Phenotypic and Functional Analysis of the Suppressive Function of Human Regulatory T Cells

Regulatory T cells (Tregs) are defined as immunosuppressive cells playing crucial roles in the establishment and maintenance of immune homeostasis. During the course of immune responses, Tregs control the balance between host defense from pathogens and the prevention of excessive immunity. Here, we describe the phenotypic analysis of Tregs, evaluated in peripheral blood mononuclear cells of healthy adults, by multiparameter flow cytometry, which allows examining the expression of peculiar markers of Treg subpopulations with different features, and provides efficient discrimination of multiple characteristics at the single-cell level. The same technique may be applied to characterize mononuclear cells extracted from different specimens, including whole blood, biological fluids or solid tissues. The immunoregulatory identity of a certain Treg subpopulation may be functionally verified by performing an in vitro suppression assay described here, testing the capability of Tregs to suppress the activation of responder cells.