Analysis of Human FOXP3+ Treg Cells Phenotype and Function

Naturally occurring regulatory T (nT _Reg ) cells play a critical role in the establishment of immunological self-tolerance in humans. Currently, the analysis of nT _Reg cell function from bulk PBMC has led to discrepancies, largely due to the failure to discriminate T _Reg cells from other antigen-experienced CD4⁺ T cells in states of inflammation. We developed a novel, multiparametric, single-cell strategy approach, which consists of isolating and expanding individual CD4⁺ CD25⁺ T cells into clones, in turn allowing us to discriminate bona fide T _Reg cells from activated, FOXP3⁺ T _Eff cells, which frequently confound bulk CD25^High T _Reg functional assays. This approach enabled us to compare their phenotype and function at the single-cell level and to uncover the functional heterogeneity that exists among the CD4⁺ FOXP3⁺ T _Reg cell population in human PBMC.