood underwent red blood cell lysis, was washed and stained with specific monoclonal antibodies. Fresh whole blood samples were stained with five panels of antibodies labelled as T cells, T&NK cells, B cells, Tregs and DCs/monos encompassing main subsets of  T cells, NK cells, B cells, Tregs, DCs and monocytes detected using 26 surface markers and the intracellular marker forkhead box P3 (FoxP3); for the Treg panel, intracellular staining was performed after fixation and permeabilization. Cells were acquired on a BD FACSCanto-II flow cytometer equipped with FACSDiva software (Becton Dickinson, Franklin Lakes, NJ).