HIGH-DIMENSIONAL ANALYSIS OF HUMAN REGULATORY T CELLS USING MASS CYTOMETRY

Abstract

Abstract Regulatory T cells (Tregs) are critical for maintenance of peripheral tolerance in a variety of tissues. Evidence that Tregs seem to be a phenotypically diverse population that varies between individuals and tissues suggests that there may be functionally-specialized subsets of Tregs which vary depending on location and/or disease state. We developed a new mass cytometry-based method to measure FOXP3 in combination with 20 other parameters to characterize the phenotype of human Tregs in different tissues, and to enable analysis of antigen-specific cells. We first developed a protocol to detect FOXP3, the Treg lineage-defining transcription factor, via mass cytometry. We found this optimized protocol is more sensitive than commercially-available methodology to detect FOXP3 by mass cytometry and is compatible with staining of other intracellular targets such as cytokines and other transcription factors. To ask how Treg populations differ depending on location and disease state, we stained mononuclear cells from adult peripheral blood, pediatric thymus and cord blood, as well as synovial fluid from pediatric subjects with juvenile idiopathic arthritis with our Treg-specific mass cytometry panel. The resulting data were analyzed using several bioinformatics approaches including viSNE and revealed diversity in the heterogeneity and phenotype of Tregs depending on their origin. The ability to stain FOXP3 using mass cytometry will facilitate the further characterization of Tregs in health versus disease and help us understand how Tregs are functionally specialized in the context of different tissues and disease.