Reciprocal Interactions Between Conventional CD4 T Cells and Regulatory T Cells Alter the Properties of Regulatory T Cells and Promote the Development of IL-17 Producing Cells.

Abstract 709

The differentiation and functional specialization of effector T cells allows for effective immune response to diverse insults. However, tight regulation of effector T cell responses is required for effective control of infections and avoidance of autoimmunity. Naïve CD4 T cells can differentiate into IFN-γ-secreting type I (Th1) cells and IL-4-secreting type II (Th2) cells. Recently, the Th1/Th2 paradigm of T helper (Th) cells differentiation has been expanded following the discovery of a third subset of effector Th cells that produce IL-17 (Th17). Regulatory T (Treg) cells have a remarkable ability to prevent naïve T cell differentiation into Th1 and Th2 cells and to suppress immune responses driven by Th1 and Th2 effector cells. The role of Treg cells in regulating IL-17 production remains undetermined. Some studies suggest that Treg cells may promote differentiation of naïve T cells into Th17 cells in the context of inflammatory cytokine milieu. The aim of our present study was to determine the role of Treg cells and conventional CD4⁺ T cells (Tconv) in the differentiation of IL-17 producing cells in the absence of exogenous cytokines and insults. Naïve Tconv cells stimulated with anti-CD3 mAb in the presence of antigen presenting cells (APCs) secreted significant amounts of IFN-γ and IL-4 but no detectable levels of IL-17, whereas Treg cells were incapable of producing any of these cytokines under the same culture conditions. Production of IFN-γ and IL-4 was significantly reduced by addition of Treg cells in the cultures of Tconv cells with anti-CD3 mAb and APC. In contrast, production of IL-17 was considerably enhanced in these co-culture conditions and the level of IL-17 displayed a positive correlation with the number of Treg cells added in the culture. To evaluate whether TCR-mediated stimulation of both Treg and Tconv cells was required for IL-17 production, we used Tconv cells and Treg cells from two different TCR transgenic mouse strains in H-2^b background, 2D2 (MOG_35-55-specific) and OT-II (OVA_323-339-specific), respectively, and co-cultured them in the presence of APCs (H-2^b). Production of IL-17 was not observed when either MOG peptide or OVA peptide alone was added in the cultures. In contrast, addition of both MOG and OVA resulted in production of IL-17, suggesting that simultaneous activation of Tconv and Treg cells was essential for induction of IL-17. To determine the source of IL-17 during co-culture of Treg and Tconv cells, we purified Treg cells from C57/B6 mice and co-cultured them with Tconv cells from the B6 congenic mouse strain B6.PL, which carry the Thy1a (Thy1.1) allele and can be easily recognized by flow cytomeric analysis using a Thy1.1-specific mAb. Detailed evaluation during co-culture revealed that a significant proportion of Thy1.1^- T cells (the source of Treg) gradually downregulated expression of Foxp3 while obtaining expression of IL-17. In contrast, there was no significant change in the expression of either Foxp3 or IL-17 in the Thy1.1⁺ population (the source of Tconv), suggesting that Treg was the main source of IL-17 when stimulated in the presence of antigen and activated Tconv cells. Several cytokines have been implicated in the induction of IL-17, in particular, TGF-β. For this reason, we investigated the potential involvement of TGF-β in this conversion process. Addition of TGF-β to Tconv cultured with APCs and anti-CD3 mAb in the absence of Treg cells resulted in upregulation of Foxp3 but not IL-17. In contrast, addition of TGF-β neutralizing antibody to Tconv cultured with APC and anti-CD3 mAb in the presence of Treg, suppressed IL-17 production. Moreover, assessment of TGF-β signaling in Tconv and Treg cells revealed a dramatically increased level of Smad3 phosphorylation in Treg compared to Tconv cells, indicating a reduced threshold of TGF-β mediated signaling in Treg cells. Taken together, our data indicate that reciprocal interactions of Treg and Tconv cells are required for conversion of Treg into IL-17 producing cells and that TGF-β-mediated signaling is required for this process. In addition, our results provide evidence that Treg may convert into proinflammatory effectors producing IL-17, under conditions that promote Tconv differentiation into Treg cells. These observations provide a new dimension to our understanding of Treg cells functions and may have important implications in therapeutic strategies using Treg cells.