Editorial: Optimizing iTreg differentiation: STATs in the balance

The pathways promoting the formation of iTregs are under intense scrutiny. In this issue of the Journal of Leukocyte Biology, Betts et al. [1] demonstrate that selective STAT3 inhibition during allogeneic stimulation in vitro increases Foxp3 expression and Foxp3 promoter demethylation in human T cells. This commentary expands discussion on the competing roles of STAT3and STAT5-dependent signaling pathways in iTreg induction. When activated in the presence of TGF1, Tconv cells up-regulate Foxp3 and develop suppressive function. These iTregs and the larger nTreg population that develops in the thymus are essential and act cooperatively to enforce peripheral tolerance [2]. Differences between the two Foxp3 subsets include nonoverlapping TCR repertoires and the methylation status of the Foxp3 promoter. Demethylation of the TSDR in the Foxp3 promoter is associated with Treg stability and heritable expression of Foxp3 [3]. The inflammatory milieu present during iTreg induction has a profound effect on the frequency and function of Tconv cells that up-regulate Foxp3. For example, GvHD requires the STAT3dependent cytokines IL-6, IL-21, and IL-23. STAT3-dependent cytokines also inhibit Foxp3 expression, whereas IL-2 and the downstream STAT5-dependent signaling pathways are required for iTreg induction. There are activating STAT5and inhibitory STAT3-binding sites in the Foxp3 promoter, which suggests that competition between STAT transcription factors is an important control checkpoint [4, 5]. Indeed, iTreg production is improved in the absence of STAT3. This view of STAT3 as a negative regulator of Foxp3 transcription is complicated by the observation that complete STAT3 deficiency in Tregs compromises their capacity to restrain Th17 responses [6]. Phosphorylated STAT3 physically interacts with Foxp3 to promote the capacity of Tregs to suppress Th17 responses while limiting the Treg expression of TGF1 and IL-6, cytokines that mediate Th17 cell differentiation. In Tregs, STAT3 couples the IL-10R to downstream gene targets and is therefore relevant to Treg stability and function. These confounding observations are best reconciled by appreciating that STAT3 and STAT5 pathways dynamically integrate different signals involved in Treg versus Th17 cell development and function. Thus, when TGFis present during T cell activation, the integration of multiple environmental cues mediated by STAT5 and STAT3 signaling pathways shapes the overall size of the iTreg/Th17 cell axis, as well as the proportional and functional capacity of each cell type. Building on their previous studies showing that JAK2 inhibition reduced human Th1and Th17-mediated alloreactivity [7], Betts et al. employed a similar allogeneic MLR system using monocyte-derived DCs as APCs (Fig. 1). T cells that received allostimulation plus a selective STAT3 inhibitor showed reduced proliferation, consistent with another established role of STAT3, namely, promoting T cell proliferation and survival [8]. Importantly, the STAT3 blockade shifted the ratio of phosphorylated STAT3 and STAT5 proteins in favor of phosphorylated STAT5, which resulted in a dose-dependent increase in iTregs that retained potent in vitro-suppressive capability. The converse was also true, in that selective STAT5 inhibition decreased iTreg production. Adoptive transfer of different Treg populations has been proposed as therapy for autoimmune diseases and GvHD. Mouse studies show that iTregs and nTregs persist after transfer and mediate tolerance. However, in vitroproduced iTregs are unstable, and loss of Foxp3 expression leaves them vulnerable to deviation down a Th1 or Th17 effector pathway [9]. In published human trials, Treg adoptive transfer immunotherapy was beneficial, although one significant limitation appears to be the stability of the transferred Tregs [10]. When Betts et al. [1] examined the promoter methylation status of the Foxp3 promoter in iTregs, produced in vitro under conditions of STAT3 inhibition, they found significant demethylation of the TSDR. This effect was correlated with a reduction in the number of cells