The cytokine, transforming growth factor-␤1 (TGF-␤1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-␤1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-␤1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor ␥t (ROR␥t), was rapidly induced by TGF-␤1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that ROR␥t binding is sufficient for activation of the minimum promoter in the HEK 293T cells. ROR␥t-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with ROR␥t through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of ROR␥t-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg.

Although it has been well established that TGF-β plays a pivotal role in immune regulation, the roles of its downstream transcription factors, Smad2 and Smad3, have not been fully clarified. Specifically, the function of Smad2 in the immune system has not been investigated because of the embryonic lethality of Smad2-deficient mice. In this study, we generated T cell-specific Smad2 conditional knockout (KO) mice and unexpectedly found that Smad2 and Smad3 were redundantly essential for TGF-β–mediated induction of Foxp3-expressing regulatory T cells and suppression of IFN-γ production in CD4+ T cells. Consistent with these observations, Smad2/Smad3-double KO mice, but not single KO mice, developed fatal inflammatory diseases with higher IFN-γ production and reduced Foxp3 expression in CD4+ T cells at the periphery. Although it has been suggested that Foxp3 induction might underlie TGF-β–mediated immunosuppression, TGF-β still can suppress Th1 cell development in Foxp3-deficient T cells, suggesting that the Smad2/3 pathway inhibits Th1 cell development with Foxp3-independent mechanisms. We also found that Th17 cell development was reduced in Smad-deficient CD4+ T cells because of higher production of Th17-inhibotory cytokines from these T cells. However, TGF-β–mediated induction of RORγt, a master regulator of Th17 cell, was independent of both Smad2 and Smad3, suggesting that TGF-β regulates Th17 development through Smad2/3-dependent and -independent mechanisms.

Transforming growth factor-beta (TGF-beta) has been shown to play an essential role in the suppression of inflammation, yet recent studies have revealed the positive roles of TGF-beta in inflammatory responses. For example, TGF-beta induces Foxp3-positive regulatory T cells (iTregs) in the presence of interleukin-2 (IL-2), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-beta inhibits the proliferation of immune cells as well as cytokine production via Foxp3-dependent and -independent mechanisms. Little is known about molecular mechanisms involved in immune suppression via TGF-beta; however, Smad2/3 have been shown to play essential roles in Foxp3 induction as well as in IL-2 and IFN-gamma suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Interaction between TGF-beta and other cytokine signaling is important in establishing the balance of immunity and tolerance.

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