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.