Analysis of mass cytometry data with viSNE reveals phenotypic heterogeneity within human Tregs (TECH1P.854)

Abstract

Abstract Regulatory T (Treg) cells play a fundamental role in the development and maintenance of immunological tolerance. Tregs are characterized by constitutive expression of the transcription factor Foxp3, in addition to a surface phenotype of CD4+CD25hiCD127lo. While this pattern of expression has proven highly useful for studying Tregs, the use of additional markers should provide greater resolution of minor subsets within the Treg compartment. Moreover, identification of a concise surface marker profile will facilitate isolation of viable Tregs by FACS. Mass cytometry is a new platform that couples flow cytometry with mass spectrometry, and enables single-cell analysis of at least 30 parameters simultaneously without the requirement for compensation. To profile Treg phenotypes in human peripheral blood, we employed a panel of metal-conjugated antibodies, against both surface and intracellular targets. High-dimensional analysis with the viSNE algorithm, a cytometry analysis tool based on the t-distributed stochastic neighbor embedding algorithm, revealed heterogeneity within the Treg compartment with respect to expression of the markers CD39, CTLA-4 and HLA-DR. Furthermore, we found these markers to be co-expressed primarily in Tregs with the highest level of Foxp3 expression. We propose a gating strategy for identification of Tregs by mass cytometry that can be used to further phenotype human regulatory T cells.