STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25- effector T cells.

Forkhead box P3 (FOXP3) is considered a specific marker for CD4(+)CD25(+) regulatory T (Treg) cells, but increasing evidence suggests that human CD4(+)CD25(-) effector T (Teff) cells can transiently express FOXP3 upon activation. We demonstrate that the signal transducer and activator of transcription 5 (STAT5)-signaling cytokines, IL-2, IL-15 and to a lesser extent IL-7, induce FOXP3 up-regulation in vitro in activated human Teff cells. In contrast, cytokines which do not activate STAT5, such as IL-4 or transforming growth factor-beta alone, do not directly induce FOXP3 expression in activated Teff cells. Moreover, expression of a constitutively active form of STAT5a is sufficient to induce FOXP3 expression in Teff cells. Expression of FOXP3 in activated Teff cells requires both TCR-mediated activation and endogenous IL-2, but is not dependent on cell division and does not induce suppressive function. The presence of STAT5-activating cytokines is also required to maintain high FOXP3 expression and suppressive activity of Treg cells in vitro. These data indicate that activation of STAT5 sustains FOXP3 expression in both Treg and Teff cells and contribute to our understanding of how cytokines affect the expression of FOXP3.

Generation of potent and stable human CD4+ T regulatory cells by activation-independent expression of FOXP3.

Therapies based on enhancing the numbers and/or function of T regulatory cells (Tregs) represent one of the most promising approaches to restoring tolerance in many immune-mediated diseases. Several groups have investigated whether human Tregs suitable for cellular therapy can be obtained by in vitro expansion, in vitro conversion of conventional T cells into Tregs, or gene transfer of the FOXP3 transcription factor. To date, however, none of these approaches has resulted in a homogeneous and stable population of cells that is as potently suppressive as ex vivo Tregs. We developed a lentivirus-based strategy to ectopically express high levels of FOXP3 that do not fluctuate with the state of T-cell activation. This method consistently results in the development of suppressive cells that are as potent as Tregs and can be propagated as a homogeneous population. Moreover, using this system, both naïve and memory CD4(+) T cells can be efficiently converted into Tregs. To date, this is the most efficient and reliable protocol for generating large numbers of suppressive CD4(+) Tregs, which can be used for further biological study and developed for antigen-specific cellular therapy applications.