Mass cytometry identifies specific Treg and NK subsets that are increased in individuals at high risk of T1D

Background: Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin producing β-cells. Immunotherapeutic prevention before disease onset has the best potential to preserve β-cell function, and thus, it is crucial to understand the autoimmune process leading to T1D. Although immune system deviations have been detected in peripheral blood of T1D patients, the preclinical period has not been fully characterized. High-dimensional mass cytometry overcomes the main limitations of fluorochrome-based flow cytometry allowing simultaneous analysis of over 30 cellular parameters at single cell resolution. In this study, we took advantage of the unique opportunities offered by mass cytometry to evaluate immune system alterations during the pre-diabetic period.
Methods: We developed a panel of 32 metal-labelled monoclonal antibodies including lineage markers and markers related to cell differentiation, activation, trafficking, transcription factors and cytokines. PBMC from children with high risk of T1D positive for multiple autoantibodies who progressed to disease (n=10) from the ABIS cohort and age and sex matched healthy individuals (n=9) were analyzed with our panel before and after stimulation with PMA and ionomycin. We applied a t-distributed stochastic neighbor embedding (t-SNE)-based approach in combination with a clustering algorithm to perform simultaneous analysis of all markers in an unbiased fashion.Results: 8 immune lineages (CD4 T cells, CD8 T cells, double negative T cells, B cells, NK cells, monocytes, myeloid dendritic cells and plasmacytoid dendritic cells) were defined based on lineage marker expression. t-SNE-clustering-based analysis identified 134 distinct subsets within these lineages. Evaluation of t-SNE map profiles and cluster frequencies revealed a memory Treg subset and 2 NK subsets that were significantly more abundant in individuals at high risk of T1D than in controls. Conclusions: High-dimensional profiling reveals alterations in subsets of Treg and NK cells during the preclinical period of T1D, suggesting increased immune activity before disease onset.